- Basu, S; Prasad, UV; Barawkar, DA; De, S; Palle, VP; Menon, S; Patel, M; Thorat, S; Singh, UP; Das Sarma, K; Waman, Y; Niranjan, S; Pathade, V; Gaur, A; Reddy, S; Ansari, S Bioorg Med Chem Lett 22: 2843-9 (2012)
- ChEMBL_342906 (CHEMBL868830) Inhibition of [14C]anandamide hydrolysis in rat brain membrane in the absence of UV irradiation
- ChEMBL_342907 (CHEMBL863817) Inhibition of [14C]anandamide hydrolysis in rat brain membrane in the presence of UV irradiation
- ChEMBL_2092230 (CHEMBL4773493) Binding affinity to Pin1 (unknown origin) preincubated for 2 hrs followed by UV irradiation for 15 mins by SDS-PAGE gel based photoaffinity labelling method
- ChEMBL_1891594 (CHEMBL4393421) Antagonistic activity against NK3R (unknown origin) expressed in CHO cells assessed as inhibition of NKB-induced intracellular calcium flux under UV-light irradiation by Calcium 4 assay
- ChEMBL_2172009 (CHEMBL5057143) Agonist activity at TRPA1 expressed in CHO cells assessed as activation of channel current in presence of 330 - 380 nm UV irradiation by whole-cell patch clamp electrophysiology
- ChEMBL_2349562 Inhibition of PI3Kalpha (unknown origin) using PIP2 as substrate incubated for 1 hr in presence of ATP under irradiation with 365 nm UV light by ADP-Glo luminescent assay
- UV-vis cuvette-based assay UV-vis cuvette-based assay.
- ChEMBL_2215583 (CHEMBL5128715) Inhibition of ABCG2 (unknown origin) expressed in human MCF7 cells membrane mediated [125I]iodoarylazidoprazosin photolabelling for 5 mins followed by UV light irradiation for 10 mins by radiometric scintillation analysis
- ChEMBL_2215589 (CHEMBL5128721) Inhibition of ABCG2 (unknown origin) expressed in human MCF7 cells membrane mediated [125I]iodoarylazidoprazosin photolabelling for 10 mins followed by UV light irradiation for 10 mins by radiometric scintillation analysis
- ChEMBL_2238600 (CHEMBL5152496) Inhibition of recombinant human GSTA1-1 using GSH and CDNB as substrate preincubated for 5 mins followed by substrate addition and measured in the absence of UV irradiation by spectrophotometric analysis
- ChEMBL_2238602 (CHEMBL5152498) Inhibition of recombinant human GSTP1-1 using GSH and CDNB as substrate preincubated for 5 mins followed by substrate addition and measured in the absence of UV irradiation by spectrophotometric analysis
- ChEMBL_2238601 (CHEMBL5152497) Inhibition of recombinant human GSTA1-1 using GSH and CDNB as substrate preincubated for 2 mins followed by substrate addition and measured upto 6 mins in the presence of UV irradiation by spectrophotometric analysis
- ChEMBL_2238603 (CHEMBL5152499) Inhibition of recombinant human GSTP1-1 using GSH and CDNB as substrate preincubated for 2 mins followed by substrate addition and measured upto 6 mins in the presence of UV irradiation by spectrophotometric analysis
- ChEBML_1693600 Inhibition of human MDR1 expressed in Sf9 cell membranes assessed as reduction in [125I]-IAAP binding incubated for 20 mins under UV light irradiation and measured after 30 mins by densitometry based photo-affinity labeling assay
- ChEBML_1693601 Inhibition of human ABCG2 expressed in Sf9 cell membranes assessed as reduction in [125I]-IAAP binding incubated for 20 mins under UV light irradiation and measured after 30 mins by densitometry based photo-affinity labeling assay
- ChEMBL_1476207 (CHEMBL3429393) Inhibition of PTP1B catalytic domain (1 to 321 amino acids) (unknown origin) expressed in Escherichia coli Rosetta (DE3) incubated for 30 mins followed by UV irradiation with 365 nm for 45 mins by pNPP assay
- ChEMBL_1476206 (CHEMBL3429392) Inhibition of PTP1B catalytic domain (1 to 321 amino acids) (unknown origin) expressed in Escherichia coli Rosetta (DE3) incubated for 30 mins in absence of UV irradiation with 365 nm for 45 mins by pNPP assay
- ChEMBL_1693601 (CHEMBL4044491) Inhibition of human ABCG2 expressed in Sf9 cell membranes assessed as reduction in [125I]-IAAP binding incubated for 20 mins under UV light irradiation and measured after 30 mins by densitometry based photo-affinity labeling assay
- ChEMBL_1901710 (CHEMBL4403932) Inhibition of DNA-PK in human A549 cells assessed as reduction in irradiation-induced autophosphorylation at S2056 residue preincubated for 1 hr followed by 8 Gy irradiation and measured after 1 hr by ELISA
- ChEMBL_2229348 (CHEMBL5142861) Inhibition of DNA-PK in human A549 cells assessed as reduction in irradiation-induced autophosphorylation at S2056 residue preincubated for 1 hr followed by 8 Gy irradiation and measured after 1 hr by ELISA
- ChEMBL_1476209 (CHEMBL3429395) Inhibition of PTP1B catalytic domain (1 to 321 amino acids) (unknown origin) expressed in Escherichia coli Rosetta (DE3) incubated for 30 mins followed by UV irradiation with 365 nm for 45 mins in presence of 1 mM DTT by pNPP assay
- Spectral Binding Assay All UV/Vis spectra of the enzyme-ligand complexes were obtained on a Cary 5000 UV/Vis spectrophotometer.
- ChEMBL_1901760 (CHEMBL4403982) Inhibition of ATM in human HT-29 cells assessed as reduction in irradiation-induced autophosphorylation at Ser1981 residue preincubated for 1 hr followed by 6 Gy irradiation and measured after 1 hr by Hoechst staining-based imaging analysis
- ChEMBL_1476208 (CHEMBL3429394) Inhibition of PTP1B catalytic domain (1 to 321 amino acids) (unknown origin) expressed in Escherichia coli Rosetta (DE3) incubated for 30 mins in absence of UV irradiation with 365 nm for 45 mins and in presence of 1 mM DTT by pNPP assay
- ChEMBL_2127680 (CHEMBL4837025) Inhibition of AF9 YEATS domain (1 to 138 residues) (unknown origin) assessed as inhibition of probe-1 induced AF9 YEATS domain labelling incubated for 10 mins followed by UV-irradiation for 20 mins by Photo-cross linking based In-gel fluorescence scanning analysis
- ChEMBL_2440853 Inhibition of PI3Kalpha (unknown origin) preincubated for 30 sec under shaking condition followed by 15 mins incubation at 25 degreeC and measured after 60 mins of substrate addition (compound prepared in presence of 365 nm UV light irradiation) by ADP-Glo reagent based assay
- ChEMBL_2440859 Inhibition of PI3Kbeta (unknown origin) preincubated for 30 sec under shaking condition followed by 15 mins incubation at 25 degreeC and measured after 60 mins of substrate addition (compound prepared in presence of 365 nm UV light irradiation) by ADP-Glo reagent based assay
- ChEMBL_2440861 Inhibition of PI3Kdelta (unknown origin) preincubated for 30 sec under shaking condition followed by 15 mins incubation at 25 degreeC and measured after 60 mins of substrate addition (compound prepared in presence of 365 nm UV light irradiation) by ADP-Glo reagent based assay
- ChEMBL_2440863 Inhibition of PI3Kgamma (unknown origin) preincubated for 30 sec under shaking condition followed by 15 mins incubation at 25 degreeC and measured after 60 mins of substrate addition (compound prepared in presence of 365 nm UV light irradiation) by ADP-Glo reagent based assay
- ChEMBL_2304356 Inhibition of human CA1 by UV spectrophotometric analysis
- ChEMBL_2304357 Inhibition of human CA2 by UV spectrophotometric analysis
- ChEMBL_2261256 (CHEMBL5216267) Inhibition of human TDO by UV based assay
- ChEMBL_2270666 Inhibition of PFKFB3 (unknown origin) by UV-vis spectrophotometry
- ChEMBL_2304358 Inhibition of AChE (unknown origin) by UV spectrophotometric analysis
- ChEMBL_595938 (CHEMBL1041787) Inhibition of Helicobacter pylori DHQ2 by UV spectroscopy
- ChEMBL_696628 (CHEMBL1641179) Inhibition of soybean lipoxygenase at by UV spectrophotometry
- ChEMBL_1586205 (CHEMBL3819920) Inhibition of TCPTP (unknown origin) by UV/Vis spectrophotometry
- Binding Assay Substrate and ligand binding assay using UV- visible absorbance analysis of CYP142 was done on a Cary UV-50 UV-visible scanning spectrophotometer (Varian, UK) using a 1-cm path length quartz cuvette, recording spectra between 250 and 800 nm.
- ChEMBL_1290921 (CHEMBL3118894) Inhibition of 5-LOX (unknown origin) by UV-Vis spectrophotometry
- ChEMBL_1449304 (CHEMBL3371450) Inhibition of human leukocyte 5-lipoxygenase by UV-visible spectrophotometry
- ChEMBL_1449305 (CHEMBL3371451) Inhibition of human platelet 12-lipoxygenase by UV-visible spectrophotometry
- ChEMBL_1449308 (CHEMBL3371454) Inhibition of rabbit reticulocyte 15-LOX by UV-visible spectrophotometry
- ChEMBL_1556337 (CHEMBL3766396) Inhibition of human COX-2 by UV-visible spectrophotometric analysis
- ChEMBL_1672830 (CHEMBL4022859) Inhibition of recombinant human GAA by UV-visible spectrophotometric method
- ChEMBL_1910991 (CHEMBL4413437) Binding affinity to Trypanosoma cruzi CYP51 by UV-vis spectrophotometry
- ChEMBL_828512 (CHEMBL2050039) Binding affinity to Mycobacterium tuberculosis CYP125A1 by UV-visible spectrophotometry
- ChEMBL_1449306 (CHEMBL3371452) Inhibition of human reticulocyte 15-lipoxygenase-1 by UV-visible spectrophotometry
- ChEMBL_1449307 (CHEMBL3371453) Inhibition of human reticulocyte 15-lipoxygenase-2 by UV-visible spectrophotometry
- ChEMBL_1495504 (CHEMBL3579888) Competitive inhibition of human recombinant COX-2 by UV-Visible spectrophotometry
- ChEMBL_1564890 (CHEMBL3784810) Inhibition of porcine tubulin polymerization by UV-Vis microplate reader analysis
- ChEMBL_1715048 (CHEMBL4125097) Inhibition of human erythrocyte CuZn-SOD by UV-Visible spectrophotometric method
- ChEMBL_1782414 (CHEMBL4253931) Binding affinity to Mycobacterium tuberculosis PtpB by UV-VIS spectrophotometric analysis
- ChEMBL_1910488 (CHEMBL4412934) Inhibition of human erythrocyte CuZn-SOD by UV-Visible spectrophotometric method
- ChEMBL_1912043 (CHEMBL4414489) Inhibition of human erythrocyte CuZn-SOD by UV-Visible spectrophotometric method
- ChEMBL_2024495 (CHEMBL4678308) Inhibition of human erythrocytes CuZn-SOD by UV-VIS spectrophotometric method
- ChEMBL_216786 (CHEMBL817062) Inhibition constant for inhibition of alpha-chymotrypsin by the UV light
- ChEMBL_2494936 Inhibition of human TS incubated for 5 mins by UV-spectrophotometry analysis
- ChEMBL_537038 (CHEMBL986187) Inhibition of pig kidney microsome aminopeptidase N by UV-visible spectrophotometer
- ChEMBL_563472 (CHEMBL961020) Inhibition of pig kidney microsome aminopeptidase N by UV-visible spectrophotometer
- ChEMBL_664208 (CHEMBL1261649) Binding affinity to Trypanosoma cruzi sterol 14-alpha-demethylase by UV-Spectrophotometry
- ChEMBL_835274 (CHEMBL2071980) Binding affinity to full-lenght/His-tagged ERBB2 UV-vis emission spectroscopy
- ChEMBL_2016850 (CHEMBL4670428) Agonist activity at PPARgamma (unknown origin) expressed in HEK293T cells incubated for 14 to 16 hrs under irradiation by hybrid Gal4 reporter gene assay
- ChEMBL_1347345 (CHEMBL3265474) Inhibition of horse serum BuChE using butyrylthiocholine as substrate by UV spectroscopic analysis
- ChEMBL_1456602 (CHEMBL3369984) Inhibition of human recombinant carboxy-terminal His-tagged LDHA by UV endpoint assay
- ChEMBL_1456606 (CHEMBL3369988) Inhibition of human recombinant carboxy-terminal His-tagged LDHB by UV endpoint assay
- ChEMBL_1921724 (CHEMBL4424569) Inhibition of soybean lipoxygenase using sodium linoleate as substrate by UV-based analysis
- ChEMBL_1928156 (CHEMBL4431228) Inhibition of human recombinant carboxy-terminal His-tagged LDHA by UV endpoint assay
- ChEMBL_1928157 (CHEMBL4431229) Inhibition of human recombinant carboxy-terminal His-tagged LDHB by UV endpoint assay
- ChEMBL_2283055 Induction of bovine serum albumin denaturation incubated for 23 mins by UV-Visible spectrophotometry
- ChEMBL_552544 (CHEMBL953988) Binding affinity to human serum albumin at 4 uM by UV-vis measurement
- ChEMBL_650320 (CHEMBL1224978) Inhibition of Escherichia coli AmpC assessed as nitrocefin hydrolysis by UV-Vis spectrophotometry
- ChEMBL_675415 (CHEMBL1273495) Inhibition of human 5-lipoxygenase after 15 mins by UV-vis spectrophotometer analysis
- ChEMBL_755432 (CHEMBL1804901) Inhibition of bovine liver DHFR using dihydrofolic acid substrate by UV-visible spectrophotometry
- ChEMBL_1821278 (CHEMBL4320938) Displacement of [3H]-QNB from muscarinic receptor in rat brain membranes incubated for 45 mins under irradiation exposure at 365 nm by radioligand binding assay
- ChEMBL_647718 (CHEMBL1220315) Inhibition of calcineurin-mediated NFAT activation in human Jurkat cells measured after 45 min of irradiation with 740 nm light by luciferase reporter gene assay
- ChEMBL_1772820 (CHEMBL4229812) Competitive inhibition of human plasma kallikrein using S2302 as substrate by UV-visible spectrophotometer
- ChEMBL_1772821 (CHEMBL4229813) Competitive inhibition of human plasmin using chromozyme PL as substrate by UV-visible spectrophotometer
- ChEMBL_1823749 (CHEMBL4323513) Binding affinity to Mycobacterium tuberculosis His6-tagged CYP121 by UV-visible scanning spectrophotometric analysis
- ChEMBL_1852472 (CHEMBL4353096) Inhibition of recombinant human AChE using acetylthiocholine as substrate by UV-visible absorption method
- ChEMBL_2220562 (CHEMBL5133896) Inhibition of human proteasome subunit beta type-5i using fluorogenic substrate by UV-spectrometry
- ChEMBL_2220563 (CHEMBL5133897) Inhibition of human proteasome subunit beta type-2i using fluorogenic substrate by UV-spectrometry
- ChEMBL_2282138 Inhibition of mushroom tyrosinase using L-DOPA as a substrate by UV-2450 spectrophotometeric assay
- ChEMBL_2300004 Inhibition of Mycobacterium tuberculosis CitA expressed in Escherichia coli cell by UV spectrophotometric based analysis
- ChEMBL_563681 (CHEMBL964242) Inhibition of pig kidney microsomal aminopeptidase N after 30 mins by UV-VIS spectrophotometry
- ChEMBL_599273 (CHEMBL1041001) Inhibition of Carboxypeptidase A assessed as amount of Cl-CPL consumed by UV spectrometer
- ChEMBL_636732 (CHEMBL1167021) Inhibition of human platelet N-terminal His6-tagged 12-lipoxygenase by UV-vis spectrometry
- ChEMBL_636733 (CHEMBL1167022) Inhibition of human reticulocyte N-terminal His6-tagged 15-lipoxygenase by UV-vis spectrometry
- ChEMBL_743326 (CHEMBL1767573) Inhibition of rat recombinant nNOS expressed in Escherichia coli by UV-vis spectrometric analysis
- ChEMBL_743327 (CHEMBL1767574) Inhibition of bovine recombinant eNOS expressed in Escherichia coli by UV-vis spectrometric analysis
- ChEMBL_647730 (CHEMBL1220327) Inhibition of calcineurin-mediated streptavidin-coupled NFAT activation in human Jurkat cells measured after 45 min of irradiation with 740 nm light by luciferase reporter gene assay
- ChEBML_1686641 Inhibition of human plasma thrombin using chromogenix AB as substrate after 30 secs by UV-spectrophotometry
- ChEBML_1686642 Inhibition of bovine plasma thrombin using chromogenix AB as substrate after 30 secs by UV-spectrophotometry
- ChEMBL_1290548 (CHEMBL3119997) Inhibition of human 5-LOX using arachidonic acid as substrate by UV-vis spectrophotometric analysis
- ChEMBL_1352302 (CHEMBL3269366) Inhibition of human 5-lipoxygenase using arachidonic acid as substrate by UV-vis spectrometric analysis
- ChEMBL_1516310 (CHEMBL3618750) Inhibition of human salivary alpha-amylase using GalG2CNP as substrate by UV-Vis spectrophotometric analysis
- ChEMBL_1556336 (CHEMBL3766395) Inhibition of human 5-LOX using arachidonic acid as substrate by UV-visible spectrophotometric analysis
- ChEMBL_1772822 (CHEMBL4229814) Competitive inhibition of human factor 10a using pefachrome F10a as substrate by UV-visible spectrophotometer
- ChEMBL_1772823 (CHEMBL4229815) Competitive inhibition of human factor 11a using pefachrome PCa as substrate by UV-visible spectrophotometer
- ChEMBL_1776929 (CHEMBL4233921) Inhibition of human recombinant AChE using acetylthiocholine as substrate by UV-visible spectrophotometric Ellman's method
- ChEMBL_1776931 (CHEMBL4233923) Inhibition of horse serum BuChE using acetylthiocholine as substrate by UV-visible spectrophotometric Ellman's method
- ChEMBL_1795343 (CHEMBL4267460) Inhibition of Trypanosoma cruzi trans-sialidase using CF3MuSA as substrate by UV/visible spectrophotometric method
- ChEMBL_1842109 (CHEMBL4342536) Inhibition of cathepsin B (unknown origin) using p-nitroanilide as substrate by UV spectroscopic method
- ChEMBL_1900696 (CHEMBL4402918) Inhibition of human leukocyte 5-LOX using arachidonic acid as substrate by UV/Vis spectrophotometry
- ChEMBL_2220564 (CHEMBL5133898) Inhibition of human proteasome subunit beta type-1i in using fluorogenic substrate by UV-spectrometry
- ChEMBL_636734 (CHEMBL1167023) Inhibition of human N-terminal His6-tagged 5-lipoxygenase arachidonic acid by UV-vis spectrometry
- ChEMBL_647222 (CHEMBL1217363) Inhibition of pig kidney microsome aminopeptidase N after 30 mins by UV-vis spectrophotometric analysis
- ChEMBL_743328 (CHEMBL1767575) Inhibition of mouse macrophage recombinant iNOS expressed in Escherichia coli by UV-vis spectrometric analysis
- ChEMBL_764478 (CHEMBL1821030) Competitive inhibition of Bacillus subtilis LuxS preincubated for 15 mins by UV-vis spectrophotometric analysis
- ChEMBL_1469977 (CHEMBL3414404) Inhibition of wild-type His-tagged human thymidylate synthase after 1 hr by UV-visible spectrophotometry
- ChEMBL_1469978 (CHEMBL3411863) Inhibition of His-tagged human thymidylate synthase K47A mutant after 1 hr by UV-visible spectrophotometry
- ChEMBL_1469979 (CHEMBL3411864) Inhibition of His-tagged human thymidylate synthase F59A mutant after 1 hr by UV-visible spectrophotometry
- ChEMBL_1469980 (CHEMBL3411865) Inhibition of His-tagged human thymidylate synthase L198A mutant after 1 hr by UV-visible spectrophotometry
- ChEMBL_1469981 (CHEMBL3411866) Inhibition of His-tagged human thymidylate synthase Y202A mutant after 1 hr by UV-visible spectrophotometry
- ChEMBL_1670581 (CHEMBL4020469) Inhibition of human AKR1B1 using glyceraldehyde as substrate measured for 3 mins by UV-vis spectrophotometer
- ChEMBL_1745495 (CHEMBL4180005) Inhibition of Trypanosoma cruzi trans-sialidase using Trifluoromethylumbelliferyl alpha-sialoside as substrate by UV/visible spectrophotometer
- ChEMBL_1855609 (CHEMBL4356338) Inhibition of human plasma kallikrein using S2302 as substrate after 10 mins by UV/Vis photometry
- ChEMBL_1979316 (CHEMBL4612451) Inhibition of human aromatase using ASD as substrate incubated for 16 hrs by UV/vis-spectrophotometry
- ChEMBL_2053951 (CHEMBL4708952) Covalent inhibition of FBPase WT (unknown origin) measured for 45 mins by UV-visible spectrometry analysis
- ChEMBL_2281790 Inhibition of alpha glucosidase (unknown origin) using p-nitrophenyl glucopyranoside as substrate by UV Vis- spectrophotometer analysis
- ChEMBL_2282423 Inhibition of carbonic anhydrase 9 (unknown origin) using 4-nitrophenylacetate as substrate by UV/visible spectrophotometer analysis
- ChEMBL_2286062 Inhibition of human PTP1B using pNPP as substrate incubated for 30 min by UV-Visible spectrophotometer assay
- ChEMBL_2437356 Inhibition of GST-tagged human PARP1 incubated for 30 mins by chemiluminescence based UV/visible spectrophotometric analysis
- ChEMBL_2437357 Inhibition of GST-tagged human PARP2 incubated for 30 mins by chemiluminescence based UV/visible spectrophotometric analysis
- ChEMBL_639005 (CHEMBL1168988) Inhibition of human uridine 5'-monophosphate synthase after overnight incubation at room temperature by UV spectroscopy
- ChEMBL_839551 (CHEMBL2090358) Inhibition of mushroom tyrosinase using L-tyrosine as substrate after 5 mins by UV-spectrophotometric analysis
- ChEMBL_839553 (CHEMBL2090360) Inhibition of mushroom tyrosinase using L-DOPA as substrate after 5 mins by UV-spectrophotometric analysis
- ChEMBL_984872 (CHEMBL2432695) Binding affinity to recombinant Trypanosoma cruzi CYP51 expressed in Escherichia coli by UV-Vis spectrophotometric analysis
- ChEMBL_1932515 (CHEMBL4478167) Inhibition of ATM phosphorylation at Ser-1981 residue in human HT-29 cells incubated for 1 hr followed by X-ray irradiation by Hoechst staining based fluorescence plate reader analysis
- ChEBML_1651002 Inhibition of human CA1 using para-nitrophenylacetate as substrate measured over 3 mins by UV-vis spectrophotometric method
- ChEBML_1651003 Inhibition of human CA2 using para-nitrophenylacetate as substrate measured over 3 mins by UV-vis spectrophotometric method
- ChEMBL_1285211 (CHEMBL3107142) Inhibition of ALDH1A1 (unknown origin) using NAD+/propionaldehyde as substrate after 15 mins by UV-fluorescence assay
- ChEMBL_1802610 (CHEMBL4274902) Inhibition of recombinant human TDO using L-Trp as substrate after 75 mins by UV absorption analysis
- ChEMBL_1885371 (CHEMBL4386953) Inhibition of tyrosinase in mouse B16F0 cells using L-DOPA as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1986667 (CHEMBL4620214) Inhibition of human carbonic anhydrase 2 using p-nitrophenyl acetate as substrate by UV-VIS spectrophotometric analysis
- ChEMBL_1986668 (CHEMBL4620215) Inhibition of human carbonic anhydrase 9 using p-nitrophenyl acetate as substrate by UV-VIS spectrophotometric analysis
- ChEMBL_1986669 (CHEMBL4620216) Inhibition of human carbonic anhydrase 5A using p-nitrophenyl acetate as substrate by UV-VIS spectrophotometric analysis
- ChEMBL_2015618 (CHEMBL4669196) Inhibition of AChE in rat brain using acetylthiocholine iodide as substrate by Ellman's UV-vis spectrophotometric method
- ChEMBL_2019646 (CHEMBL4673459) Inhibition of Plasmodium falciparum enoyl-ACP reductase using crotonoyl-CoA as substrate by UV-Vis spectrophotometric method
- ChEMBL_2053920 (CHEMBL4708921) Covalent inhibition of FBPase WT (unknown origin) measured 15 to 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053921 (CHEMBL4708922) Covalent inhibition of FBPase C38S mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053922 (CHEMBL4708923) Covalent inhibition of FBPase C92S mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053923 (CHEMBL4708924) Covalent inhibition of FBPase C116S mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053924 (CHEMBL4708925) Covalent inhibition of FBPase C128S mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053925 (CHEMBL4708926) Covalent inhibition of FBPase C179S mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053926 (CHEMBL4708927) Covalent inhibition of FBPase C281S mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053927 (CHEMBL4708928) Covalent inhibition of FBPase S124A mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053928 (CHEMBL4708929) Covalent inhibition of FBPase N125A mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053929 (CHEMBL4708930) Covalent inhibition of FBPase D127A mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053930 (CHEMBL4708931) Covalent inhibition of FBPase R243A mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053931 (CHEMBL4708932) Covalent inhibition of FBPase R254A mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2053932 (CHEMBL4708933) Covalent inhibition of FBPase Y258A mutant (unknown origin) measured upto 45 mins by UV-visible spectrometry analysis
- ChEMBL_2107117 (CHEMBL4815792) Inhibition of human carbonic anhydrase 2 using p-nitrophenyl acetate as substrate by UV-VIS spectrophotometric analysis
- ChEMBL_2107118 (CHEMBL4815793) Inhibition of human carbonic anhydrase 9 using p-nitrophenyl acetate as substrate by UV-VIS spectrophotometric analysis
- ChEMBL_2236365 (CHEMBL5150261) Inhibition of human ALDH1A1 using propionaldehyde as substrate in presence of NAD+ by UV-visible spectrophometric analysis
- ChEMBL_2267270 Inhibition of human LSD1 expressed in Escherichia coli using pLys4Met peptide as substrate by UV-visible spectrophotometric analysis
- ChEMBL_2281792 Inhibition of alpha glucosidase (unknown origin) using p-nitrophenyl glucopyranoside as substrate by UV based microplate reader analysis
- ChEMBL_2347785 Inhibition of Alpha-glucosidase (unknown origin) using p-nitrophenyl glucopyranoside as substrate by UV based microplate reader analysis
- ChEMBL_723551 (CHEMBL1676791) Inhibition of Pseudomonas aeruginosa beta-lactamase IMP-1 assessed as hydrolysis of nitrocefin by UV spectrophotometric analysis
- ChEMBL_723554 (CHEMBL1676794) Inhibition of Pseudomonas aeruginosa beta-lactamase VIM-2 assessed as hydrolysis of nitrocefin by UV spectrophotometric analysis
- ChEMBL_801785 (CHEMBL1947348) Inhibition of C57BL/6 mouse eNOS assessed as conversion of oxyhemoglobin to methemoglobin by UV spectrophotometer analysis
- ChEMBL_839908 (CHEMBL2090109) Inhibition of Bacillus anthracis MBL Bla2 using nitrocefin as substrate after 12 hrs by UV-spectrophotometric analysis
- Tyrosinase Assay Mushroom tyrosinase using either L-DOPA or L-tyrosine as substrate. In spectrophotometric experiments, enzyme activity was monitored by dopachrome formation at 475 nm with a UV-Vis spectrophotometer (Spectro UV-Vis Double beam; UVD-3500, Labomed, Inc.) at 30 C.
- ChEMBL_1738020 (CHEMBL4153770) Inhibition of ATM autophosphorylation at Ser1981 in human HT29 cells preincubated for 1 hr followed by X ray irradiation and measured after 1 hr by Hoechst 33342 dye-based immunofluorescence assay
- ChEBML_216785 Difference between inhibition of alpha-chymotrypsin by the UV light and inhibition of alpha-chymotrypsin by the ambient light
- ChEMBL_1366787 (CHEMBL3297111) Inhibition of human lysosomal beta-glucocerebrosidase using 2,4-dinitrophenyl-beta-D-glucopyranoside as substrate by UV spectrophotometric analysis
- ChEMBL_1464056 (CHEMBL3406241) Inhibition of human CA-1 using p-nitro-phenylacetate as substrate after 3 mins by UV-Vis spectrophotometry
- ChEMBL_1464057 (CHEMBL3406242) Inhibition of human CA-2 using p-nitro-phenylacetate as substrate after 3 mins by UV-Vis spectrophotometry
- ChEMBL_1507873 (CHEMBL3598884) Inhibition of human acetylcholinesterase incubated for 60 mins using ATChI substrate by DTNB dye based UV-visible spectrophotometry
- ChEMBL_1586211 (CHEMBL3819926) Inhibition of human recombinant PTP1B expressed in Escherichia coli using p-nitrophenyl phosphate as substrate by UV spectroscopy
- ChEMBL_1651002 (CHEMBL4000257) Inhibition of human CA1 using para-nitrophenylacetate as substrate measured over 3 mins by UV-vis spectrophotometric method
- ChEMBL_1651003 (CHEMBL4000258) Inhibition of human CA2 using para-nitrophenylacetate as substrate measured over 3 mins by UV-vis spectrophotometric method
- ChEMBL_1670976 (CHEMBL4021005) Inhibition of recombinant PTP1B (unknown origin) using p-NPP as substrate after 30 mins by UV-spectroscopic analysis
- ChEMBL_1715047 (CHEMBL4125096) Inhibition of Trypanosoma cruzi MHOM/Pe/2011/Arequipa DTU 5 epimastigotes Fe-SOD by UV-Visible spectrophotometric method
- ChEMBL_1748418 (CHEMBL4182928) Inhibition of NDM1 in Escherichia coli BL21 (DE3) using MEPM substrate incubated for 15 mins by UV spectroscopy
- ChEMBL_1823748 (CHEMBL4323512) Binding affinity to Mycobacterium tuberculosis CYP121 expressed in Escherichia coli HMS174 (DE3) by UV-visible scanning spectrophotometric analysis
- ChEMBL_1862497 (CHEMBL4363353) Inhibition of human dihydrofolate reductase assessed as inhibition constant by UV-Vis spectra based Benesi Hilebrand equation analysis
- ChEMBL_1899994 (CHEMBL4402109) Inhibition of His-tagged p97 (unknown origin) in presence of ATP and PNP by UV-transparent microplate assay
- ChEMBL_1908254 (CHEMBL4410612) Inhibition of human dihydrofolate reductase using dihydrofolate as substrate in presence of NADPH by UV-vis spectrophotometry analysis
- ChEMBL_2220555 (CHEMBL5133889) Inhibition of human constitutive proteasome subunit beta type-5 in human erythrocytes using fluorogenic substrate by UV-spectrometry
- ChEMBL_2220560 (CHEMBL5133894) Inhibition of human constitutive proteasome subunit beta type-2 in human erythrocytes using fluorogenic substrate by UV-spectrometry
- ChEMBL_2220561 (CHEMBL5133895) Inhibition of human constitutive proteasome subunit beta type-1 in human erythrocytes using fluorogenic substrate by UV-spectrometry
- ChEMBL_2346632 Inhibition of XO (unknown origin) using xanthine as substrate measured after 2 to 3 mins by UV-spectrophotometric analysis
- ChEMBL_621471 (CHEMBL1100032) Binding affinity to thrombin in presence of 100 mJ/cm'2 UV light by surface plasmon resonance assay
- ChEMBL_642470 (CHEMBL1175843) Inhibition of human quinone reductase 2 expressed in Escherichia coli BL21(DE3) by UV-vis microplate reader analysis
- ChEMBL_675414 (CHEMBL1273494) Inhibition of human N-terminal His6-tagged platelet 12-lipoxygenase after 15 mins by UV-vis spectrophotometer analysis
- ChEMBL_693673 (CHEMBL1637941) Inhibition of Klebsiella pneumoniae HP205 cephalosporinase CMY-36 by UV spectrophotometer in presence of 100 uM of cephalothin
- ChEMBL_839906 (CHEMBL2090107) Inhibition of Bacillus anthracis MBL Bla2 using nitrocefin as substrate preincubated for 20 mins by UV-spectrophotometric analysis
- Cellular Assay ATM and ATR have distinct and overlapping responses to DNA damage. They must participate together and responses must be co-ordinated. Both pathways may be activated by ionising radiation, however only ATR is activated by UV. Since UV treatment is not practical for use in a high throught-put cell assay, the UV mimetic 4NQ0 (Sigma) was chosen to activate the ATR DNA damage response pathway.
- ChEMBL_1437303 (CHEMBL3384806) Inhibition of soybean LOX assessed as reduction in conversion of sodium linoleate to 13-hydroperoxylinoleic acid by UV spectroscopy
- ChEMBL_1468440 (CHEMBL3412604) Inhibition of human recombinant His-tagged HPPD expressed in Escherichia coli BL21(DE3) by UV/visible plate reader analysis
- ChEMBL_1540763 (CHEMBL3745377) Inhibition of human recombinant MAO-A using p-tyramine as substrate after 15 mins by UV/Vis spectrophotometer analysis
- ChEMBL_1540764 (CHEMBL3745378) Inhibition of human recombinant MAO-B using p-tyramine as substrate after 15 mins by UV/Vis spectrophotometer analysis
- ChEMBL_1556334 (CHEMBL3766393) Inhibition of N-terminal His6-tagged human 12-LOX using arachidonic acid as substrate by UV-visible spectrophotometric analysis
- ChEMBL_1750749 (CHEMBL4185509) Inhibition of recombinant human O-GlcNAcase using pNP-GlcNAc as substrate measured for 5 mins by UV-VIS spectrophotometer
- ChEMBL_1766161 (CHEMBL4201408) Inhibition of Plasmodium falciparum DXR assessed as reduction in NADPH oxidation in presence of DOXP by UV-visible spectrophotometry
- ChEMBL_1855608 (CHEMBL4356337) Inhibition of human plasmin using tosyl-Gly-Pro-Lys-pNA as substrate after 10 mins by UV/Vis photometry
- ChEMBL_1988113 (CHEMBL4621660) Inhibition of human DHFR in presence of DHF and NADPH by UV-vis spectrometry by Lineweaver-Burk plot analysis
- ChEMBL_2075537 (CHEMBL4731071) Binding affinity to ornithine aminotransferase (unknown origin) assessed as inhibition constant by SSDH coupled enzyme based UV-Vis spectrophotometry
- ChEMBL_2078862 (CHEMBL4734653) Binding affinity to recombinant human IDO1 expressed in Escherichia coli BL21 incubated for 4 hrs by UV-Visible Spectroscopy
- ChEMBL_2116452 (CHEMBL4825393) Inhibition of human 12-LOX assessed as enzymatic rate using arachidonic acid as substrate by UV/Vis spectrophotometric analysis
- ChEMBL_2116453 (CHEMBL4825394) Inhibition of human 5-LOX assessed as enzymatic rate using arachidonic acid as substrate by UV/Vis spectrophotometric analysis
- ChEMBL_2116454 (CHEMBL4825395) Inhibition of mouse 15-LOX2 assessed as enzymatic rate using arachidonic acid as substrate by UV/Vis spectrophotometric analysis
- ChEMBL_216785 (CHEMBL817061) Difference between inhibition of alpha-chymotrypsin by the UV light and inhibition of alpha-chymotrypsin by the ambient light
- ChEMBL_2264458 Inhibition of recombinant human AChE using acetylthiocholine as substrate measured after 3 mins by DTNB reagent based UV/Vis spectroscopy
- ChEMBL_2321054 Inhibition of Stenotrophomonas maltophilia L1 preincubated for 30 mins followed by nitrocefin solution measured by UV based microtitre plate analysis
- ChEMBL_2375555 Inhibition of Mycobacterium tuberculosis InhA-catalysed chemical reaction in presence of NADH measured for 1 min by UV/Visible spectrophotometry
- ChEMBL_52406 (CHEMBL665909) Inhibition of cPLA2 measuring Calcium ionophore A-23,187-induced arachidonic acid release from bovine platelets with HPLC/UV detection
- ChEMBL_52407 (CHEMBL665910) Inhibition of cPLA2 measuring Calcium ionophore A-23,187-induced arachidonic acid release from bovine platelets with HPLC/UV detection.
- ChEMBL_675412 (CHEMBL1273492) Inhibition of human N-terminal His6-tagged reticulocyte 15-lipoxygenase-1 after 15 mins by UV-vis spectrophotometer analysis
- ChEMBL_675413 (CHEMBL1273493) Inhibition of human N-terminal His6-tagged epithelial 15-lipoxygenase-2 after 15 mins by UV-vis spectrophotometer analysis
- ChEMBL_761394 (CHEMBL1817024) Inhibition of human 5-lipoxygenase assessed as conjugated diene product formation using arachidonic acid by UV-vis spectrophotometer analysis
- ChEMBL_821127 (CHEMBL2039530) Inhibition of ACE from rat kidney using hippuryl-histidyl-leucine as substrate after 30 mins by UV/VIS spectrophotometry
- ChEMBL_839550 (CHEMBL2090357) Inhibition of bovine milk XO assessed as decrease in uric acid formation after 5 mins by UV-spectrophotometric analysis
- ChEMBL_839907 (CHEMBL2090108) Inhibition of recombinant Pseudomonas aeruginosa MBL IMP-1 using nitrocefin as substrate after 12 hrs by UV-spectrophotometric analysis
- ChEMBL_864915 (CHEMBL2175865) Binding affinity to Bacillus thermoproteolyticus thermolysin by UV-VIS spectrophotometric analysis in presence of 2-furanacryloyl-Gly-Leu-NH2
- ChEMBL_950925 (CHEMBL2349998) Inhibition of human KAT2 using L-kynurenine as substrate after 15 to 20 hrs by UV-visible spectra analysis
- ChEMBL_2093866 (CHEMBL4775129) Inhibition of DNA-PK in human A549 cells assessed as reduction in radiation-induced autophosphorylation at S2056 residue preincubated for 1 hr followed by 8 Gy irradiation and measured after 1 hr by ELISA
- ChEBML_1560521 Inhibition of porcine microsomal aminopeptidase N preincubated for 30 mins using L-Leu-p-nitroanilide as substrate by UV-VIS spectrophotometer
- ChEMBL_1290549 (CHEMBL3119998) Inhibition of N-terminal His6-tagged human epithelial 15-LOX2 using arachidonic acid as substrate by UV-vis spectrophotometric analysis
- ChEMBL_1290552 (CHEMBL3116601) Inhibition of N-terminal His6-tagged human reticulocyte 15-LOX1 using arachidonic acid as substrate by UV-vis spectrophotometric analysis
- ChEMBL_1290553 (CHEMBL3116602) Inhibition of N-terminal His6-tagged human platelet 12-LOX using arachidonic acid as substrate by UV-vis spectrophotometric analysis
- ChEMBL_1352303 (CHEMBL3269367) Inhibition of N-terminal His6-tagged human platelet 12-lipoxygenase using arachidonic acid as substrate by UV-vis spectrometric analysis
- ChEMBL_1432452 (CHEMBL3385734) Inhibition of alpha-mannosidase (unknown origin) using p-nitrophenyl-alpha-D-mannopyranoside substrate incubated for 10 mins by UV spectrophotometry
- ChEMBL_1457647 (CHEMBL3368715) Inhibition of BChE in rat serum using butyrylthiocholine iodide substrate incubated for 15 mins by UV spectroscopy based Ellman's method
- ChEMBL_1490921 (CHEMBL3533591) Drug metabolism assessed as human recombinant UGT1A1-mediated formation of scutellarein-6,7-diglucuronide after 25 mins by HPLC/UV analysis
- ChEMBL_1491709 (CHEMBL3536901) Drug metabolism assessed as human recombinant UGT1A10-mediated formation of scutellarein-6,7-diglucuronide after 25 mins by HPLC/UV analysis
- ChEMBL_1550167 (CHEMBL3756508) Competitive inhibition of Escherichia coli beta-galactosidase assessed as p-nitrophenyl-beta-D-galactopyranoside substrate hydrolysis by UV/Vis spectroscopy
- ChEMBL_1556331 (CHEMBL3766390) Inhibition of N-terminal His6-tagged human 12/15-LOX using arachidonic acid as substrate by UV-visible spectrophotometric analysis
- ChEMBL_1556335 (CHEMBL3766394) Inhibition of N-terminal His6-tagged human 15-LOX-2 using arachidonic acid as substrate by UV-visible spectrophotometric analysis
- ChEMBL_1586204 (CHEMBL3819919) Inhibition of flag-tagged PTP1B (1 to 321 residues) (unknown origin) expressed in bacterial expression system by UV/Vis spectrophotometry
- ChEMBL_1775774 (CHEMBL4232766) Irreversible inhibition of human arginase 1 using thioarginine as substrate measured up to 360 mins by UV micro plate method
- ChEMBL_1775776 (CHEMBL4232768) Irreversible inhibition of human arginase 2 using thioarginine as substrate measured up to 360 mins by UV micro plate method
- ChEMBL_1869808 (CHEMBL4370874) Inhibition of human IDO1 expressed in Escherichia coli using L-tryptophan as substrate after 1 hr by UV absorption analysis
- ChEMBL_1911234 (CHEMBL4413680) Inhibition of recombinant human 5-LOX expressed in Escherichia coli using arachidonic acid as substrate by UV-vis spectrophotometric method
- ChEMBL_1972532 (CHEMBL4605350) Displacement of [3H]-QNB from truncated EGFP-fused human muscarinic M1 receptor expressed in HEK293 cells by UV-visible spectroscopy
- ChEMBL_1992561 (CHEMBL4626296) Inhibition of Mycobacterium tuberculosis PTPA using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_2033475 (CHEMBL4687633) Inhibition of rat CD39 expressed in CHO cells using ATP as substrate incubated for 10 mins by UV absorbance method
- ChEMBL_2033476 (CHEMBL4687634) Inhibition of rat NTPDase2 expressed in CHO cells using ATP as substrate incubated for 10 mins by UV absorbance method
- ChEMBL_2060794 (CHEMBL4716047) Displacement of [3H]-QNB from truncated EGFP-fused human muscarinic M1 receptor expressed in HEK293 cells by UV-visible spectroscopy
- ChEMBL_2116448 (CHEMBL4825389) Inhibition of human 15-LOX-2 assessed as enzymatic rate using arachidonic acid as substrate by UV/Vis spectrophotometric analysis
- ChEMBL_2116451 (CHEMBL4825392) Inhibition of human 15-LOX-1 assessed as enzymatic rate using arachidonic acid as substrate by UV/Vis spectrophotometric analysis
- ChEMBL_2116455 (CHEMBL4825396) Inhibition of human recombinant COX-1 assessed as enzymatic rate using arachidonic acid as substrate by UV/Vis spectrophotometric analysis
- ChEMBL_2116456 (CHEMBL4825397) Inhibition of human recombinant COX-2 assessed as enzymatic rate using arachidonic acid as substrate by UV/Vis spectrophotometric analysis
- ChEMBL_2346379 Inhibition of human DHFR using DHF as substrate assessed as inhibition constant in presence of NADPH by UV-Vis spectrometry analysis
- ChEMBL_693672 (CHEMBL1637940) Inhibition of Salmonella enterica serotype Newport AM17274 cephalosporinase CMY-31 by UV spectrophotometer in presence of 100 uM of cephalothin
- ChEMBL_792733 (CHEMBL1930560) Inhibition of Wistar rat kidney ALR1 using D-glucuronate as substrate after 10 mins by UV/VIS double spectrophotometric analysis
- ChEMBL_839905 (CHEMBL2090106) Inhibition of recombinant Pseudomonas aeruginosa MBL IMP-1 using nitrocefin as substrate preincubated for 20 mins by UV-spectrophotometric analysis
- UV/VIS The experimental studies were carried out in 20 mM sodium phosphate buffer at pH7.4, at a temperature of 298 K.
- ChEMBL_1352304 (CHEMBL3269368) Inhibition of N-terminal His6-tagged human epithelial 15-lipoxygenase-2 using arachidonic acid as substrate by UV-vis spectrometric analysis
- ChEMBL_1438115 (CHEMBL3388490) Inhibition of purified His6-tagged recombinant human HPPD assessed as inhibition of maleylacetoacetate formation after 30 mins by UV/visible spectrophotometry
- ChEMBL_1457646 (CHEMBL3368714) Inhibition of AChE in rat cortex homogenates using acetylthiocholine iodide substrate incubated for 15 mins by UV spectroscopy based Ellman's method
- ChEMBL_1491697 (CHEMBL3536530) Drug metabolism assessed as human recombinant UGT1A1-mediated formation of scutellarein-7-O-glucuronide after 25 mins by HPLC/UV analysis
- ChEMBL_1522059 (CHEMBL3627219) Inhibition of human 15-LOX1 assessed as conversion of linoleic acid to 13(S)-HpODE after 10 mins by UV analysis
- ChEMBL_1560521 (CHEMBL3776793) Inhibition of porcine microsomal aminopeptidase N preincubated for 30 mins using L-Leu-p-nitroanilide as substrate by UV-VIS spectrophotometer
- ChEMBL_1579453 (CHEMBL3810522) Inhibition of monophenolase activity of mushroom tyrosinase using L-tyrosine as substrate incubated for 10 mins by UV-Visible spectrophotometric analysis
- ChEMBL_1579454 (CHEMBL3810523) Inhibition of diphenolase activity of mushroom tyrosinase using L-DOPA as substrate incubated for 10 mins by UV-Visible spectrophotometric analysis
- ChEMBL_1621946 (CHEMBL3864298) Inhibition of electric eel AChE using ATC as substrate preincubated for 15 mins followed by substrate addition by UV-spectrophotometric analysis
- ChEMBL_1621947 (CHEMBL3864299) Inhibition of horse serum BChE using BTC as substrate preincubated for 15 mins followed by substrate addition by UV-spectrophotometric analysis
- ChEMBL_1772938 (CHEMBL4229930) Inhibition of human supersome CYP2C9 using diclofenac as substrate assessed as diclofenac 4'-hydroxylation after 30 mins by HPLC-UV detection
- ChEMBL_1772939 (CHEMBL4229931) Inhibition of human supersome CYP2C19 using perazine as substrate assessed as perazine N-demethylation after 30 mins by HPLC-UV detection
- ChEMBL_1772940 (CHEMBL4229932) Inhibition of human supersome CYP2D6 using bufuralol as substrate assessed as bufuralol 1'-hydroxylation after 30 mins by HPLC-UV detection
- ChEMBL_1772941 (CHEMBL4229933) Inhibition of human supersome CYP3A4 using testosterone as substrate assessed as testosterone 6beta-hydroxylation after 30 mins by HPLC-UV detection
- ChEMBL_1795347 (CHEMBL4267464) Inhibition of recombinant human 5-LOX expressed in Escherichia coli BL21 using arachidonic acid as substrate by UV-vis spectrophotometric method
- ChEMBL_1869810 (CHEMBL4370876) Inhibition of human TDO expressed in Escherichia coli at using L-tryptophan as substrate after 1 hr by UV absorption analysis
- ChEMBL_1900392 (CHEMBL4402614) Inhibition of monophenolase activity of mushroom tyrosinase assessed as reduction in dopachrome formation using L-Tyrosine substrate by UV-Vis spectrophotometry
- ChEMBL_1900397 (CHEMBL4402619) Inhibition of diphenolase activity of mushroom tyrosinase assessed as reduction in dopachrome formation using L-DOPA substrate by UV-Vis spectrophotometry
- ChEMBL_1900688 (CHEMBL4402910) Competitive inhibition of recombinant human 5-LOX expressed in Escherichia coli BL21 cells using Arachidonic acid as substrate by UV absorbance
- ChEMBL_1913632 (CHEMBL4416215) Inhibition of human HMGR using HMGCoA as substrate measured after 15 mins in presence of NADPH by UV microplate reader analysis
- ChEMBL_2078867 (CHEMBL4734658) Inhibition of N-terminal His-tagged human IDO1 expressed in Escherichia coli using L-Trp as substrate by UV absorbance method
- ChEMBL_2130246 (CHEMBL4839675) Inhibition of wild type human N-terminal His-tagged 12-LOX using arachidonic acid as substrate by UV-Vis spectroscopy analysis
- ChEMBL_2275090 Inhibition of human N-terminal his-tagged IDO1 expressed in Escherichia coli using L-tryptophan as substrate by UV absorption based analysis
- ChEMBL_2379888 Inhibition of human IDO1 using L-tryptophan as substrate assessed as reduction in N-formyl kynurenine formation by UV-visible spectroscopic analysis
- ChEMBL_2380282 Inhibition of XO (unknown origin) using xanthine as substrate preincubated for 3 hrs followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_762857 (CHEMBL1820674) Competitive inhibition of recombinant Escherichia coli DAH7P synthase using PEP as substrate by Michaelis-Menten equation analysis using UV-visible spectrophotometry
- ChEMBL_1352297 (CHEMBL3269361) Inhibition of recombinant N-terminal His6-tagged human reticulocyte 12/15-lipoxygenase using arachidonic acid as substrate by UV-vis spectrometric analysis
- ChEMBL_1438923 (CHEMBL3382434) Inhibition of Sprague-Dawley rat brain AChE using acetylthiocholin iodide as substrate preincubated for 25 mins by Ellman's/UV-vis spectroscopy analysis
- ChEMBL_1460521 (CHEMBL3395431) Inhibition of Mycobacterium tuberculosis recombinant InhA assessed as NADH oxidation to NAD+ using 2-trans-dodecenoyl-CoA substrate by UV/visible spectrophotometry
- ChEMBL_1546972 (CHEMBL3748075) Inhibition of N-terminal His-tagged human recombinant IDO1 expressed in Escherichia coli using tryptophan as substrate by UV-visible absorption spectroscopy
- ChEMBL_1586212 (CHEMBL3819927) Inhibition of human recombinant TCPTP (1 to 341 residues) expressed in Escherichia coli using p-nitrophenyl phosphate as substrate by UV spectroscopy
- ChEMBL_1661999 (CHEMBL4011680) Inhibition of Wistar rat kidney ALR1 assessed as reduction in NADPH oxidation using D-glycuronate as substrate by UV-visible spectrophotometric method
- ChEMBL_1772713 (CHEMBL4229705) Inhibition of human serum C2-mediated lysis of antibody-sensitized sheep red blood cells after 30 mins by UV-Vis spectrophotometric method
- ChEMBL_1772937 (CHEMBL4229929) Inhibition of human supersome CYP1A2 using caffeine as substrate assessed as caffeine 3-N-demethylation after 30 mins by HPLC-UV detection
- ChEMBL_1814803 (CHEMBL4314377) Binding affinity to human FBPase expressed in Escherichia coli BL21 (DE3) assessed as dissociation constant at 100 uM by UV-visible spectroscopy
- ChEMBL_1888958 (CHEMBL4390712) Inhibition of alpha-glucosidase (unknown origin) incubated for 20 mins before pNPG substrate addition and measured after 30 mins by UV spectrometry
- ChEMBL_2078863 (CHEMBL4734654) Inhibition of human N-terminal His-tagged IDO1 expressed in Escherichia coli M15 incubated for 1 hr by UV-Vis spectrophotometric method
- ChEMBL_2242991 (CHEMBL5157201) Inhibition of xanthine oxidase (unknown origin) using xanthine as substrate preincubated for 15 mins followed by substrate addition by UV spectrophotometric analysis
- ChEMBL_2282442 Inhibition of topoisomerase 1 (unknown origin) assessed as relaxation of supercoiled kDNA incubated for 30 mins ethidium bromide staining based UV transilluminator analysis
- ChEMBL_2286066 Inhibition of ALR2 (unknown origin) using D,L-glyceraldehyde as substrate incubated for 10 mins in presence of NADPH by UV-spectrophotometric assay
- ChEMBL_2350383 Inhibition of Saccharomyces cerevisiae alpha-glucosidase using p-nitrophenyl-alpha-D-glucopyranoside as substrate incubated for 15 mins by UV-visible spectrophotometric analysis
- ChEMBL_764381 (CHEMBL1820933) Inhibition of bovine milk xanthine oxidase assessed as decrease of uric acid formation preincubated for 5 mins by UV-visible spectrophotometric analysis
- ChEMBL_801784 (CHEMBL1947347) Inhibition of rat recombinant nNOS expressed in baculovirus-infected Sf9 cells assessed as conversion of oxyhemoglobin to methemoglobin by UV spectrophotometer analysis
- Enzyme Assay The kinetic studies involving a spectrophotometric product were conducted by monitoring the change in UV/visible absorbance with a Cintra 10 spectrophotometer.
- ChEMBL_1502709 (CHEMBL3590705) Inhibition of phosphorylated NPM1 recruitment to chromatic in 6 Gy irradiated human A549 cells assessed as increase in protein solubility as extraction of pT199NPM1 pretreated 30 mins and measured after 90 mins of irradiation by Western blot analysis
- ChEMBL_2015028 (CHEMBL4668606) Light-dependent inhibition of recombinant full length human HDAC2 expressed in baculovirus infected Sf9 insect cells preincubated for 1 hr in presence of LED light irradiation followed by substrate addition and measured for 30 mins by fluorescence method
- ChEMBL_1667719 (CHEMBL4017607) Inhibition of human LSD1 assessed as reduction in H2O2 production using pLys4Met H3 peptide as substrate by peroxidase coupled UV-visible spectrophotometric method
- ChEMBL_1698554 (CHEMBL4049536) Inhibition of Arabidopsis thaliana HPPD expressed in Escherichia coli BL21(DE3) using HPPA as substrate after 10 mins by UV-Vis spectrometric analysis
- ChEMBL_1739641 (CHEMBL4155391) Inhibition of human carbonic anhydrase 9 expressed in Escherichia coli BL21 D3 strain using p-nitrophenyl acetate as substrate by UV/visible spectrophotometry
- ChEMBL_1739642 (CHEMBL4155392) Inhibition of human carbonic anhydrase 2 expressed in Escherichia coli BL21 D3 strain using p-nitrophenyl acetate as substrate by UV/visible spectrophotometry
- ChEMBL_1761650 (CHEMBL4196897) Inhibition of tau (unknown origin) aggregation expressed in Escherichia coli (DE3) incubated for overnight by Th-S fluorescence staining based UV-Vis spectrophotometer
- ChEMBL_1769994 (CHEMBL4222106) Inhibition of PGAM1 in human MDA-MB-231 cells using 3-PGA as substrate after 2 hrs in by UV-vis spectrophotometric method
- ChEMBL_1862249 (CHEMBL4363105) Competitive inhibition of red kidney bean PAP using varying levels of pNPP as substrate measured at pH 6.2 by UV-vis spectrophotometric analysis
- ChEMBL_1888586 (CHEMBL4390263) Inhibition of ALR1 (unknown origin) using sodium d-glucoronate as substrate preincubated for 10 mins followed by substrate addition by UV-spectrophotometry analysis
- ChEMBL_1888587 (CHEMBL4390264) Inhibition of ALR2 (unknown origin) using sodium dl-glyceraldehyde as substrate preincubated for 10 mins followed by substrate addition by UV-spectrophotometry analysis
- ChEMBL_2266620 Inhibition of His-tagged Escherichia coli DXR using DXP as substrate incubated for 1 min in presence of NADPH by UV-Vis spectrophotometric assay
- ChEMBL_2268716 Inhibition of EGFR (unknown origin) phosphorylation using kit-tyr4 peptide as substrate incubated for 1 hr in presence of ATP by UV-Vis spectrophotometer
- ChEMBL_2288187 Inhibition of human PGAM1 in human MDA-MB-231 cells assessed as decrease in PGAM1 activity incubated for 2 hrs by UV/Vis spectrophotometry
- ChEMBL_2346370 Inhibition of wild type Plasmodium falciparum DHFR using DHF as substrate assessed as inhibition constant in presence of NADPH by UV-Vis spectrometry analysis
- ChEMBL_2347966 Inhibition of human carbonic anhydrase II esterase activity using p-nitrophenol acetate as substrate and measured for 3 mins by UV/visible spectrophotometric analysis
- ChEMBL_2350542 Inhibition of N-terminus hexa-histidine tagged recombinant human IDO1 expressed in Escherichia coli M15 incubated for 1 hr by UV-visible spectrophotometric analysis
- ChEMBL_744350 (CHEMBL1772256) Competitive inhibition of red kidney bean PAP using para-nitrophenol as substrate at pH 4.9 preincubated for 10 mins by UV-visible spectrophotometry
- ChEMBL_744351 (CHEMBL1772257) Uncompetitive inhibition of red kidney bean PAP using para-nitrophenol as substrate at pH 4.9 preincubated for 10 mins by UV-visible spectrophotometry
- ChEMBL_932803 (CHEMBL3072691) Inhibition of wild type C-South African Human immunodeficiency virus 1 protease using chromogenic peptide H-1048 as substrate by UV spectrophotometric analysis
- ChEMBL_962697 (CHEMBL2388424) Inhibition of human recombinant His-tagged thymidylate synthase assessed as N5,N10-methylenetetrahydrofolate oxidation to dihydrofolate after 20 mins by UV spectrophotometric analysis
- ChEMBL_1473077 (CHEMBL3421187) Inhibition of recombinant human ALDH1A2 using propionaldehyde as substrate preincubated for 2 mins with NAD+ followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1473079 (CHEMBL3421189) Inhibition of recombinant human ALDH1A3 using propionaldehyde as substrate preincubated for 2 mins with NAD+ followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1473081 (CHEMBL3421191) Inhibition of recombinant human ALDH2 using propionaldehyde as substrate preincubated for 2 mins with NAD+ followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1473083 (CHEMBL3421193) Inhibition of recombinant human ALDH1B1 using propionaldehyde as substrate preincubated for 2 mins with NAD+ followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1473085 (CHEMBL3421195) Inhibition of recombinant human ALDH3A1 using benzaldehyde as substrate preincubated for 2 mins with NAD+ followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1473980 (CHEMBL3419576) Inhibition of recombinant human ALDH1A1 using propionaldehyde as substrate preincubated for 2 mins with NAD+ followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1507810 (CHEMBL3598674) Reactivation of sarin-inhibited human acetylcholinesterase assessed as dissociation constant incubated for 60 mins using ATChI substrate by DTNB dye based UV-visible spectrophotometry
- ChEMBL_1507812 (CHEMBL3598676) Reactivation of VX-inhibited human acetylcholinesterase assessed as dissociation constant incubated for 60 mins using ATChI substrate by DTNB dye based UV-visible spectrophotometry
- ChEMBL_1574573 (CHEMBL3800856) Inhibition of ALR2 from rat lens using D,L-glyceraldehyde as substrate measured as absorption of NADPH for 4 mins by UV/vis spectrophotmetry
- ChEMBL_1628554 (CHEMBL3871139) Inhibition of recombinant human tyrosinase expressed in baculovirus infected Sf9 cells assessed as diphenolase activity using L-DOPA as substrate by UV-vis spectrophotometry
- ChEMBL_1670580 (CHEMBL4020468) Inhibition of human AKR1B10 expressed in Escherichia coli BL21(DE3) using pyridine-3-aldehyde as substrate measured for 3 mins by UV-vis spectrophotometer
- ChEMBL_1709503 (CHEMBL4119552) Competitive inhibition of Aeromonas hydrophila recombinant CphA expressed in Escherichia coli BL21(DE3) using meropenem as substrate by UV/Vis multi-plate spectrophotometric method
- ChEMBL_1709505 (CHEMBL4119554) Competitive inhibition of Pseudomonas aeruginosa recombinant AIM1 expressed in Escherichia coli BL21(DE3) using cefuroxime as substrate by UV/Vis multi-plate spectrophotometric method
- ChEMBL_1748419 (CHEMBL4182929) Non-competitive inhibition of NDM1 in Escherichia coli BL21 (DE3) using MEPM substrate incubated for 15 mins by UV spectroscopy based L-B plot
- ChEMBL_1839209 (CHEMBL4339424) Inhibition of recombinant human AKR1C4 expressed in Escherichia coli BL21 (D3) using S-tetralol as substrate in presence of NADP+ by UV-spectrophotometric method
- ChEMBL_1839213 (CHEMBL4339428) Inhibition of recombinant human AKR1C1 expressed in Escherichia coli BL21 (D3) using S-tetralol as substrate in presence of NADP+ by UV-spectrophotometric method
- ChEMBL_1839215 (CHEMBL4339430) Inhibition of recombinant human AKR1C2 expressed in Escherichia coli BL21 (D3) using S-tetralol as substrate in presence of NADP+ by UV-spectrophotometric method
- ChEMBL_1839216 (CHEMBL4339431) Inhibition of recombinant human AKR1C3 expressed in Escherichia coli BL21 (D3) using S-tetralol as substrate in presence of NADP+ by UV-spectrophotometric method
- ChEMBL_1885372 (CHEMBL4386954) Inhibition of mushroom tyrosinase diphenolase activity using L-dopa as substrate preincubated for 10 mins followed by substrate addition by UV-Visible spectrophotometric analysis
- ChEMBL_1911522 (CHEMBL4413968) Inhibition of rat CPA3 using AAFP as substrate preincubated for 45 mins measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1920696 (CHEMBL4423541) Inhibition of recombinant human thymidine phosphorylase expressed in Escherichia coli Rosetta (DE3) cells using thymidine as substrate after 1 min by UV/visible spectrophotometry
- ChEMBL_2062458 (CHEMBL4717711) Inhibition of recombinant human IDO1 assessed as N-formylkynurenine formation using L-tryptophan as substrate measured after 1 hr by UV-vis spectrophotometric analysis
- ChEMBL_2062462 (CHEMBL4717715) Inhibition of recombinant human TDO assessed as N-formylkynurenine formation using L-tryptophan as substrate measured after 1 hrs by UV-vis spectrophotometric analysis
- ChEMBL_2069786 (CHEMBL4725039) Inhibition of rabbit ACE using Abz-FRK(DNP)-P as substrate measured for every 30 sec for 5 mins by UV-fluorescence based assay
- ChEMBL_2128589 (CHEMBL4838018) Inhibition of recombinant Trypanosoma brucei brucei alternative oxidase using ubiquinol as substrate preincubated for 2 mins followed by substrate addition by UV-Vis spectrophotometry
- ChEMBL_2196078 (CHEMBL5108594) Non competitive type inhibition of human TS assessed as inhibition constant using varying levels of mTHF as substrate by UV-Vis spectroscopy based analysis
- ChEMBL_2267271 Inhibition of human GST-tagged LSD1/CoREST (305 to 482 residues) expressed in Escherichia coli using pLys4Met peptide as substrate by UV-visible spectrophotometric analysis
- ChEMBL_2330723 Pseudo-irreversible inhibition of human BChE using butyrylthiocholine iodide as substrate incubated for 20 mins followed by substrate addition by Ellman's method based UV analysis
- ChEMBL_2330724 Pseudo-irreversible inhibition of human AChE using acetylthiocholine iodide as substrate incubated for 20 mins followed by substrate addition by Ellman's method based UV analysis
- ChEMBL_794981 (CHEMBL1936308) Inhibition of human recombinant mPGES-1 in assessed as conversion of PGH2 into PGE2 at 20 degC after 5 mins by HPLC-UV analysis
- ChEMBL_893548 (CHEMBL3049525) Inhibition of Homo sapiens (human) recombinant protoporphyrinogen oxidase expressed in Escherichia coli JM109 assessed as oxidation of protoporphyrinogen IX substrate by UV-visible spectrophotometry
- ChEMBL_1502715 (CHEMBL3590711) Inhibition of phosphorylated NPM1 recruitment to chromatic in 6 Gy irradiated human A549 cells assessed as extraction of pT199NPM1 pretreated 30 mins and measured after 90 mins of irradiation by Western blot analysis in presence of high NP-40 lysis buffer
- ChEMBL_1473164 (CHEMBL3418216) Inhibition of human ALDH1A1 G458N mutant using propionaldehyde as substrate preincubated for 2 mins with NAD+ followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1477654 (CHEMBL3428033) Inhibition of ALR2 in calf lenses using DL-glyceraldehyde as substrate treated with compound for 10 mins prior to substrate addition by UV spectrophotometer analysis
- ChEMBL_1507811 (CHEMBL3598675) Reactivation of O-ethylsarin-inhibited human acetylcholinesterase assessed as dissociation constant incubated for 60 mins using ATChI substrate by DTNB dye based UV-visible spectrophotometry
- ChEMBL_1566186 (CHEMBL3788092) Inhibition of human recombinant dihydrofolate reductase using dihydrofolate as substrate measured every 30 secs over 6 mins by UV/visible spectrophotometer in presence of NADPH
- ChEMBL_1696005 (CHEMBL4046895) Inhibition of bovine liver beta-galactosidase using 4-nitrophenyl-beta-D-galactopyranoside substrate measured over 240 mins at 10 mins intervals by UV/Vis spectroscopy
- ChEMBL_1703416 (CHEMBL4054649) Inhibition of xanthine oxidase (unknown origin) assessed as reduction in uric acid formation using xanthine as substrate after 4 mins by UV-Vis spectrophotometric assay
- ChEMBL_1703430 (CHEMBL4054663) Inhibition of xanthine oxidase (unknown origin) assessed as reduction in uric acid formation using xanthine as substrate after 30 mins by UV-Vis spectrophotometric assay
- ChEMBL_1732054 (CHEMBL4147590) Inhibition of Mycobacterium tuberculosis InhA V203A mutant expressed in Escherichia coli BL21(DE3) pLysS cells using C8-CoA as substrate by UV-vis spectrophotometric analysis
- ChEMBL_1732056 (CHEMBL4147592) Inhibition of Mycobacterium tuberculosis InhA I215A mutant expressed in Escherichia coli BL21(DE3) pLysS cells using C8-CoA as substrate by UV-vis spectrophotometric analysis
- ChEMBL_1732069 (CHEMBL4147605) Inhibition of Mycobacterium tuberculosis InhA I215A mutant expressed in Escherichia coli BL21(DE3) pLysS cells assessed as enzyme-inhibitor complex by UV-vis spectrophotometric analysis
- ChEMBL_1732070 (CHEMBL4147606) Inhibition of Mycobacterium tuberculosis InhA V203A mutant expressed in Escherichia coli BL21(DE3) pLysS cells assessed as enzyme-inhibitor complex by UV-vis spectrophotometric analysis
- ChEMBL_1751423 (CHEMBL4186183) Binding affinity to GP2b/3a in rat blood assessed as conformational change by measuring soluble GP2b/3a levels after 12 hrs by UV-spectrophotometric analysis
- ChEMBL_1779688 (CHEMBL4236680) Inhibition of recombinant TDO (unknown origin) assessed as reduction in N-formylkynurenine formation using L-Tryptophan as substrate after 75 mins by UV absorption method
- ChEMBL_1992558 (CHEMBL4626293) Inhibition of PTP1B (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992559 (CHEMBL4626294) Inhibition of SHP2 (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992560 (CHEMBL4626295) Inhibition of SHP1 (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992562 (CHEMBL4626297) Inhibition of TCPTP (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992563 (CHEMBL4626298) Inhibition of LYP (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992564 (CHEMBL4626299) Inhibition of HePTP (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992565 (CHEMBL4626300) Inhibition of FAP1 (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992566 (CHEMBL4626301) Inhibition of DEP1 (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992567 (CHEMBL4626302) Inhibition of Laforin (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992569 (CHEMBL4626304) Inhibition of MKP3 (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992570 (CHEMBL4626305) Inhibition of MKP5 (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992574 (CHEMBL4626309) Inhibition of CD45 (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992575 (CHEMBL4626310) Inhibition of CDC14A (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992576 (CHEMBL4626311) Inhibition of STEP (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992577 (CHEMBL4626312) Inhibition of VHR (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992578 (CHEMBL4626313) Inhibition of PTPS (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992579 (CHEMBL4626314) Inhibition of PTPA (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992580 (CHEMBL4626315) Inhibition of PTPB (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992581 (CHEMBL4626316) Inhibition of PTPM (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992582 (CHEMBL4626317) Inhibition of PTPG (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992583 (CHEMBL4626318) Inhibition of PTPE (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_2313210 Inhibition of human recombinant MAO-B expressed in Pichia pastoris assessed as inhibition constant using MMTP as substrate by Morrison equation based UV-Visible spectrophotometric analysis
- ChEMBL_2379887 Inhibition of human IDO1 using L-tryptophan as substrate assessed as reduction in N-formyl kynurenine formation by measuring inhibition constant by UV-visible spectroscopic analysis
- ChEMBL_2473639 Inhibition of Plasmodium falciparum DXR expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL cells using DOXP as substrate in presence of NADPH by UV-visible spectrophotometry
- ChEMBL_2473644 Inhibition of Mycobacterium tuberculosis DXR expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL cells using DOXP as substrate in presence of NADPH by UV-visible spectrophotometry
- ChEMBL_754219 (CHEMBL1798863) Inhibition of nNOS in Sprague-Dawley rat brain homogenates assessed as conversion of oxyhemoglobin to methemoglobin measured for 10 mins by UV-visible spectrophotometer analysis
- ChEMBL_761393 (CHEMBL1817023) Inhibition of human platelet-type N-terminally His6-tagged 12-lipoxygenase assessed as conjugated diene product formation using arachidonic acid by UV-vis spectrophotometer analysis
- ChEMBL_761395 (CHEMBL1817025) Inhibition of human reticulocyte N-terminally His6-tagged 15-lipoxygenase-1 assessed as conjugated diene product formation using arachidonic acid by UV-vis spectrophotometer analysis
- ChEMBL_761396 (CHEMBL1817026) Inhibition of human reticulocyte N-terminally His6-tagged 15-lipoxygenase-2 assessed as conjugated diene product formation using arachidonic acid by UV-vis spectrophotometer analysis
- ChEMBL_822742 (CHEMBL2040059) Inhibition of recombinant MMP2 using succinylated gelatin as substrate incubated for 10 mins prior to substrate addition measured after 1 hr by UV/VIS spectrophotometry
- ChEBML_1715600 Inhibition of calf lens ALR2 using D,L-glyceraldehyde as substrate preincubated for 5 mins followed by NADPH addition measured after 10 mins by UV spectrophotometric method
- ChEMBL_1477653 (CHEMBL3428032) Inhibition of ALR1 in calf kidney using sodium D-glucoronate as substrate treated with compound for 10 mins prior to substrate addition by UV spectrophotometer analysis
- ChEMBL_1502123 (CHEMBL3586998) Inhibition of Bacteroides fragilis CcrA overexpressed in Escherichia coli BL21 (DE3) using cefazolin as substrate incubated for 30 mins prior to testing by UV spectrophotometric analysis
- ChEMBL_1502125 (CHEMBL3587107) Inhibition of Stenotrophomonas maltophilia L1 overexpressed in Escherichia coli BL21 (DE3) using imipenem as substrate incubated for 30 mins prior to testing by UV spectrophotometric analysis
- ChEMBL_1565722 (CHEMBL3782520) Competitive inhibition of human MAOB overexpressed in Pichia pastoris using MMTP as substrate preincubated for 5 mins followed by substrate addition by UV/vis spectrophotometric analysis
- ChEMBL_1633217 (CHEMBL3876009) Inhibition of Escherichia coli K12 thymidine phosphorylase expressed in Escherichia coli using thymidine as substrate addition measured up to 20 mins by Uv-vis spectrophotometric method
- ChEMBL_1658997 (CHEMBL4008609) Inhibition of recombinant human DHFR assessed as reduction in conversion of DHF to THF measured every 30 secs for 6 mins by UV-Vis spectrophotometric method
- ChEMBL_1718003 (CHEMBL4133003) Inhibition of electric eel AChE using acetylthiocholine iodide as substrate preincubated for 15 mins followed by substrate addition measured for 5 mins by UV spectrophotometric analysis
- ChEMBL_1718355 (CHEMBL4133355) Inhibition of human AChE using acetylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured over 5 mins by UV-Vis spectrophotometric analysis
- ChEMBL_1761647 (CHEMBL4196894) Inhibition of amyloid beta 42 (unknown origin) expressed in Escherichia coli BL21 (DE3) aggregation incubated for overnight by Th-S fluorescence staining based UV-Vis spectrophotometer
- ChEMBL_1802620 (CHEMBL4274912) Competitive inhibition of pig purple acid phosphatase Fe(3)Fe(2) state assessed as enzyme-inhibitor complex using pNPP as substrate by UV/Vis spectrophotometric method
- ChEMBL_1855632 (CHEMBL4356361) Inhibition of human plasmin assessed as reduction in plasmin-mediated thrombin/factor 13a-induced clot fibrinolysis measured over 50 to 200 min by UV absorbance method
- ChEMBL_1927845 (CHEMBL4430917) Inhibition of human N-terminal His6-tagged 15-LOX-1 L407A mutant expressed in sf9 cells using arachidonic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1927850 (CHEMBL4430922) Inhibition of human N-terminal His6-tagged 15-LOX-1 F414W mutant expressed in sf9 cells using arachidonic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1927851 (CHEMBL4430923) Inhibition of human N-terminal His6-tagged 15-LOX-1 E356Q mutant expressed in sf9 cells using arachidonic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1927852 (CHEMBL4430924) Inhibition of human N-terminal His6-tagged 15-LOX-1 Q547L mutant expressed in sf9 cells using arachidonic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1992557 (CHEMBL4626292) Inhibition of Mycobacterium tuberculosis PTPB expressed in Escherichia coli BL21 (DE3) using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992568 (CHEMBL4626303) Inhibition of PTP-MEG2 (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992573 (CHEMBL4626308) Inhibition of PTP-PEST (unknown origin) expressed in Escherichia coli BL21 using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_2078868 (CHEMBL4734659) Inhibition of N-terminal 6His-tagged recombinant human IDO2 (15 to 420 residues) expressed in Escherichia coli using L-Trp as substrate by UV absorbance method
- ChEMBL_2078870 (CHEMBL4734661) Inhibition of recombinant human N-terminal His-tagged TDO (2 to 406 residues) expressed in Escherichia coli using L-Trp as substrate by UV absorbance method
- ChEMBL_2228735 (CHEMBL5142248) Inhibition of TDO (unknown origin) assessed as reduction in N-formylkynurenine formation using L-tryptophan as substrate incubated for 75 mins by UV absorbance based analysis
- ChEMBL_2271780 Inhibition of ATR in human HCT-116 cells preincubated for 1 hr followed by UV radiation and measured after 1 hr by Bradford assay based immunoblotting analysis
- ChEMBL_2271781 Inhibition of ATM in human HCT-116 cells preincubated for 1 hr followed by UV radiation and measured after 1 hr by Bradford assay based immunoblotting analysis
- ChEMBL_2284607 Inhibition of baker's yeast alpha-glucosidase using PNP as substrate preincubated for 30 mins followed by sucrose addition and measured after 1 mins by UV spectrophotometric analysis
- ChEMBL_2315468 Binding affinity to NDM1 (unknown origin) expressed in Escherichia coli BL21(DE3) assessed as inhibition constant by measuring rate of substrate hydrolysis by UV-Vis spectrophotometric analysis
- ChEMBL_2346620 Inhibition of XO (unknown origin) using xanthine as substrate pre incubated for 15 mins followed by substrate addition measured after 30 mins by UV-VIS spectrophotometer analysis
- ChEMBL_2443691 Inhibition of bovine plasma VAP-1 using 6-(5-Pheny1-2H-tetrazol-2-yl)hexan-1-amine as substrate measured after 30 mins by UV-HPLC analysis
- ChEMBL_513183 (CHEMBL977313) Binding affinity to photoactivated rhodopsin in bovine retinal rod outer-segment membranes assessed as induction of extra receptor MII state stabilization by UV/visible difference spectroscopy
- ChEMBL_794980 (CHEMBL1936307) Inhibition of mPGES-1 in human A549 cell microsomes assessed as conversion of PGH2 into PGE2 at 0 degC after 5 mins by HPLC-UV analysis
- ChEMBL_797888 (CHEMBL1942575) Inhibition of porcine kidney microsomal aminopeptidase N using L-Leu-p-nitroanilide as substrate pre-incubated for 30 mins prior substrate addition by UV-VIS spectrophotometry
- ChEMBL_931045 (CHEMBL3069621) Inhibition of trypsin (unknown origin) using BApNA as substrate incubated for 15 min prior to substrate addition measured after 30 min by UV/VIS spectrophotometric analysis
- ChEMBL_1462490 (CHEMBL3398963) Inhibition of CYP3A4 in pooled mixed-gender human liver microsomes using testosterone substrate in presence of NADPH pre-incubated for 3 mins by HPLC-UV detection method
- ChEMBL_1462491 (CHEMBL3398964) Inhibition of CYP3A4 in pooled mixed-gender human liver microsomes using triazolam substrate in presence of NADPH pre-incubated for 3 mins by HPLC-UV detection method
- ChEMBL_1462492 (CHEMBL3398965) Inhibition of CYP3D6 in pooled mixed-gender human liver microsomes using dextromethorphan substrate in presence of NADPH pre-incubated for 3 mins by HPLC-UV detection method
- ChEMBL_1626872 (CHEMBL3869393) Inhibition of acetylcholinesterase (unknown origin) using acetylthiocholine iodide as substrate incubated for 10 mins followed by substrate addition measured after 20 mins by UV-Vis spectrophotometric analysis
- ChEMBL_1626873 (CHEMBL3869394) Inhibition of butyrylcholinesterase (unknown origin) using butyrylthiocholine chloride as substrate incubated for 10 mins followed by substrate addition measured after 20 mins by UV-Vis spectrophotometric analysis
- ChEMBL_1656606 (CHEMBL4006076) Inhibition of recombinant human N-terminal His-tagged ST6Gal-1 (44 to 406 residues) using CMP-Neu5Ac as substrate after 20 mins by UV/RP-HPLC method
- ChEMBL_1699068 (CHEMBL4050050) Inhibition of human serum BuChE using butyrylthiocholine iodide as substrate pretreated for 5 mins followed by substrate addition measured for 5 mins by UV-Vis spectrophotometric method
- ChEMBL_1715599 (CHEMBL4125648) Inhibition of bovine kidney ALR1 using D-glucoronic acid as substrate preincubated for 5 mins followed by NADPH addition measured after 10 mins by UV spectrophotometric method
- ChEMBL_1715600 (CHEMBL4125649) Inhibition of calf lens ALR2 using D,L-glyceraldehyde as substrate preincubated for 5 mins followed by NADPH addition measured after 10 mins by UV spectrophotometric method
- ChEMBL_1718356 (CHEMBL4133356) Inhibition of human serum BuChE using butyrylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured over 5 mins by UV-Vis spectrophotometric analysis
- ChEMBL_1759789 (CHEMBL4194797) Inhibition of APN in porcine kidney microsomes using L-leucine-p-nitroanilide as substrate preincubated for 30 mins followed by substrate addition by UV-visible spectrophotometric method
- ChEMBL_1781508 (CHEMBL4253025) Inhibition of bovine milk xanthine oxidase using xanthine as substrate preincubated for 15 mins followed by substrate addition measured after 30 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1897720 (CHEMBL4399755) Binding affinity to recombinant ferric state of IDO1 (unknown origin) expressed in Escherichia coli assessed as dissociation constant incubated for 30 mins by UV-Visible spectrophotometric method
- ChEMBL_1897721 (CHEMBL4399756) Binding affinity to recombinant ferrous state of IDO1 (unknown origin) expressed in Escherichia coli assessed as dissociation constant incubated for 30 mins by UV-Visible spectrophotometric method
- ChEMBL_1899849 (CHEMBL4401964) Inhibition of human factor Xa using Z-D-Arg-Gly-Arg-pNA.2HCl as substrate preincubated for 15 mins followed by substrate addition by UV absorption analysis
- ChEMBL_1900393 (CHEMBL4402615) Mixed type inhibition of monophenolase activity of mushroom tyrosinase assessed as reduction in dopachrome formation using L-Tyrosine substrate by UV-Vis spectrophotometry based Dixon plot analysis
- ChEMBL_1900394 (CHEMBL4402616) Competitive type inhibition of monophenolase activity of mushroom tyrosinase assessed as reduction in dopachrome formation using L-Tyrosine substrate by UV-Vis spectrophotometry based Dixon plot analysis
- ChEMBL_1900398 (CHEMBL4402620) Mixed type inhibition of diphenolase activity of mushroom tyrosinase assessed as reduction in dopachrome formation using L-DOPA substrate by UV-Vis spectrophotometry based Dixon plot analysis
- ChEMBL_1900399 (CHEMBL4402621) Competitive type inhibition of diphenolase activity of mushroom tyrosinase assessed as reduction in dopachrome formation using L-DOPA substrate by UV-Vis spectrophotometry based Dixon plot analysis
- ChEMBL_1911512 (CHEMBL4413958) Inhibition of human CPA1 using AAFP as substrate preincubated for 45 mins to 2 hrs measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1911513 (CHEMBL4413959) Inhibition of human CPA2 using AAFP as substrate preincubated for 45 mins to 2 hrs measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1911514 (CHEMBL4413960) Inhibition of human CPA4 using AAFP as substrate preincubated for 45 mins to 2 hrs measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1911515 (CHEMBL4413961) Inhibition of human CPB1 using AAFA as substrate preincubated for 45 mins to 2 hrs measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1980733 (CHEMBL4613995) Binding affinity to recombinant human C-terminal 4His-tagged CYP46A1 delta(2 to 50) mutant expressed in Escherichia coli in absence of substrate by UV-spectrophotometric method
- ChEMBL_1980734 (CHEMBL4613996) Binding affinity to recombinant human C-terminal 4His-tagged CYP46A1 delta(2 to 50) mutant expressed in Escherichia coli in presence of substrate by UV-spectrophotometric method
- ChEMBL_2045678 (CHEMBL4700377) Inhibition of EGFR (unknown origin) using Tyrosine 4 peptide and ATP as substrate incubated for 1 hr by Z'-lyte kinase assay based UV-VIS spectrophotometric method
- ChEMBL_2067169 (CHEMBL4722422) Inhibition of sheep COX1 using arachidonic acid as substrate preincubated for 5 mins followed by substrate addition and measured after 5 mins by UV-visible spectrophotometric method
- ChEMBL_2315465 Binding affinity to VIM-1 (unknown origin) expressed in Escherichia coli BL21(DE3) assessed as inhibition constant by measuring rate of substrate hydrolysis by UV-Vis spectrophotometric analysis
- ChEMBL_761403 (CHEMBL1817033) Inhibition of mouse N-terminally His6-tagged 12-lipoxygenase expressed in SF9 insect cells assessed as 12-HPETE formation using arachidonic acid by UV-vis spectrophotometer analysis
- ChEMBL_761404 (CHEMBL1817034) Inhibition of mouse N-terminally His6-tagged 15-lipoxygenase expressed in SF9 insect cells assessed as 15-HPETE formation using arachidonic acid by UV-vis spectrophotometer analysis
- ChEMBL_761713 (CHEMBL1816807) Inhibition of Plasmodium falciparum Enoyl-ACP reductase using crotonyl-CoA as substrate peincubated for 5 mins measured after 10 mins of substrate addition by UV-vis spectrophotometry
- ChEMBL_767113 (CHEMBL1826162) Inhibition of human DHFR assessed as NADPH consumption for conversion of dihydrofolic acid to tetrahydrofolic acid after 6 mins every 5 sec by UV-Visible Spectrophotometer method
- ChEMBL_879811 (CHEMBL2210539) Inhibition of recombinant MMP-2 using succinylated gelatin as substrate incubated for 10 mins before addition of substrate measured after 60 mins by UV-Vis spectrophotometric analysis
- ChEMBL_959387 (CHEMBL2384178) Inhibition of human recombinant carboxy-terminal his-tagged LDHB (1 to 333) expressed in insect cells using pyruvate as substrate after 10 mins by UV endpoint analysis
- ChEMBL_959391 (CHEMBL2384303) Inhibition of human recombinant carboxy-terminal his-tagged LDHA (1 to 331) expressed in Escherichia coli using pyruvate as substrate after 10 mins by UV endpoint analysis
- Enzymatic Assay The potency of the five compounds was tested against recombinant GMPS in an enzymatic assay by monitoring UV absorbance at 290 nm and by determining an IC50.
- ChEMBL_1281313 (CHEMBL3101286) Inhibition of Pin-1 (unknown origin) using Suc-Ala-Glu-Pro-Phe-pNA as substrate preincubated for 300 seconds followed by substrate addition by UV/Vis spectrophotometric analysis
- ChEMBL_1505754 (CHEMBL3594914) Inhibition of HDAC1 (unknown origin) using Boc-Lys (acetyl)-AMC as substrate preincubated for 30 mins before substrate addition measured after 20 mins by UV-vis spectrophotometer analysis
- ChEMBL_1505755 (CHEMBL3594915) Inhibition of HDAC3 (unknown origin) using Boc-Lys (acetyl)-AMC as substrate preincubated for 30 mins before substrate addition measured after 20 mins by UV-vis spectrophotometer analysis
- ChEMBL_1505756 (CHEMBL3594916) Inhibition of HDAC6 (unknown origin) using Boc-Lys (acetyl)-AMC as substrate preincubated for 30 mins before substrate addition measured after 20 mins by UV-vis spectrophotometer analysis
- ChEMBL_1561176 (CHEMBL3777369) Inhibition of CE2 in human liver microsomes using fluorescein diacetate as substrate preincubated for 10 mins followed by substrate addition measured after 30 mins by LC-UV analysis
- ChEMBL_1635957 (CHEMBL3878855) Inhibition of recombinant human DHFR using DHF as substrate measured every 5 sec over 6 mins in presence of NADPH by UV-Visible spectrophotometric method relative to control
- ChEMBL_1704436 (CHEMBL4055669) Inhibition of recombinant full length human N-terminal GST-tagged PARP1 expressed in baculovirus infected Sf9 cells using biotinylated substrate after 1 hr by UV/Vis spectrophotometric analysis
- ChEMBL_1720392 (CHEMBL4135392) Inhibition of bovine xanthine oxidase assessed as reduction in uric acid formation using xanthine as substrate preincubated for 10 mins followed by substrate addition by UV spectrophotometric method
- ChEMBL_1758984 (CHEMBL4193992) Inhibition of human AChE pre-incubated for 6 mins before ATCI substrate addition and measured after 12 mins by DTNB reagent dependent UV-Vis spectrophotometry based Ellman's method
- ChEMBL_1758985 (CHEMBL4193993) Inhibition of human BChE pre-incubated for 6 mins before ATCI substrate addition and measured after 12 mins by DTNB reagent dependent UV-Vis spectrophotometry based Ellman's method
- ChEMBL_1788862 (CHEMBL4260596) Inhibition of human coagulation factor 10a using Z-D-Arg-Gly-Arg-pNA.2HCl as substrate preincubated for 15 mins followed by substrate addition by UV absorption assay
- ChEMBL_1802621 (CHEMBL4274913) Competitive inhibition of red kidney bean purple acid phosphatase Fe(3)Fe(2) state assessed as enzyme-inhibitor complex using pNPP as substrate by UV/Vis spectrophotometric method
- ChEMBL_1930270 (CHEMBL4433521) Inhibition of human SIRT1 deacetylase activity using acetylated H3K9 as substrate preincubated for 15 mins followed by substrate addition and measured after 3 mins by UV-HPLC analysis
- ChEMBL_1992587 (CHEMBL4626322) Inhibition of Mycobacterium tuberculosis PTPB I203A mutant expressed in Escherichia coli BL21 (DE3) using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992588 (CHEMBL4626323) Inhibition of Mycobacterium tuberculosis PTPB I207A mutant expressed in Escherichia coli BL21 (DE3) using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992589 (CHEMBL4626324) Inhibition of Mycobacterium tuberculosis PTPB I207K mutant expressed in Escherichia coli BL21 (DE3) using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1992590 (CHEMBL4626325) Inhibition of Mycobacterium tuberculosis PTPB F161S mutant expressed in Escherichia coli BL21 (DE3) using p-nitrophenyl phosphate as substrate measured after 30 mins by UV-vis spectrophotometric method
- ChEMBL_1994392 (CHEMBL4628287) Inhibition of IDO1 in IFN-gamma stimulated human HeLa cells assessed as reduction in kynurenine formation using L-tryptophan as substrate incubated for 24 hrs by UV-spectrophotometry
- ChEMBL_2102228 (CHEMBL4810624) Inhibition of human TDO2 expressed in HEK293E cells assessed as reduction in N-formylkynurenine formation using tryptophan as substrate measured after 24 hrs by HPLCC based UV spectrometry
- ChEMBL_822740 (CHEMBL2040057) Inhibition of pig kidney APN using L-Leu-p-nitroanilide as substrate incubated for 5 mins prior to substrate addition measured after 30 mins by UV/VIS spectrophotometry
- ChEMBL_963630 (CHEMBL2395149) Inhibition of pig kidney APN using L-Leu-p-nitroanilide as substrate incubated for 5 mins prior to substrate addition measured after 30 mins by UV-Vis spectrophotometry
- Esterase Activity Assay Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion using spectrophotmeter (CHEBIOS UV-VIS).
- ChEBML_1651523 Inhibition of human platelet cytosolic phospholipase alpha-2 using 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycerol as substrate after 60 mins by UV-HPLC method
- ChEBML_1662514 Inhibition of bovine xanthine oxidase assessed as reduction in uric acid formation using xanthine as substrate pretreated for 5 mins followed by substrate addition measured by UV-vis spectrophotometric method
- ChEBML_1691736 Inhibition of thrombin (unknown origin) using CH3SO2-D-Cha-Gly-Arg-pNA.AcOH as substrate measured every 20 sec for 30 mins in presence of NaCl by UV-Visible spectrophotometric method
- ChEMBL_1332321 (CHEMBL3226921) Displacement of [125I]-iodoarylazidoprazosin from human P-gp expressed in baculovirus-infected insect Sf9 cells incubated for 10 mins followed by UV light illumination for 20 mins by autoradiography
- ChEMBL_1448377 (CHEMBL3376861) Inhibition of Thermus flavus MDH pre-incubated for 4 mins before reaction initiation in presence of OAA and NAPH in absence of 0.01% Triton X100 by UV-Vis spectrophotometry
- ChEMBL_1449197 (CHEMBL3379871) Inhibition of Thermus flavus MDH pre-incubated for 4 mins before reaction initiation in presence of OAA and NAPH in presence of 0.01% Triton X100 by UV-Vis spectrophotometry
- ChEMBL_1502606 (CHEMBL3592746) Inhibition of Pin4 (unknown origin) pre-incubated for 10 mins before addition of Suc-Ala-Glu-Pro-Phe-pNA substrate in presence of alpha-chymotrypsin by UV/Vis spectrophotometry
- ChEMBL_1782401 (CHEMBL4253918) Inhibition of human PTP1B using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1825933 (CHEMBL4325697) Binding affinity to His-tagged CYP3A4 (unknown origin) expressed in Escherichia coli DH5alpha assessed as type 2 spectral shift by measuring dissociation constant by UV-vis spectrophotometric titration analysis
- ChEMBL_1897723 (CHEMBL4399758) Binding affinity to recombinant ferrous state of IDO1 C129Y mutant (unknown origin) expressed in Escherichia coli assessed as dissociation constant incubated for 30 mins by UV-Visible spectrophotometric method
- ChEMBL_1927846 (CHEMBL4430918) Inhibition of human N-terminal His6-tagged 15-LOX-1 I417A mutant expressed in sf9 cells using arachidonic acid or linoleic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1927847 (CHEMBL4430919) Inhibition of wild type human N-terminal His6-tagged 15-LOX-1 expressed in sf9 cells using arachidonic acid or linoleic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1927849 (CHEMBL4430921) Inhibition of human N-terminal His6-tagged 15-LOX-1 F414I mutant expressed in sf9 cells using arachidonic acid or linoleic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1927853 (CHEMBL4430925) Inhibition of human N-terminal His6-tagged 15-LOX-1 R404L mutant expressed in sf9 cells using arachidonic acid or linoleic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_1927854 (CHEMBL4430926) Inhibition of human N-terminal His6-tagged 15-LOX-1 R402L mutant expressed in sf9 cells using arachidonic acid or linoleic acid as substrate by UV-Visible spectrophotometric analysis
- ChEMBL_2075518 (CHEMBL4731052) Binding affinity pig brain GABA-AT assessed as inactivation constant using GABA as substrate in presence of NADP+ and alpha-ketoglutarate by SSDH coupled enzyme based UV-Vis spectrophotometry
- ChEMBL_2198745 (CHEMBL5111261) Inhibition of human BChE using butyrylthiocholineiodide as substrate preincubated for 20 mins followed by substrate addition and measured every 30 sec for 3 mins by DTNB-based UV analysis
- ChEMBL_2261255 (CHEMBL5216266) Inhibition of human recombinant N-terminal His tagged IDO1 expressed in Escherichia coli using L-Trp as substrate by measuring amount of N-formylkynurenine formed by UV based method
- ChEMBL_851741 (CHEMBL2156083) Inhibition of recombinant CYP3A4 NF-14 expressed in Escherichia coli assessed as formation of 6-beta-hydroxytestosterone using testosterone as substrate after 5 mins by HPLC based UV method
- ChEMBL_879874 (CHEMBL2211411) Inhibition of APN in porcine kidney microsome assessed as inhibition of L-Leu-p-nitroanilide substrate hydrolysis incubated for 5 mins before substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_959385 (CHEMBL2384176) Inhibition of human recombinant carboxy-terminal his-tagged MDH-2 (20 to 338) expressed in Escherichia coli using oxaloacetic acid as substrate after 10 mins by UV endpoint analysis
- ChEMBL_959386 (CHEMBL2384177) Inhibition of human recombinant carboxy-terminal his-tagged MDH-1 (3 to 334) expressed in Escherichia coli using oxaloacetic acid as substrate after 10 mins by UV endpoint analysis
- ChEMBL_1622575 (CHEMBL3864927) Inhibition of recombinant human N-terminus IDO1 expressed in Escherichia coli assessed as N-formylkynurenine formation using L-tryptophan as substrate measured after 180 mins by UV absorbance based assay
- ChEMBL_1624269 (CHEMBL3866681) Inhibition of FLAP in human monocytes assessed as suppression of A23187-stimulated 5-LO product formation preincubated for 15 mins followed by A23187 after 10 mins RP-HPLC/UV analysis
- ChEMBL_1651523 (CHEMBL4000778) Inhibition of human platelet cytosolic phospholipase alpha-2 using 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycerol as substrate after 60 mins by UV-HPLC method
- ChEMBL_1662514 (CHEMBL4012195) Inhibition of bovine xanthine oxidase assessed as reduction in uric acid formation using xanthine as substrate pretreated for 5 mins followed by substrate addition measured by UV-vis spectrophotometric method
- ChEMBL_1684865 (CHEMBL4035344) Inhibition of human liver microsomes CYP1A2 expressed in Escherichia coli DH5alpha coexpressing NADPH-P450 reductase using 7-ethoxyresorufin O-deethylation as substrate in presence of NADPH by UV absorbance analysis
- ChEMBL_1684866 (CHEMBL4035345) Inhibition of human liver microsomes CYP1B1 expressed in Escherichia coli DH5alpha coexpressing NADPH-P450 reductase using 7-ethoxyresorufin O-deethylation as substrate in presence of NADPH by UV absorbance analysis
- ChEMBL_1703421 (CHEMBL4054654) Inhibition of bovine milk xanthine oxidase assessed as reduction in uric acid formation using xanthine as substrate preincubated for 5 mins followed by substrate addition by UV-Vis spectrophotometric assay
- ChEMBL_1703426 (CHEMBL4054659) Inhibition of bovine milk xanthine oxidase assessed as reduction in uric acid formation using xanthine as substrate preincubated for 3 hrs followed by substrate addition by UV-Vis spectrophotometric assay
- ChEMBL_1704437 (CHEMBL4055670) Inhibition of recombinant human N-terminal GST-tagged PARP2 (2 to 583 residues) expressed in baculovirus infected Sf9 cells using biotinylated substrate after 1 hr by UV/Vis spectrophotometric analysis
- ChEMBL_1752600 (CHEMBL4187360) Time dependent inhibition of recombinant human PTP1B (1 to 322 residues) expressed in Escherichia coli using P-NPP as substrate preincubated with enzyme for 30 mins by UV-spectrophotometric method
- ChEMBL_1782399 (CHEMBL4253916) Inhibition of Mycobacterium tuberculosis PtpB using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1782400 (CHEMBL4253917) Inhibition of Mycobacterium tuberculosis PtpA using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1782403 (CHEMBL4253920) Inhibition of LYP (unknown origin) using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1823691 (CHEMBL4323455) Inhibition of human serum BChE using butyrylthiocholine iodide as substrate preincubated for 30 mins followed by substrate addition and measured after 2.5 mins by DTNB reagent based UV-spectrophotometric method
- ChEMBL_1900395 (CHEMBL4402617) Inhibition of monophenolase activity of mushroom tyrosinase assessed as inhibition constant for free enzyme-inhibitor complex by measuring reduction in dopachrome formation using L-Tyrosine substrate by UV-Vis spectrophotometry
- ChEMBL_1900400 (CHEMBL4402622) Inhibition of diphenolase activity of mushroom tyrosinase assessed as inhibition constant for free enzyme-inhibitor complex by measuring reduction in dopachrome formation using L-DOPA substrate by UV-Vis spectrophotometry
- ChEMBL_2074990 (CHEMBL4730524) Inhibition of 5-LOX (unknown origin) expressed in Escherichia coli BL21-(DE3) pre-incubated for 15 mins before AA substrate addition and measured after 10 mins by HPLC/UV assay
- ChEMBL_2125111 (CHEMBL4834344) Inhibition of APN in pig kidney microsomes assessed as reduction in p-nitroanilide release using L-leu-p-nitroanilide as substrate incubated for 30 mins by UV absorption based analysis
- ChEMBL_2198746 (CHEMBL5111262) Inhibition of human AChE using acetylthiocholine iodide as substrate preincubated for 20 mins followed by substrate addition and measured every 30 sec for 3 mins by DTNB-based UV analysis
- ChEMBL_2286015 Inhibition of human Aldose reductase using glyceraldehyde as substrate preincubated for 5 mins in presence of NADPH followed by substrate addition and measured after 3 mins by UV-visible spectrophotometric assay
- ChEMBL_879813 (CHEMBL2210541) Inhibition of APN in human ES2 cell surface assessed as inhibition of L-Leu-p-nitroanilide substrate hydrolysis incubated for 5 mins before substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1553874 (CHEMBL3767388) Inhibition of human factor 12a using S-2302 as substrate assessed as para-nitroaniline release preincubated for 10 mins followed by substrate addition measured after 30 mins by HPLC-UV analysis
- ChEMBL_1628730 (CHEMBL3871356) Inhibition of human 15-LOX-1 assessed as reduction in conversion of linoleic acid to 13(S)-HpODE incubated for 10 mins followed by linoleic acid addition by UV-absorbance analysis
- ChEMBL_1630617 (CHEMBL3873323) Inhibition of human recombinant 5-LOX expressed in Escherichia coli BL21 assessed as decrease in 5-HPETE production preincubated with enzyme followed by arachidonic acid substrate addition by UV-vis spectrophotometry
- ChEMBL_1699069 (CHEMBL4050051) Inhibition of recombinant human AChE expressed in HEK293 cells using acetylthiocholine iodide as substrate pretreated for 5 mins followed by substrate addition measured for 5 mins by UV-Vis spectrophotometric method
- ChEMBL_1759793 (CHEMBL4194801) Inhibition of APN in porcine kidney microsomes using L-leucine-p-nitroanilide as substrate preincubated for 30 mins followed by substrate addition measured after 15 mins by UV-visible spectrophotometric method
- ChEMBL_1782405 (CHEMBL4253922) Inhibition of PTP-PEST (unknown origin) using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1793232 (CHEMBL4265151) Inhibition of recombinant human Ptp1B using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition and measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1793235 (CHEMBL4265154) Inhibition of recombinant human LYP using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition and measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1862193 (CHEMBL4363049) Inhibition of recombinant human full length N-terminal GST- tagged PARP1 expressed in baculovirus infected sf9 cells using Colorimetric HRP as substrate incubated for 30 mins by UV/Vis spectrophotometer analysis
- ChEMBL_1890173 (CHEMBL4391927) Inhibition of human recombinant AChE using acetylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition and measured over 2 mins by UV-Vis spectrophotometric analysis based Ellman's method
- ChEMBL_1890174 (CHEMBL4391928) Inhibition of human recombinant BChE using butyrylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition and measured over 2 mins by UV-Vis spectrophotometric analysis based Ellman's method
- ChEMBL_1970870 (CHEMBL4603688) Agonist activity at human TLR7 expressed in HEK-Blue cells assessed as induction of NFkappaB activation by measuring increase in SEAP level by SEAP reporter gene-based UV-vis absorbance method
- ChEMBL_1970871 (CHEMBL4603689) Agonist activity at human TLR8 expressed in HEK-Blue cells assessed as induction of NFkappaB activation by measuring increase in SEAP level by SEAP reporter gene-based UV-vis absorbance method
- ChEMBL_2056654 (CHEMBL4711655) Inhibition of electric eel AChE using acetylthiocholine iodide as substrate preincubated for 15 mins followed by substrate addition and measured for 5 mins by Ellman's reagent based UV-Vis spectrophotometric method
- ChEMBL_2078864 (CHEMBL4734655) Inhibition of human IDO1 in IFN-stimulated human MDA-MB-231 cells preincubated for 48 hrs followed by L-tryptophan addition and measured after 5 hrs by UV-Vis spectrophotometric method
- ChEMBL_2196079 (CHEMBL5108595) Mixed type inhibition of human TS assessed as inhibitor dissociation constant by measuring changes in slope of the double reciprocal plot using dTMP as substrate by UV-Vis spectroscopy based analysis
- ChEMBL_2275091 Inhibition of human recombinant N-terminal his6-tagged IDO1 expressed in Escherichia coli BL21 using L-tryptophan as substrate incubated for 1 hr by methylene blue dye based UV-Vis spectrophotometer analysis
- ChEMBL_2455451 Inhibition of recombinant human SHIP2 (418 to 884 residues) using PIP3 as substrate preincubated for 15 mins followed by substrate addition and measured after 15 mins by UV/visible microplate spectrophotometric analysis
- ChEMBL_2472799 Inhibition of human recombinant PDHK2 assessed as residual PDH activity in presence of ATP using sodium pyruvate/Coenzyme A/thiamine pyrophosphate as substrates incubated for 90 mins by UV-transparent microplate analysis
- ChEMBL_822741 (CHEMBL2040058) Inhibition of APN in human ES2 cell surface using L-Leu-p-nitroanilide as substrate incubated for 5 mins prior to substrate addition measured after 1 hr by UV/VIS spectrophotometry
- ChEMBL_963631 (CHEMBL2395150) Inhibition of APN in human ES-2 cells using L-Leu-p-nitroanilide as substrate incubated for 5 mins prior to substrate addition measured after 1 hr by UV-Vis spectrophotometry
- Enzyme Assay The enzyme assay were performed spectrophotometrically by following the absorbance at 340nm using a hewlett Packard 8453 UV spectrophotometer equipped with a multi-cell transporter and thermostated with a circulating bath.
- ChEBML_1703428 Inhibition of bovine milk xanthine oxidase assessed as reduction in uric acid formation using xanthine as substrate preincubated for 3 hrs followed by substrate addition measured after 1 min by UV spectrophotometric assay
- ChEMBL_1281078 (CHEMBL3100317) Inhibition of rat kidney NADPH-dependent aldose reductase assessed as DL-glyceraldehyde conversion to glycerol preincubated for 20 mins followed by NADPH addition measured after 5 mins by UV-Visible spectrophotometric analysis
- ChEMBL_1561177 (CHEMBL3777370) Inhibition of CE1 in human liver microsomes using 2-(2-Benzoyl-3-methoxyphenyl) benzothiazole as substrate preincubated for 10 mins followed by substrate addition measured after 30 mins by LC-UV analysis
- ChEMBL_1624744 (CHEMBL3867156) Inhibition of hog pancreas alpha-amylase using starch as substrate preincubated for 10 mins followed by substrate addition measured after 10 mins by dinitrosalicylic acid color reagent-based UV-Vis spectrophotometric analysis
- ChEMBL_1683516 (CHEMBL4033995) Inhibition of HDAC1/2 in human HeLa cell nuclear extract using COLOR DE LYS as substrate pretreated for 5 mins followed by substrate addition measured after 30 mins by UV-absorption method
- ChEMBL_1755382 (CHEMBL4190142) Inhibition of C-terminal His-tagged recombinant human CYP17A1delta19H mutant expressed in Escherichia coli JM109 cells assessed as decrease in progesterone hydroxylation in presence of cytochrome P450 reductase by HPLC-UV method
- ChEMBL_1793230 (CHEMBL4265149) Inhibition of recombinant Mycobacterium tuberculosis PtpA using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition and measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1793231 (CHEMBL4265150) Inhibition of recombinant Mycobacterium tuberculosis PtpB using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition and measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1793233 (CHEMBL4265152) Inhibition of recombinant Yersinia enterocolitica YopH using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition and measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1793234 (CHEMBL4265153) Inhibition of recombinant human PTP-PEST using p-nitrophenyl phosphate as substrate preincubated for 10 mins followed by substrate addition and measured every minute for 10 mins by UV-VIS spectrophotometric analysis
- ChEMBL_1892980 (CHEMBL4394901) Inhibition of recombinant human adenosine kinase expressed in Escherichia coli BL21 (DE3) using adenosine as substrate in presence of ATP and PEP by pyruvate kinase-lactate dehydrogenase-coupled UV-vis spectrophotometric assay
- ChEMBL_1930271 (CHEMBL4433522) Inhibition of human SIRT3 assessed as reduction in deacetylase activity using acetylated H3K9 as substrate preincubated for 15 mins followed by substrate addition and measured after 15 mins by UV-HPLC analysis
- ChEMBL_2350313 Inhibition of Saccharomyces cerevisiae alpha-glucosidase using p-nitrophenyl-alpha-D-glucopyranoside as substrate preincubated for 5 mins followed by alpha-glucosidase addition and measured after 15 mins by UV-visible spectrophotometric analysis
- ChEBML_1696006 Inhibition of bovine liver beta-galactosidase pre-incubated for 30 mins with 4-nitrophenyl-beta-D-galactopyranoside substrate before enzyme addition measured over 45 mins at 27 secs intervals by UV/Vis based spectroscopy
- ChEMBL_1509999 (CHEMBL3608052) Inhibition of 15-LOX-1 in human L1236 cells assessed as reduction in conversion of arachidonic acid to 15-HETE pre-incubated 5 min before arachidonic acid substrate addition by HPLC-UV spectroscopy
- ChEMBL_1703428 (CHEMBL4054661) Inhibition of bovine milk xanthine oxidase assessed as reduction in uric acid formation using xanthine as substrate preincubated for 3 hrs followed by substrate addition measured after 1 min by UV spectrophotometric assay
- ChEMBL_1823692 (CHEMBL4323456) Inhibition of human erythrocyte AChE at 10 uM using acetylthiocholine iodide as substrate preincubated for 30 mins followed by substrate addition and measured after 2.5 mins by DTNB reagent based UV-spectrophotometric method
- ChEMBL_1884887 (CHEMBL4386469) Inhibition of human recombinant N-terminal His6-tagged 15-LOX1 expressed in Escherichia coli BL21(DE3) using linoleic acid as substrate preincubated for 10 mins followed by substrate addition by UV absorption analysis
- ChEMBL_1893114 (CHEMBL4395035) Inhibition of human recombinant N-terminal His6-tagged 15-LOX1 expressed in Escherichia coli BL21(DE3) using linoleic acid as substrate preincubated for 8 mins followed by substrate addition by UV absorption analysis
- ChEMBL_2047513 (CHEMBL4702212) Inhibition of C-terminal His-tagged recombinant human MIF expressed in Escherichia coli BL21 (DE3) using 4-HPP as substrate preincubated for 10 mins followed by substrate addition by UV absorption spectrum analysis
- ChEMBL_2286014 Inhibition of Aldose reductase (unknown origin) using D,L-glyceraldehyde as substrate preincubated for 5 mins in presence of NADPH followed by substrate addition and measured after 3 mins by UV-visible spectrophotometric assay
- ChEMBL_2443702 Inhibition of porcine kidney DAO using 6-(5-phenyltetrazol-2-yl)hexan-1-amine as substrate preincubated with enzyme for 15 mins followed by substrate addition measured after 30 mins by UV-HPLC analysis
- ChEMBL_1509997 (CHEMBL3608050) Inhibition of human 15-LOX-1 expressed in Sf9 cells assessed as reduction in conversion of arachidonic acid to 15-HETE pre-incubated 5 min before arachidonic acid substrate addition by HPLC-UV spectroscopy
- ChEMBL_1510006 (CHEMBL3608059) Inhibition of rat 15-LOX-1 expressed in Sf9 cells assessed as reduction in conversion of arachidonic acid to 15-HETE pre-incubated 5 min before arachidonic acid substrate addition by HPLC-UV spectroscopy
- ChEMBL_1510008 (CHEMBL3608061) Inhibition of pig 15-LOX-1 expressed in Sf9 cells assessed as reduction in conversion of arachidonic acid to 15-HETE pre-incubated 5 min before arachidonic acid substrate addition by HPLC-UV spectroscopy
- ChEMBL_1696006 (CHEMBL4046896) Inhibition of bovine liver beta-galactosidase pre-incubated for 30 mins with 4-nitrophenyl-beta-D-galactopyranoside substrate before enzyme addition measured over 45 mins at 27 secs intervals by UV/Vis based spectroscopy
- ChEMBL_1809198 (CHEMBL4308557) Inhibition of human recombinant 5-LOX expressed in Escherichia coli BL21 assessed as conversion of arachidonic acid to 5-HPETE using arachidonic acid as substrate in presence of ATP by UV-vis spectrophotometry analysis
- ChEMBL_1911527 (CHEMBL4413973) Inhibition of human MMP9 using fluorogenic Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide as substrate preincubated for 45 mins measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1911529 (CHEMBL4413975) Inhibition of human MMP10 using fluorogenic Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide as substrate preincubated for 45 mins measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1911530 (CHEMBL4413976) Inhibition of human MMP12 using fluorogenic Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide as substrate preincubated for 45 mins measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1911531 (CHEMBL4413977) Inhibition of human MMP13 using fluorogenic Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide as substrate preincubated for 45 mins measured at 30 sec intervals for 15 mins by UV/vis-spectrophotometry
- ChEMBL_1914052 (CHEMBL4416635) Inhibition of C-terminal His10-tagged Staphylococcus aureus seg50 SerRS expressed in Lemo21(DE3) using Ap4A as substrate in presence of serine by leuconostoc mesenteroides hexokinase/glucose 6-Phosphate dehydrogenase coupled UV-visible spectrophotometry
- ChEMBL_1914053 (CHEMBL4416636) Inhibition of human N-terminal polyHis-tagged SerRS expressed in Escherichia coli BL21 (DE3) using Ap4A as substrate in presence of serine by leuconostoc mesenteroides hexokinase/glucose 6-Phosphate dehydrogenase coupled UV-visible spectrophotometry
- ChEMBL_2067170 (CHEMBL4722423) Inhibition of recombinant human COX2 expressed in baculovirus infected Sf21 cells using arachidonic acid as substrate preincubated for 5 mins followed by substrate addition and measured after 5 mins by UV-visible spectrophotometric method
- ChEMBL_2242993 (CHEMBL5157203) Competitive inhibition of xanthine oxidase (unknown origin) assessed as inhibitory constant of enzyme-substrate complex using xanthine as substrate at varying concentrations preincubated for 15 mins followed by substrate addition by UV spectrophotometric analysis
- ChEMBL_2286026 Inhibition of recombinant Aldose reductase in rat lens using D,L-glyceraldehyde as substrate preincubated for 1 min in presence of NADPH followed by substrate addition and measured after 3 mins by UV-spectrophotometric assay
- ChEMBL_1352309 (CHEMBL3269373) Inhibition of recombinant N-terminal His6-tagged human reticulocyte 12/15-lipoxygenase using arachidonic acid as substrate assessed as equilibrium constant of dissociation from catalytic site measured 15-HpETE formation by UV-vis spectrometric analysis
- ChEMBL_1352310 (CHEMBL3269374) Inhibition of recombinant N-terminal His6-tagged human reticulocyte 12/15-lipoxygenase using arachidonic acid as substrate assessed as equilibrium constant of dissociation from secondary site measured 15-HpETE formation by UV-vis spectrometric analysis
- ChEMBL_1825929 (CHEMBL4325693) Binding affinity to heme in His-tagged CYP3A5 (unknown origin) expressed in Escherichia coli DH5alpha assessed as changes in absorbance spectra of heme-compound complex by measuring dissociation constant by UV-vis spectrophotometric titration analysis
- ChEMBL_1914051 (CHEMBL4416634) Inhibition of C-terminal His10-tagged Escherichia coli B ER2560 SerRS expressed in Lemo21(DE3) using Ap4A as substrate in presence of serine by leuconostoc mesenteroides hexokinase/glucose 6-Phosphate dehydrogenase coupled UV-visible spectrophotometry
- ChEMBL_2107524 (CHEMBL4816199) Inhibition of G6PD (unknown origin) assessed as reduction in 6-phospho-D-glucono-1,5-lactone and NADPH production using glucose-6-phosphate and NADP+ as substrate incubated for 15 mins by UV absorption photometry assay
- ChEMBL_2242992 (CHEMBL5157202) Mixed-type inhibition of xanthine oxidase (unknown origin) assessed as inhibitory constant of enzyme-substrate complex using xanthine as substrate at varying concentrations preincubated for 15 mins followed by substrate addition by UV spectrophotometric analysis
- ChEMBL_966861 (CHEMBL2401005) Inhibition of C-terminal His6-tagged wild type Francisella tularensis SCHU S4 FabI expressed in Escherichia coli BL21 (DE3) using CrCoA as substrate by UV-vis spectrophotometric analysis in presence of non pre-equilibrated NAD+
- Protease Inhibition Assay Sensitivity of HIV-1 protease activity to protease inhibitors was determined by a peptide substrate cleavage assay. The products were analyzed by using HPLC with UV detection (225 nm) for quantification of the products.
- ChEBML_1688196 Inhibition of recombinant human PTP1B (1 to 322 residues) expressed in Escherichia coli using pNPP as substrate preincubated for 60 mins followed by substrate addition measured at 30 secs interval for 10 mins by UV-spectrophotmetric method
- ChEMBL_2060801 (CHEMBL4716054) Inhibition of N-terminal His6-tagged Desulfovibrio desulfuricans G20 CutC encoding 52 residues assessed as reduction in acetaldehyde using choline as substrate in presence of NADH-dependent yeast alcohol dehydrogenase by UV-Vis absorbance based assay
- ChEMBL_2074995 (CHEMBL4730529) Inhibition of 5-LOX in human PMNL assessed as reduction in 5-HETE and LTB4 formation pre-incubated for 15 mins before addition of AA and A23187 and measured after 10 mins by HPLC/UV assay
- ChEMBL_2443690 Inhibition of bovine plasma VAP-1 using 6-(5-Pheny1-2H-tetrazol-2-yl)hexan-1-amine as substrate preincubated with enzyme for 15 mins followed by substrate addition measured after 30 mins by UV-HPLC analysis
- ChEMBL_2443705 Inhibition of recombinant human MAO-B using 4-(5-phenyl-2H-tetrazol-2-yl)butan-1-amine as substrate preincubated with enzyme for 15 mins followed by substrate addition measured after 30 mins by UV-HPLC analysis
- ChEMBL_2443707 Inhibition of human plasma VAP-1 using 6-(5-Pheny1-2H-tetrazol-2-yl)hexan-1-amine as substrate preincubated with enzyme for 15 mins followed by substrate addition measured after 120 mins by UV-HPLC analysis
- PPIase Assay PPIase activity assay were performed at 283 K in quartz cuvettes with a path length of 1 cm under vigorous stirring with a Hewlett-Packard 8453A UV-vis spectrophotometer in 35 mM HEPES buffer (pH 7.8).
- ChEMBL_1688196 (CHEMBL4038766) Inhibition of recombinant human PTP1B (1 to 322 residues) expressed in Escherichia coli using pNPP as substrate preincubated for 60 mins followed by substrate addition measured at 30 secs interval for 10 mins by UV-spectrophotmetric method
- ChEMBL_1808810 (CHEMBL4308169) Inhibition of human TRPV6 expressed in HEK293 cells assessed as reduction in Cd2+ influx preincubated for 5 mins followed by CdCl2 addition using 365 nm UV-light irradiated compound by calcium-5 fluorescence dye-based FLIPR assay
- Enzyme Inhibition Assay Enzyme reaction was monitored by measuring the NADH absorption at 340 nm on a UV-Vis spectrometer. The formation of NADH was measured for 30 s. Data were graphically analyzed by Lineweaver-Burk double reciprocal plots
- ChEMBL_1624267 (CHEMBL3866679) Inhibition of FLAP in human neutrophils assessed as suppression of A23187-stimulated 5-LO product formation using arachidonic acid as substrate preincubated for 15 mins followed by A23187/arachidonic acid addition after 10 mins RP-HPLC/UV analysis
- ChEMBL_1630267 (CHEMBL3872973) Inhibition of Leishmania major recombinant N-terminal hexa-His tagged PTR1 catalytic activity expressed in Escherichia coli BL21(DE3) using biopterin as substrate measured for 60 sec in presence of NADPH by UV-visible spectrophotometric method relative to control
- ChEMBL_1677681 (CHEMBL4027824) Displacement of [125I]-IAAP from human P-gp expressed in high five insect cell membrane vesicles preincubated for 10 mins followed by photocrosslinking for 10 mins by exposing under UV light for 10 mins by SDS-PAGE based method
- ChEMBL_1774622 (CHEMBL4231614) Inhibition of FLAP in A23187-stimulated human neutrophils assessed as reduction in 5-LO product formation preincubated for 15 mins followed by A23187 and arachidonic acid addition and measured after 10 mins by UV based RP-HPLC analysis
- ChEMBL_1774623 (CHEMBL4231615) Inhibition of FLAP in A23187-stimulated human monocytes assessed as reduction in 5-LO product formation preincubated for 15 mins followed by A23187 and arachidonic acid addition and measured after 10 mins by UV based RP-HPLC analysis
- ChEMBL_1979320 (CHEMBL4612455) Inhibition of mPGES1 in human A549 cells assessed as reduction in IL-1beta induced PGE2 release using PGH2 as substrate preincubated for 15 mins followed by substrate addition and measured after 1 min by RP-UV-HPLC analysis
- ChEMBL_2034043 (CHEMBL4688201) Inhibition of N-terminal His tagged human Pin1 expressed in Escherichia coli BL21 (DE3) using Suc-Ala-Glu-Pro-Phe-4-nitroanilide as substrate preincubated with enzyme for 10 mins followed by substrate addition by UV-Vis spectrophotometer analysis
- ChEMBL_2062459 (CHEMBL4717712) Inhibition of human IDO1 in IFN-gamma-induced human MDA-MB-231 cells assessed as N-formylkynurenine formation using L-tryptophan as substrate pretreated for 4 hrs followed substrate addition measured after 5 hrs by UV-vis spectrophotometric analysis
- ChEMBL_2071491 (CHEMBL4727025) Inhibition of recombinant human N-terminal Met and 6-His-tagged Glyoxalase-1 (Ala2 to Met184 residues) using glutathione and methylglyoxal as substrates preincubated for 15 mins followed by enzyme addition by double beam UV-vis spectrophotometric method
- DHFR Inhibition Assay Enzyme activity assays were performed by monitoring the change in UV absorbance at 340 nm. Enzyme assays were performed at least four times. IC50 values and their standard deviations were calculated in the presence of varying ligand concentration.
- ChEMBL_1624270 (CHEMBL3866682) Inhibition of FLAP in human monocytes assessed as suppression of A23187-stimulated 5-LO product formation using 10 uM arachidonic acid as substrate preincubated for 15 mins followed by A23187/arachidonic acid addition after 10 mins by RP-HPLC/UV analysis
- ChEMBL_1774624 (CHEMBL4231616) Inhibition of 5-LO in A23187-stimulated human neutrophils assessed as reduction in 5-LO product formation preincubated for 15 mins followed by A23187 and 10 uM arachidonic acid addition and measured after 10 mins by UV based RP-HPLC analysis
- ChEMBL_2157952 (CHEMBL5042702) Inhibition of PI3K alpha (unknown origin) using PIP2 as substrate irradiated with 365 nm of UV exposure for 2 mins and incubation in dark at room temperature for 1 hr in presence of ATP measured by ADP-Glo luminescent assay
- ChEMBL_2195999 (CHEMBL5108515) Inhibition of Mycobacterium tuberculosis Eis assessed as Eis-mediated kanamycin acetylation preincubated for 10 mins followed by substrate addition and measured for 2 to 5 mins using acetyl-CoA as substrate in presence of kanamycin by UV-Vis spectroscopy analysis
- Inhibition Assay The rate of the enzymatic reaction was determined by measuring product formation by a spectrophotometric assay developed by Xun and co-worker with modification. The assay was performed on a Cary 50 Bio or Cary 5000 UV-visible spectrophotometer (Varian).
- UV-Thermal Melt UV thermal melt experiments were carried out in a quartz cuvette loaded with sample containing enzyme and each test compound using a spectrophotometer. For each measurement, absorbance data at 230 nm was collected as the temperature was scanned from 25 to 80 C at a ramp rate of 0.2 C /min. The melting temperature (Tm) for each sample was calculated as the maximum deflection point of the first derivative of the melting transition. The Tm values are converted into Kd values using the equation described in J. Med. Chem., 46, 4669.
- ChEMBL_1892973 (CHEMBL4394894) Inhibition of wild-type N-terminal TEV cleavage site-fused/His-tagged Mycobacterium tuberculosis H37Rv adenosine kinase expressed in Escherichia coli BL21 (DE3) using adenosine as substrate in presence of ATP and PEP by pyruvate kinase-lactate dehydrogenase coupled UV-vis spectrophotometric method
- ChEMBL_2293100 Inhibition of P32-[alpha-GTP] cap radiolabelled RNA binding to recombinant eIF4E (unknown origin) assessed as decrease in cross linked RNA-eIF4E complex formation preincubated with UV radiation for 30 mins followed by RNase addition and measured after 30 mins by SDS-PAGE based autoradiographic analysis
- ChEMBL_1914617 (CHEMBL4417200) Inhibition of of N-terminal (His)6 and Xpress-tagged LTA4H (unknown origin) expressed in Escherichia coli BL21(DE3) cells assessed as reduction in aminopeptidase activity by measuring p-nitroanilide release using L-Alanine-p-nitroanilide incubated for 10 mins by UV plate reader based assay
- ChEMBL_1914636 (CHEMBL4417219) Inhibition of of N-terminal (His)6 and Xpress-tagged LTA4H (unknown origin) expressed in Escherichia coli BL21(DE3) cells assessed as reduction in aminopeptidase activity by measuring p-nitroanilide release using L-arginine-p-nitroanilide incubated for 10 mins by UV plate reader based assay
- ChEMBL_2067368 (CHEMBL4722621) Inhibition of C-terminal His-tagged recombinant human MIF tautomerase activity expressed in Escherichia coli BL21 using 4-hydroxyphenyl pyruvic acid as substrate preincubated for 15 mins followed by substrate addition and measured for 10 mins in presence of 0.5 mM EDTA by UV absorbance analysis
- ChEMBL_1930269 (CHEMBL4433520) Inhibition of recombinant human N-terminal His6-tagged/SUMO-fused SIRT2 (38 to 356 residues) expressed in Escherichia coli BL21 assessed as reduction in deacetylase activity using acetylated H3K9 as substrate preincubated for 15 mins followed by substrate addition and measured after 6 mins by UV-HPLC analysis
- ChEMBL_2067370 (CHEMBL4722623) Inhibition of C-terminal His-tagged recombinant human MIF tautomerase activity expressed in Escherichia coli BL21 using 4-hydroxyphenyl pyruvic acid as substrate preincubated for 15 mins followed by substrate addition and measured for 10 mins in absence of EDTA by UV absorbance based Cheng-Prusoff equation analysis
- ChEMBL_2067369 (CHEMBL4722622) Inhibition of C-terminal His-tagged recombinant human MIF tautomerase activity expressed in Escherichia coli BL21 using 4-hydroxyphenyl pyruvic acid as substrate preincubated for 15 mins followed by substrate addition and measured for 10 mins in presence of 0.5 mM EDTA by UV absorbance based Cheng-Prusoff equation analysis
- ChEMBL_941535 (CHEMBL2330526) Inhibition of human recombinant N-terminus 6X-histidine-tagged indoleamine 2,3-dioxygenase expressed in Escherichia coli assessed as inhibition of L-tryptophan to N-formylkynurenine formation measured as residual enzyme activity incubated for 15 mins prior to substrate addition measured after 30 mins by UV-vis spectrophotometric analysis relative to DMSO-treated control
- Enzyme Inhibition Assay Enzymes were incubated with inhibitors at eight inhibitor concentrations bracketing the Ki, prepared by serial dilution along with control lacking the inhibitor. Substrate was added, and initial rate of substrate hydrolysis were determined using a UV/MAX Kinetic Microplate Reader. Ki values were determined with BatchKi or true Ki values with DYNAFIT.
- Heme Binding Assay Heme binding was tracked by difference spectroscopy in the Soret region of the UV−visible spectrum. Successive aliquots of 0.5 mM hemin in 0.1 M NaOH were added to both the sample cuvette, which contained 10 μM apo-HutZ, and the referencecuvette. Spectra were recorded 3 min after the addition of each heme aliquot.
- UV-Visible Titration Assay All UV-visible spectra were measured on a Varian Cary 50 spectrometer with temperature control from a Peltier cooler. The holo-enzyme was titrated in 0.1M potassium phosphate buffer starting at pH 6.8, 25 °C, and finishing at pH 5.1 or 10.2. Each titration was conducted in a 2-ml cuvette with the protein at ~8 uM heme-containing monomer, where the heme concentration was determined via the pyridine hemochrome assay. Solutions were continuously stirred, and the pH was monitored using a pH meter with a glass electrode (Corning, pH 430). Small volumes of HCl (1 M) or NaOH (1 M) were added to the solution; the pH was measured, and UVvisible spectra were recorded. All spectra were adjusted for dilution.
- NF-keppaB GFP Assay NFκB/Jurkat/GFP transcriptional reporter cell line was obtained from SBI System Biosciences. NF-κB/Jurkat/GFP Reporter cells (5×105 cells) were plated at a concentration of 1 million cells/ml into each well of a 24-well plate. TNF-α (5 ng/ml) was added. Compounds were added via serial dilutions to corresponding wells. After 24 hours, 100 μl of the cells were transferred to a well of a Costar UV plate (96 well, No lid, w/ UV Transparent Flat Bottom, Corning, N.Y., Cat#3635) and the intensity of GFP fluorescence was measured (Excitation 485+/−20, Emission 528+/−20) in a Synergy HT Multi-Detection Microplate Reader (BioTech, Winooski, Vt.). The intensities of GFP measured were plotted against the amount of TNF-α.
- Glutathione Reductase Activity Assay Enzymatic activity was measured by Beutler's method with a Shimadzu Spectrophotometer UV-(1208), at 25°C. The assay system contained 100 mM Tris-HCl buffer pH 8.0, including 0.5 mM EDTA, 3.3 mM GSSG and 0.1 mM NADPH. One enzyme unit is defined as the oxidation of 1 μmol NADPH per min under the assay condition at 25°C.
- Steady State Kinetics Assays Steady state kinetics assays were performed on a Cary 100 UV−vis spectrophotometer in 10 mM phosphate-buffered saline (pH 7.4) with 2 nM ADC-7. The Vmax and Km of the β-lactam substrate nitrocefin (NCF) for ADC-7 were determined from initial steady-state velocities using this colorimetric indicator (NCF, Δε482 = 17400 M^−1 cm^−1).
- Enzyme Assay The aim of this study was to evaluate TK-112690 in vivo as an inhibitor of uridine phosphorylase (UPase) enzyme activity. The range of TK-112690 doses studied for ability to prevent metabolic breakdown of uridine, through the in vitro inhibition of mouse and human small intestinal UPase enzyme, was 0, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 5000 and 10000 uM). Detection of UPase activity was determined by HPLC analysis using UV detection of uracil concentration (UPase catabolizes uridine into uracil and ribose-1-phosphate).The UPase enzyme material was prepared from homogenized mouse and human being small intestinal tissue. TK-112690 was dissolved in water (50 mg/ml) and analyzed for UPase inhibition in aqueous solution containing 5 mM uridine, 0.01 M Tris, 0.01 M phosphate, 1 mM EDTA, and 1 mM DTT. Reactions were performed at 37 C. at pH of 7.3.TK-11260 inhibition of mouse and human UPase was analyzed by reverse phase HPLC using UV detection.
- GLO1 Activity Assay Assays were carried out in 100 mM sodium phosphate, pH 7.0 buffer using 96-well Clear UV Plate (Corning UV Transparent Microplates; catalog #3635). A fresh solution of glutathione (pre-substrate 1, 100 mM) as well as methylglyoxal (pre-substrate 2, 100 mM) was prepared in deionized water. The substrate was prepared by adding 14.5 ml of buffer and 0.99 ml of each pre-substrate components. The substrate mixture was vortexed vigorously for 15 s, then allowed to sit at room temperature for 20 min. Initial well volume was 50 μl containing GLO1 (40 ng) and inhibitor. This protein and inhibitor mixture was incubated for 15–20 min before the addition of substrate. To this was then added substrate (150 μl), yielding a maximum amount of 5% DMSO per well. The enzyme activity was measured using a BioTek Synergy H4 plate reader by measuring absorbance at 240 nmevery 1 min for 8 min.
- TDO2 Enzymatic Assay The TDO2 enzymatic assay was performed by UV absorption using a recombinant TDO2 and L-Tryptophan substrate. The UV absorption signal at 321 nm is correlated with the amount of N-formylkynurenine reaction product of TDO2. The compounds were diluted in 10% DMSO and 5 μL of the dilution was added to a 100 μL reaction so that the final concentration of DMSO is 0.5% in all of reactions. All of the reactions were conducted at room temperature. The 100 μL reaction mixture in TDO2 Assay Buffer contains 50 nM TDO2, the indicated amount of the inhibitor, 200 μM tryptophan, and the coupled reaction components. The reaction mixture incubated for 75 min prior to reading the UV absorption signal. For the negative control (blank), 5 μl of the assay buffer was added instead of the TDO2.Absorption signals were measured using a Tecan Infinite M1000 plate reader. Binding experiments were performed in duplicate at each concentration. The percent activity in the presence of each compound was calculated according to the following equation: % activity=[(A−Ab)/(At−Ab)]×100, where A=the absorption signal in the presence of the compound, Ab=the absorption signal of blank, At=the absorption signal in the absence of the compound. The percent inhibition was calculated according to the following equation: % inhibition=100−% activity. The IC50 was calculated using non-linear regression in Graph Pad Prism 4.
- Esterase Activity Assay Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV-VIS) according to the method described by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229].The inhibitory effects of compounds 1-14 and AZA were examined. All compounds were tested in triplicate at each concentration used.
- Protease Inhibition Assay The IC50 values for the compounds were determined using purified HIV-1 Protease. Inhibition of the cleavage of the peptide H-Val-Ser-Gln-Asn-(L-beta-napthylalanine)-Pro-Ile-Val-OH was assessed at 30 °C, pH = 5.5 with [Enzyme] = 30 pM for 1 h, using HPLC with UV detection for quantification of the products. For the IC50 data a substrate concentration of 0.4 mg/mL was used, and data was fit to a four parameter sigmoidal equation.
- Study with Mouse and Human Intestinal Tissue Homogenates The aim of this study was to evaluate TK-112690 in vivo as an inhibitor of uridine phosphorylase (UPase) enzyme activity. The range of TK-112690 doses studied for ability to prevent metabolic breakdown of uridine, through the in vitro inhibition of mouse and human small intestinal UPase enzyme, was 0, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 5000 and 10000 μM). Detection of UPase activity was determined by HPLC analysis using UV detection of uracil concentration (UPase catabolizes uridine into uracil and ribose-1-phosphate).The UPase enzyme material was prepared from homogenized mouse and human being small intestinal tissue. TK-112690 was dissolved in water (50 mg/ml) and analyzed for UPase inhibition in aqueous solution containing 5 mM uridine, 0.01 M Tris, 0.01 M phosphate, 1 mM EDTA, and 1 mM DTT. Reactions were performed at 37° C. at pH of 7.3.TK-11260 inhibition of mouse and human UPase was analyzed by reverse phase HPLC using UV detection. HPLC analysis was performed at ambient temperature with a Water 2695 Alliance system equipped with a C18 ECONOSIL 5 U ALLtech column. 20 μl of UPase reaction samples were auto-injected onto column. Mobile phase consisted of water eluting for first 2.5 ml and acetonitrile gradient to 100% in 12.5 ml (flow rate 0.5 ml/min). The outlet fluent was monitored by UV absorption in the range of 240-320 nm. UPase enzymatic activity was based on the AUC of the uracil peaks.
- AChE and BuChE Inhibition Assay This spectrophotometric assay is based on the reaction of 5,5-dithio-bis(2-nitrobenzoic)acid (DTNB) with thiocholine to yield a colored product. Shimadzu (Kyoto, Japan) UV-1700 UV-vis spectrophotometer was used to record the measurements. The enzyme solutions were prepared at 2.5 units/mL concentrationin 1% gelatin solution. BChE/AChE and compound solutions (50 μL each) in 2% DMSO at 10^-1-10^-6 mM concentration were added to 3 mL phosphate buffer (pH 8 &plusnm; 0.1)followed by incubation for 5 min at 25 °C. The reaction was commenced by the addition of acetylthiocholine iodide (ATC) (10 μL) and DTNB (50 μL) to the enzyme-inhibitor mixture. The yellow anion production was measured at 412 nm for 10 min. Using similar method, same enzyme solution without the inhibitor was processed which served as a control. The blank reading contained 10 μL substrate, 50 μL DTNB, 50 μL (2%) DMSO, and 3 mL buffer. The reaction was performed in triplicate.
- Kinetic Studies Inhibitor concentrations varied from 0.25 to 5 times the Ki value in 50 mM sodium phosphate, 100 mM sodium chloride buffer, pH 7.0, at 30 °C. 2-Chloro-4-nitrophenyl α-d-maltotrioside (CNP-G3) was used as the substrate in a similar range of concentrations. Initial reaction rates for the release of chloronitrophenolate were measured and the further progress of substrate cleavage monitored continuously. Reactions were performed on a Varian Cary 4000 UV/Vis spectrophotometer at 400 nm and 30 °C.
- Topoisomerase IIα Relaxation Assay Relaxation of negatively supercoiled plasmid DNA by human topoisomerase IIα was assayed in 20 µL of reaction buffer (10 mM Tris-HCl, pH 7.9, 150 mM NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol and ATP) containing 250 ng of supercoiledpHOT1 plasmid DNA and 1 unit of enzyme. After incubation at 37 °C for 30 min, the reactions were terminated and analysed by agarose gel electrophoresis. The ethidium bromide-stained gel was photographed over UV light for densitometry analysis.
- Enzyme Inhibition Assay The enzyme reaction was started by the addition of enzyme to the reaction mixture containing substrate and test compounds. The reaction was stopped by spotting an aliquot onto a silica gel plate that had been prespotted with thymine and thymidine. The plate was developed in ethyl acetate-water-formic acid (60:35:5), and the spots were visualized under UV light (254 nm) and cut out for radioactivity determination in toluene-based scintillation cocktail. IC50 values were determined from dose response curves, and were means of minimum three experiments.
- Esterase Activity Assay CA activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25 °C using a spectrophotometer (Shimadzu, UVmini-1240 UV-VIS spectrophotometer) according to the method described by Verpoorte et al.37 The enzymatic reaction contained 1.4mL 0.05M Tris-SO4 buffer (pH 7.4), 1mL 3mM 4-NPA, 0.5mL H2O and 0.1mL enzyme solution (total volume, 3.0 mL). A reference measurement was obtained bypreparing the mixture without the enzyme solution. All measurements were recorded in triplicate.
- NOS Enzyme Inhibition Assay Nitric oxide formation from NOS was monitored by the hemoglobin capture assay. The assay was initiated by addition of enzyme and was monitored at 401 nm on a Perkin-Elmer Lambda 10 UV-vis spectrophotometer. The apparent Ki values were obtained by measuring percent inhibition in the presence of 10 uM L-arginine with at least four different concentrations of inhibitor. The IC50 values were determined by linear regression analysis of the percent inhibition data. Inhibition constants Ki were derived from the equation, Ki = IC50 / (1+ [S]/Km).
- NOS Enzyme Inhibition Assay Nitric oxide formation from NOS was monitored by the hemoglobin capture assay. The assay was initiated by addition of enzyme and was monitored at 401 nm on a Perkin-Elmer Lambda 10 UV-vis spectrophotometer. The apparent Ki values were obtained by measuring percent inhibition in the presence of 10 uM L-arginine with at least four different concentrations of inhibitor. The IC50 values were determined by linear regression analysis of the percent inhibition data. Inhibition constants Ki were derived from the equation, Ki = IC50 / (1+ [S]/Km).
- Protease Inhibition Assay IC50 values are determined by inhibition of the cleavage of the peptidic substrate [V-S-Q-N-(beta-naphthylalanine)-P-I-V]. The IC50 value represents the concentration of inhibitor required to inhibit cleavage of substrate by 50%. The assay is performed by addition of purified enzyme to a mixture of inhibitor and the above substrate at pH 5.5. The reaction medium is incubated for 30 min at 37 °C followed by quenching with H3-PO4 and then analyzed by reverse phase HPLC UV (220 nm) detection.
- QR2 Inhibition Assay The activity of recombinant human QR2 under steady-state conditions was evaluated on a MolecularDevices SpectraMax Plus 384 UV-visible spectrophotometer by monitoring the decrease in absorbance of the enzyme co-substrate NMeH at 360 nm. The inhibition assays were initiated by adding the enzyme to a reaction mixture containing NMeH, menadione and different concentrations of inhibitor. The inhibitory concentration that produces 50% inhibition (IC50 value) for each inhibitor was calculated by curve-fitting. All initial velocity data were measured in triplicate, and the mean +/- SD.
- Activity Assay GR activity was determined by the method of Carlberg and Mannervik [Carlberg et al., FL:Academic Press, 72:248-254] with a Shimadzu Spectrophotometer UV-(1208) at 25°C. The assay system contained 40 mM Tris-HCl buffer, pH 8.0, including 0.8 mM EDTA, 1 mM GSSG and 0.1 mM NADPH in 1 mL total reaction volume. The activity was measured by monitoring the decrease in absorbance at 340 nm due to the oxidationof NADPH at 25°C. One enzyme unit is defined as the oxidation of 1 mmol NADPH per min under the assay conditions.
- CA Activity Assay CA activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV-VIS) according to the method described by Verpoorte et al [Verpoorte et al., J. Biol. Chem., 242:4221-4229]. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL 3 mM 4-NPA, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette withoutenzyme solution.
- Cholinesterases Inhibition Assay The stock solutions of the target compounds were prepared in a mixture of DMSO (1 ml) and ethanol (9 ml) and diluted with 0.1 MKH2PO4/K2HPO4 buffer (pH 8.0) to obtain final concentrations. 20 ml of substrate (acetylthiocholine iodide 0.075 m) was added to the test solution to obtain final concentration of 466 mm. All experiments were performed based on the previously described method.[Nadri et al., Bioorg. Med. Chem., 18:6360] Spectrophotometric measurements were performed on a UV Unico double-beam spectrophotometer. The same method was also taken for BuChE inhibition assay.
- Enzyme Inhibition Assay Experimental Procedure for Kinetic Analyses: Enzymatic reactions were carried out in PBS buffer (pH 7.4) using pNP-GlcNAc as a substrate (0.5 mM) and monitored continuously at 37 C. at 400 nm using a Cary 3E UV-VIS spectrophotometer equipped with a Peltier temperature controller. Reactions were pre-heated in a 500 L quartz cuvette for approximately 5 minutes followed by addition of 10 L enzyme via syringe (final enzyme concentration 0.002 mg/mL). Reaction velocities were determined by linear regression of the linear region of the reaction progress curve between the first and third minutes.
- Esterase Activity Assay Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV-VIS) according to the method described in the literature [Verpoorte et al., J. Biol. Chem., 242:4221-4229; Innocenti et al., Bioorg. Med. Chem. Lett., 18:2267-2271]. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL of 0.05 M tris-SO4 buffer (pH 7.4), 1 mL of 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution.
- Esterase Activity Assay Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of3 min at 25°C using a spectrophotometer (CHEBIOS UV-VIS) according to the method described in the literature [Verpoorte et al., J. Biol. Chem., 242:4221-4229; Innocenti et al., Bioorg. Med. Chem. Lett., 18:2267-2271]. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL of 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL of 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution.
- Human Topoisomerase IIalpha Decatenation Inhibition Assay Human Topoisomerase IIα assays were carried out by adding the compounds to 185 ng of kinetoplast DNA (kDNA, from TopoGen) in the buffer supplied by the manufacturer before the addition of 2 U of the enzyme. The samples were incubated for 15 minutes at 37° C. before the addition of 4 μL of a stop buffer containing 5% sarkosyl, 0.25% bromophenol blue, and 25% glycerol. The reactions were then analyzed by electrophoresis in 1% agarose gels containing 0.5 g/mL ethidium bromide before being photographed under UV light.
- IC50 Activity Assays The assays were performed at 25 degrees Celsius in a 100mM sodium phosphate buffer (pH 7.0), with the AR protein amount reaching the Vmax and 0.2mM NADPH. The final reaction volume was 500uL per reaction. All compounds were dissolved in dimethyl sulphoxide, and the corresponding solution was added to the cell and incubated for 5 min at 25 degrees Celsius prior to the addition of the substrate. The reaction was initiated by the addition of 1mM glyceraldehyde, and the decrease in optical density at 340nm was monitored for 3 min at 25 degrees Celsius in a UV-vis spectrophotometer.
- Inhibition Activity Assay Screening Method:Name of method: Activity Evaluation of DPP4, Fluorescence.Instrument: Microplate reader, Envision (PerkinElmer, USA).Material: human DPP4, which was, in this experiment, obtained by using baculovirus expression system in insect cells. Substrate was Gly-Pro-AMC.Process:DPP4 can specifically hydrolyze the substrate, Gly-Pro-AMC to produce a product, AMC, which, excited by UV light at 355 nm, can produce emission light at 460 nm. Linear change of fluorescence values were dynamically measured at 460 nm wavelengths per unit time, thereby calculating DPP4 activity. MERK-0431 was used as a control compound in the experiment.
- Paraoxonase Activity Assay Paraoxonase enzyme activity was determined at 25 °C with paraoxon (1 mM) in 50 mM glycine-NaOH (pH 10.5) containing 1 mM CaCl2. The enzyme assay was based on the estimation of p-nitrophenol at 412 nm. Assays were performed using a spectrophotometer (CHEBIOS UV-VIS, Fullerton, CA). The molar extinction coefficient of paranitrophenol (Ɛ = 18 290/M/cm at pH 10.5) is used for thecalculation of enzyme activity. One enzyme unit was defined as the amount of enzyme that catalyzes the hydrolysis of 1 µmol of paraoxon at 25 °C (J. Chromatogr. B. 2006, 836:15-21).
- Thermal Denaturation Assay UV thermal melt experiments were carried out in a quartz cuvette loaded with sample containing enzyme and each test compound using a spectrophotometer. For each measurement, absorbance data at 230 nm was collected as the temperature was scanned from 25 to 80 C at a ramp rate of 0.2 C /min. The melting temperature (Tm) for each sample was calculated as the maximum deflection point of the first derivative of the melting transition. The Tm values are converted into Kd values using the equation: Tm =3.08 (log Ka) + 31.2, where Ka= 1/Kd.
- hKAT-I Activity Assay A typical reaction mixture contains kynurenine, alpha-ketobutyrate, PLP and hKAT-I in the presence of test compound. The reaction mixture was incubated for 20 min at 38 deg C, and the reaction was stopped by adding an equal volume of 1 M formic acid. The product KYNA was detected by high-performance liquid chromatography and quantified on the basis of the absorbance at 330 nm as measured by a Hitachi L-7400 UV detector. The assays for each inhibitor were performed four times, and the data were analyzed using the SigmaPlot enzyme kinetics module (SPSS Inc.).
- ICL Enzyme Assay Isocitrate lyase activity was determined at 37°C by measuringthe formation of glyoxylate phenylhydrazone at 324 nm [Bai et al., Drug Dev. Res., 67:818-23]. The reaction mixture contained 100 μL of 0.5 mM potassium phosphate buffer, 1.2 μL of 1 mM magnesium chloride, 24 μL of 100 mM 2-mercaptoethanol, 7 μL of 4 mM phenylhydrazine hydrochloride, 6 μL of 50 mM trisodium isocitric acid, and ICL enzyme (usually 3-6 μL). This mixture was made up to 200 μL with MilliQ water (water that has been purified and deionized to a high degree by a water purification system manufactured by Millipore Corporation). At the end of the 10th minute this reaction mixture was made up to 1 mL and ultraviolet (UV) absorbance was measured at 324 nM, which served as a control. For the test compounds, 3 μL of 100 mM 3-nitropropionic acid (3-NPA) was used, and in the case of the candidate molecules, 10 μL of 10 mM concentration was added to the abovementioned reaction mixture. At the end of the 10th minute this reaction mixture was made up to 1 mL and UV absorbance was measured at 324 nM, which served as the test.
- Antimalarial Testing In Vitro (IC50) and Measurement of Inhibition Constant (Ki) The concentration of inhibitor that inhibited 50% of the parasite growth (IC50) was determined from the sigmoidal curve obtained by plotting the percentages of [3H]-hypoxanthine incorporation against drug concentrations. The activity of pfDHFR-TS was determined spectrophotometrically using a UV-Vis spectrophotometer. The Ki values of the inhibitors for the wild-type and mutant enzymes were determined by fitting to the equation IC50 = Ki(1 + ([S]/Km)), where IC50 is the concentration of inhibitor that inhibits 50% of the enzyme activity under the standard assay condition and Km is the Michaelis constant for the substrate H2folate.
- Enzyme Inhibition Assay The enzyme reaction was started by the addition of enzyme to the reaction mixture containing substrate and test compounds. The reaction was stopped by spotting an aliquot onto a silica gel plate that had been prespotted with thymine and thymidine. The plate was developed in ethyl acetate-water-formic acid (60:35:5), and the spots were visualized under UV light (254 nm) and cut out for radioactivity determination in toluene-based scintillation cocktail. Kinetic constants Km and Ki were determined from the Lineweaver-Burk and Dixon plots. Data based on results from at least four independent experiments were evaluated by the nonlinear regression method (GOSA, Bio-Log).
- Esterase Activity Assay CA activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV-Vis) according to the method described by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229]. The enzymatic reaction contained 1.4 mL 0.05 M Tris-SO4 buffer(pH 7.4), 1 mL 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution (total volume, 3.0 mL). A reference measurement was obtained by preparing themixture without the enzyme solution. All measurements were made in triplicate.
- Fluorescence Direct Binding Assay 1) Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content2) Titration measurements of the protein against ligand by at least 12 titration steps to obtain a good s-curve fit3) Repeat the same titration measurements with the ligand alone to enable correction4) Check the stability of the protein once by titration against DMSO alone5) Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of an UV-spectrophotometer6) Use Excel template for the correction of the measured raw data7) Use GraphPad Prism software for the quadratic binding fit and the KD evaluation.
- QR2 Assay and IC50 Value Determination The activity of QR2 under steady-state conditions was evaluated on SpectraMax Plus 384 UV/vis spectrophotometer by monitoring the increase in absorbance of the product formazan at 610 nm which is produced from MTT. IC50 values were determined in 96-well plates. Each assay mixture contained QR2, NMeH, and MTT in a reaction buffer containing test compounds. Inhibitor concentrations ranged from 0.4 to 500 uM, and 11 different inhibitor concentrations were chosen for each IC50 value determination. Assays at each inhibitor concentration were performed in triplicate, and the average rate values and standard deviation at each rate were used to determine the IC50 values.
- XO inhibitory activity Bovine milk XO activity was assayed spectrophotometrically by measuring the uric acid formation at 293 nm using a UV-visible spectrophotometer at 25°C. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.6), 75 µM xanthine, and 0.08 units of XO. Inhibition of XO activity of compounds was measured by following the decrease in the uric acid formation at 293 nm at 25°C. The enzyme was pre-incubated for 5 min, with test compound, dissolved in DMSO (1% v/v). The reaction was initiated by the addition of xanthine. Allopurinol (1 mM) was used as a positive control.
- Enzyme Assay The assay was performed in a 100 μL final volume in 96-well UV plates (Costar, 3635) with a reaction buffer composed of 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 3 mM EDTA and 1 mM dithiothreitol, 4% v/v DMSO plus or minus inhibitory compound and 0.75 μg of purified IMPDH II enzyme per well (from 1 mg/mL stock concentration). The final volume of the enzyme stock solution per well was 0.75 μL which was insignificant to cause any change in the final assay buffer composition. The reaction was initiated by the addition of 300 μM of IMP and 500 μM of NAD+ and the assay was allowed to proceed at 37 °C for 50 min before stopping by the addition of 15 mM GMP. The NADH generated was measured by reading the absorbance at 340 nm. At this wavelength, a background of <0.1 optical density (OD) was observed with negligible crosstalk between wells. Molecular Devices Spectramax 384 plus (Molecular Devices, LLC, Sunnyvale, CA) was used for recording the absorbance, which allows for selection of up to six wavelengths at a time for absorbance detection in the UV-vis range (190-1000 nm). Mycophenolic acid (10 μM) used as a positive control and DMSO as a vehicle control.
- Fluorescence Direct Binding Protocol Process 1) Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content 2) Titration measurements of the protein against ligand by at least 12 titration steps to obtain a good s-curve fit 3) Repeat the same titration measurements with the ligand alone to enable correction 4) Check the stability of the protein once by titration against DMSO alone 5) Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of a UV-spectrophotometer 6) Use Excel template for the correction of the measured raw data 7) Use GraphPad Prism software for the quadratic binding fit and the Kd evaluation.
- APN Inhibition Assay IC50 values against APN from Porcine Kidney were determined by using L-Leu-p-nitroanilide as a substrate. All the solutions of the inhibitors were prepared in the assay buffer( pH 7.5). All the inhibitors were preincubated with APN for 5 min at room temperature. The assay mixture, which contained the inhibitor solution (concentration dependent on the inhibitor), the enzyme solution (4 mg/mL final concentration), and the assay buffer, was adjusted to 200 μL, and then incubated at 37 ℃ for 30 min. The hydrolysis of the substrate was monitored by following the change in the absorbance measured at 405 nm with the UV-vis spectrophotometer Pharmacia LKB, Biochrom 4060.
- Enzyme Inhibition Assay Freshly prepared Zn2-NDM-1 (50 nM) or Zn2-VIM-2 were first incubated with various concentrations of Bi(III) compounds for 1 h at 25° C. The assay was performed in a 1 cm quartz cuvette using the kinetic mode of Varian Cary 50 UV-visible spectrophotometer at 25° C. The final assay buffer contains 50 mM HEPES/Na pH 7.0, 100 mM NaCl and 100 μM MER. The decrease in absorbance at 300 nm due to ring-opening of MER was monitored every second for a duration of 10 mins until the reaction was completed. The initial rate were extracted and calculated from each reaction curves.
- Esterase Activity Assay Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV-vis) according to the method described in the literature [Verpoorte et al., J. Biol. Chem., 242:4221-4229; Innocenti et al., Bioorg. Med. Chem. Lett., 18:2267-2271]. The enzymatic reaction, in a total volume of 3 mL, contained 1.4 mL of 0.05 M tris-SO4 buffer (pH 7.4), 1 mL of 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without any enzyme solution.
- Esterase Assay Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25 C using a spectrophotometer (Shimadzu UV-VIS) according to the method described by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229]. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL 3 mM NPA, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. All compounds were tested in triplicate at each concentration used.
- PqsA Spectrophotometric Assay Reactions were performed at 37 °C in a 0.5 mL volume in a Varian Cary 100 UV-visible spectrophotometer with Cary WinUV software. Reaction mixtures contained 100 mM HEPES, pH 8.0, 0.2 mM dithiothreitol, 2 mM MgCl2, ~3.3 μg PqsA protein (~60 nM final), and depending on which substrate varied, 1 mM ATP (disodium salt), 0.5 mM coenzyme A (trilithium salt), 0.5 mM sodium anthranilate, and inhibitor (usually as a DMSO stock solution). Reactions were blanked and preincubated with all components except anthranilate at 37 °C for 1 min, then initiated by addition of anthranilate. Formation of anthraniloyl-CoA was monitoredby absorbance at 365 nm (ε = 5.5 mM-1cm-1) for 1 min.
- CA Activity Assay The CA activity was assayed by following the change in absorbance at 348 nm of 4-NPA to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (Shimadzu UV-VIS) according to the method described by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229] The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05M Tris-SO4 buffer (pH 7.4), 1mL 3 mM 4-NPA, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of the sulphonamide derivativeswere examined. All compounds were tested in triplicate at each concentration used. Different concentrations of the compounds were used.
- CA Inhibition Assay CA activity was assayed by following the change in absorbance at 348 nm of 4-NPA to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer(Shimadzu UV-VIS) according to the method described by Verpoorte et al. [Verpoote et al., J. Biol. Chem., 242:4221-4229] The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05M Tris-SO4 buffer (pH 7.4), 1 mL, 3 mM NPA, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of compounds 4-11 were examined. All compounds were tested in triplicate at each concentration used. Different inhibitor concentrations were used.
- Esterase Activity Assay The carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of the 4-nitrophenylacetate (NPA) to the 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV-VIS, Rome, Italy) according to the method described in the literature [Verpoorte et al., J. Biol. Chem., 242:4221-4229; Innocenti et al., Bioorg. Med. Chem. Lett., 18:2267-2271]. The enzymatic reaction, in a total volume of 3 mL, contained 1.4 mL of 0.05 M tris-SO4 buffer (pH 7.4), 1 mL of 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution.
- FAS Activity Assay FAS activity was determined by monitoring the decrease in absorbence at 340 nm resulting from the oxidation of NADPH using an Amersham Pharmacia Ultrospec 4300 pro UV-Vis spectrophotometer at 37°C. The assay mixture contained 100 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA (ethylenediaminetetraacetic acid), 2.5 μM acetyl-CoA, 10 μM malonyl-CoA, 32 μM NADPH, and 10 μg chicken liver FAS in a total volume of 2.0 mL [Tian et al., J. Biol. Chem., 260:11375-87]. Fast inhibition was determined by adding thioether to the reaction system before FAS initiated the reaction. The initial velocity was measured to calculate the remaining activity (RA) of FAS, and each assay was repeated three times.
- GAR Transformylase Inhibition Assay GARFTase activity was determined by measuring the rate of 5,8-dideazatetrahydrofolate production in the presence of varying concentrations of antifolate inhibitors using an established spectrophotometric protocol.39 A 150 μL total volume containing 30 μM α,β-GAR, 5.4 μM 10-CHODDF and antifolate (stock solution dissolved in DMSO) in 0.1 M HEPES (pH 7.5) was preincubated at 37 °C in a UV. To initiate the assay, 150 μL of 20 nM His-GARFTase or buffer was added, and the plate was shaken for 5 s. Absorbance changes at 295 nm wererecorded at 15 s intervals over 20 min using a Synergy H1M plate reader (BioTek). For each antifolate, triplicate assays were performed at 20 different drug concentrations.
- Isothermal Titration Calorimetry ITC experiments were performed at 25° C. using a MicroCal PEAQ-ITC microcalorimeter (Malvern). PAH proteins were prepared in a HEPES buffer (HEPES 20 mM Hepes, 0.2M NaCl, 1 mM DTT, pH 7.0) containing 5% DMSO, in order to match the final DMSO concentration of the compounds dilutions. The protein concentration used (30 μM) was determined using ultraviolet (UV) absorbance at 280 nm. The compounds (500 mM) were prepared using the exact same buffer. Twelve injections of 3 μL of compound/ligand were titrated into the sample cell (containing the protein) over 31 min with a stirring speed of 750 rpm. Data was baseline adjusted by subtracting the background obtained from equivalent injections of compound into the buffer solution.
- Lipoxygenase Inhibition Assay A mixture of test compound (10 μL; 1 mM) in MeOH, type-1B lipoxygenase (EC 1.13.11.12; from soyabean) (20 μL; 70 units) in 0.1 M aqueous phosphate buffer (pH 8.0) in a total volume of 160 μL was incubated for 10 min at 25°C. Then the reaction was initiated by the addition of a solution of linoleic acid as substrate (10 μL; 20 μM), resulting in the formation of (9Z,11E,13S)-13-hydroperoxyoctadeca-9,11-dienoate. The change in UV absorbance at 234 nm was followed over a period of 6 min. All reactions were performed in triplicate, and analyzed with a 96-well plate reader (SpectraMax Plus 384; Molecular Devices).
- Optical Titration Assay Optical titrations to determine the binding affinity (KD values) of ligands were carried out on a Cary 60 UV−visible spectrophotometer (Varian, UK) according to a previously described procedure [McLean et al., J. Inorg. Biochem., 91:527-541]. Ligands were prepared as stock solutions typically 0.1−100 mM) in DMSO and added as 0.2 μL aliquots to cuvettes containing either a solution of CYP144A1 (∼4-8 μM) in 100 mM KPi (pH 7.0), containing 10 mM KCl, or to buffer alone. DMSO concentrations did not exceed 1% v/v of the final titration volume (1 mL), and the absorbance spectrum of CYP144A1 was not affected within this range. Spectra were recorded continuously between 800 and 250 nm at 25 °C.
- CA Activity Assay CA activity was assayed by following the change in absorbance at 348 nm of 4-NPA to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer(Shimadzu UV-VIS) according to the method described by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229] The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4-mL 0.05-M Tris SO4 buffer (pH 7.4), 1-mL 3-mM 4-NPA, 0.5-mL H2O and 0.1-mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of the phenolic compounds were examined. All compounds were tested in triplicate at each concentration used. Different concentrations of the compounds were used.
- CA Activity Assay Carbonic anhydrase activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (Shimadzu UV-VIS) according to the method described by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229]. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05M Tris-SO4 buffer (pH 7.4), 1mL 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of the sulphonamide derivatives were examined. All compounds were tested in triplicate at each concentration used. Different concentrations of the compounds were used.
- Spectral Titration of TCCYP51 As a cysteine-coordinated hemoprotein, sterol 14-alpha-demethylase responds spectrally to any perturbations in the area surrounding the heme iron. These spectral responses can be used to estimate the apparent dissociation constants (Kd) of the enzyme/ligand complexes. The titration experiments were carried out at in a cuvette containing TCCYP51 in the wavelength range 350-500 nm using a Shimadzu UV-2401PC spectrophotometer. The tested compounds were added in 1 ul aliquots to the test cuvette until the maximum in spectral response was reached. Equal volumes of DMSO were added to the reference cuvette. The apparent Kd values were determined from the equilibrium titration curves by plotting absorbance changes against the concentration of free ligand and fitting the data to a rectangular hyperbola using SigmaPlot Statistics.
- Tryptophan Fluorescence Quenching Assay (Kd) Binding was also measured by a competition assay with a noncovalently bound inhibitor (Kd=4.6 uM) that quenched tryptophan fluorescence upon binding in the protease active site. Then 3.5 uM protein was titrated by this competitive inhibitor (from 0 to 40 uM) in the absence or presence of 50 uM test compound. An aliquot of each dilution was transferred to a UV-star Greiner 96-well microplate. After 1 h of incubation at room temperature, fluorescence was measured at 25 deg C on a Biotek Synergy4 microplate reader with exe=280 nm (bandwidth 10 nm) and em=340 nm (bandwidth 20 nm). Fluorescence intensities were corrected for inner-filter effect. Kd values of compounds were inferred from their effect on the Kd of the reference compound.
- BChE Inhibition Assay BChE inhibitions by the carbamate inhibitors were assayed by the Ellman method [Ellman et al., Biochem. Pharm., 7:88-95]. BChE-catalyzed hydrolysis of BTCh in the presence of the carbamate inhibitors and DTNB was followed continuously at 410 nm on a UV-visible spectrometer. The temperature was maintained at 25.0°C by a refrigerated circulating water bath. All inhibition reactions were performed in sodium phosphate buffer (1 mL, 0.1 M, pH 7.0) containing NaCl (0.1 M), acetonitrile (2% by volume), triton X-100 (0.5% by weight), substrate (ATCh for AChE or BTCh for BChE) (50 μM), DTNB, and varying concentrations of inhibitor. Requisite volumes of stock solution of substrateand inhibitors in acetonitrile were injected into the reaction buffer via a pipet. BChE was dissolved in sodium phosphate buffer (0.1 M, pH 7.0).
- Biochemical Assay Briefly, samples containing 20 nM purified L1 EN and compounds at the concentrations indicated above were incubated at room temperature for 60 minutes prior to adding 2 nM of plasmid. The reaction buffer was as follows: 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.1 mg/mL bovine serum albumin, and 4 mM DTT. Samples were incubated at 37° C. for 30 minutes before the reactions were stopped by heat inactivation at 70° C. for 10 minutes or addition of 50 mM EDTA. Samples were then run on a 1% agarose gel containing ethidium bromide in 1X TAE buffer for 90 minutes at 120V and the products visualized with a UV imager. Slower migration of plasmid relative to uncut substrate indicated L1 EN nicking activity. Linear plasmid resulted from multiple nicks near each other on opposite strands.
- Cholinesterase Activity Assay Reactions were performed in a mediumcontaining substrate (0.05-0.4 mM) combined with 0.125 mM DTNB in 100 mM 3-(N-morpholino)propanesulfonic acid buffer, pH 8.0 at 25 °C, initiated by the addition of enzyme, and monitored spectrophotometrically at 412 nm in a UV-visible 1700 Shimadzu PC spectrophotometer. The activity was determined by measuring the increase in absorbance at 412 nm for 7 min (εTNB = 14.2 mM^-1 cm^-1). Activities of the treated samples were compared to control to estimate percentage of inhibition (I%). Dose-response curves were plotted using the GraphPad Prism 5 software and IC50 values were determined. Data are expressed as mean ± SEM. An enzyme kinetic assay was performed for compound 2 at different butyrylthiocholine (BTC) concentrations (0.05-0.3 mM). Donepezil-HCl (Sigma) was tested as a reference compound.
- Enzyme Assay MIF tautomerase activity was measured by UV-Visible recording spectrophotometry (SHIMADZU, UV1600U). A fresh stock solution of L-dopachrome methyl ester was prepared at 2.4 mM through oxidation of L-3,4-dihydroxyphenylalanine methyl ester with sodium periodate. 1 μL of MIF solution (800-900 ng/mL) and 1 μL of a DMSO solution with various concentrations of the enzymatic inhibitor were added into a plastic cuvette (10 mm, 1.5 mL) containing 0.7 mL assay buffer (50 mM potassium phosphate, pH 7.2). Then L-dopachrome methyl ester solution (0.3 mL) was added to the assay buffer mixture. Activity was determined at room temperature and the spectrometric measurements were made at λ=475 nm for 20 seconds by monitoring the rate of decolorization of L-dopachrome methyl ester in comparison to a standard solution.
- Verification on Inhibitory Activities of TAM Receptor Inhibiting Compounds on Tyro 3, Axl, and Mer For evaluation of inhibitory activities of the compounds according to the present invention on Tyro 3, Axl, and Mer, the following test was carried out.The specific test method followed the method provided by Cisbio. The compounds of the examples were prepared at various concentrations, followed by addition of Tyro 3, Axl, or Mer and substrate peptides, and then ATP was added to initiate a reaction. After 1 hour, the reaction was stopped by addition of a solution containing EDTA. Thereafter, the amount of phosphorylated peptides was measured. Here, the phosphorylated peptides were treated with an europium (Eu)-labeled antibody recognizing phosphorylated peptides, and then after 1 hour, excited by irradiation of the light of a wavelength of 320 or 340 nm using an Envision Reader. The amount of light emitted at 665 nm was measured to investigate the degrees of inhibition of TAM receptors. The TAM receptor inhibitory activities (IC50 values) of the respective compounds were analyzed using the GraphPad Prism program, and tabulated in Tables 1 to 3 below. LDC1267 (CAS no. 1361030-48-9, Calbiochem), which is a TAM receptor inhibitor compound, was used as a positive control.
- ALR1 in vitro Inhibition Assay The assay results were obtained at 340 nm and ALR1 inhibitory activity was measured with the absorbance change at this respective wavelength. Each well contains 200 µL of assay mixture containing potassium phosphate buffer 100 mM at pH 6.2 with test sample 20 µL of 10 mM, 70 µL enzyme preparation and 40 µL of10 mM sodium D-glucoronic acid as a substrate. This mixture was incubated at 37 °C for 5 min and 50 µL of 0.1 mM NADPH as a cofactor was added and the read was obtained at 340 nm. The assay mixture was incubated again at 37 °C for 10 min and the read was taken at the respective UV range in ELIZA plate reader. As positive and negative control, 10 mM valproic acid (20 µL) and buffer solution (20 µL) were used [Biol. Chem., 240:877-882].
- ALR2 in vitro Inhibition Assay The activity of ALR2 enzyme was determined at 340 nm in UV spectrophotometer that depends upon the measurement of NADPH consumption. Each well of the 96-well plate contains 200 µL of assay mixture consisting of potassium phosphate buffer 100 mMat 6.2 pH, 20 µL of 10 mM test sample, 70 µL of enzyme preparation and substrate D,L-glyceraldehyde (40 µL), all mixture was incubated at 37 °C for 5 min and then 0.1 mM NADPH (50 µL) was added as a cofactor in the enzyme reaction and read was measured at 340 nm and followed by incubation at 37 °C for 10 min and thenabsorbance was measured in Eliza plate reader at respective wavelength. Negative and positive control used were 10 m M buffer (20 µL) and 10 mM sulindac (20 µL) [Med. Chem. Commun., 5:1371-1380].
- Cholinesterase Inhibition Assay Test compounds were prepared in DMSO (maximum concentration used 1% v/v), and 10 μL of each (0.001-25 μm final concentration range) was incubated for 5 min at room temperature with 160 μL of 1.5 mm DTNB, 50 μL 0.22 U/mL AChE [in 50 mm Tris-HCl, pH 8.0, 0.1% w/v bovine serum albumin (BSA)], or 50 μL 0.12 U/mL BuChE (in 50 mm Tris-HCl, pH 8.0, 0.1% w/v BSA). After the incubation period, 30 μLof ATChI (15 mm, prepared in ultrapure water) or BuTChI (15 mm, prepared in ultrapure water) was added to 96-well plates. Test compound inhibition of ChE was measured via UV absorbance (412 nm wavelength) using a microplate reader (SpectraMax M5) at various time intervals (t = 0, 1, 2, 3, 4, and 5 min).
- E. coli Topoisomerase I Relaxation Activity Inhibition Assay The relaxation activity of E. coli topoisomerase I was assayed in a buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/mL gelatin, and 0.5 mM MgCl2. Half microliter from the appropriate stock solutions of compounds dissolved in the solvent (DMSO) or the solvent alone (control) was mixed with 9.5 μL of the reaction buffer containing 10 ng of enzyme before the addition of 10 μL of reaction buffer containing 200 ng of supercoiled pBAD/Thio plasmid DNA purified by cesium chloride gradient as substrate. Following incubation at 37° C. for 30 min, the reactions were terminated by the addition of 4 μL of a stop buffer (50% glycerol, 50 mM EDTA, and 0.5% (v/v) bromophenol blue), and analyzed by agarose gel electrophoresis. The gels were stained in ethidium bromide and photographed under UV light.
- Esterase Assay CA activity was assayed by following the change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°Cusing a spectrophotometer (CHEBIOS UV-VIS) according to the method described by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229] The enzymatic reaction contained 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL 3 mM 4-NPA, 0.5 mL H2O and 0.1 mL enzyme solution (total volume, 3.0 mL). A reference measurement was obtained by preparing the mixture without the enzyme solution. All measurements were made in triplicate. The Ki values were determined from a series of experiments using three different bromophenols concentrations and 4-NPA as the substrate at five different concentrations to construct Lineweaver-Burk curves [Lineweaver et al., J. Am. Chem. Soc., 57:685-693].
- FabZ Inhibition Assay The enzymatic activities of FtFabZ and YpFabZ were determined via the reportedspectrophotometric method using the substrate analogue crotonoyl-CoA.7 Initial reaction velocities were determined by monitoring the decreasing absorbance at 280 nm, resulting from the conversion of crotonoyl-CoA to beta-hydroxybutyryl-CoA. The conversion of hydroxybutyryl-CoA to crotonoyl-CoA was not used in our assay. An extinction coefficient of 3600 s-1 cm-1, corresponding to the hydration of the enoyl double bond, was used to convert absorbance readings to substrate concentrations.20 Reactions were performed using a Beckman-Coulter DU730 UV/vis spectrophotometer. Reaction volumes of 100 uL were prepared in a buffer solution consisting of 2.0 ug of FtFabZ or 0.5 ug of YpFabZ, 20 mM Tris, pH 7.0. Allreactions were performed at 25 C. Inhibition reactions for IC50 calculations wereperformed under similar conditions with a fixed concentration of 100 uM crotonoyl-CoA.
- AO Enzyme Assay Guinea pig AO activity was assayed spectrophotometrically using phenanthridine as a substrate at 322 nm. All spectrophotometric determinations were carried out at 25 °C using a PClinked perkin Elemer Lambda 25 UV/Vis spectrophotometer. The varying substrate concentrations (4-60 µM) were separately added into Sorenson's phosphate buffer (final concentration of 50 mM, pH 7.0) containing 0.1 mM of EDTA. Then, the reaction was started by addition of the enzyme and monitored for up to 2 min. The enzyme was also assayed in the presence of different concentrations of the compounds as potential inhibitors (1-200 µM) and constant concentration of substrate (40 µM) to calculation of IC50 values. The results were compared with the inhibitory effects of 1-100 µM menadione, a specific AO inhibitor. Before enzymatic reaction, all of the solutions were equilibrated at 25 °C, except the enzyme fraction which was kept on ice bath prior to addition to the incubation solution. All compounds were dis
- Biological Assay Assay A, a 384-well format on a Corning 3653 assay plate is used, and monitored by UV-vis spectroscopy in continuous mode at rt. Compounds were prepared in DMSO as 4 mM stocks, diluted using an 11-point half-log scheme on a Biomek FX (Beckman Coulter), and incubated at rt for 30 minutes with the reaction mixture containing 50 mM HEPES, pH 7.4, 140 mM KCl, 3.5 mM MgCl2, 0.8 mM fructose, 2 mM TCEP, 0.8 mM PEP, 0.7 mM NADH, 0.01% Triton X-100, 30 U/mL pyruvate kinase-lactate dehydrogenase, and 10 nM purified KHK-C. The compound concentration in each well ranged from 1 nM to 100 μM. The reaction was initiated with the addition of 0.2 mM ATP. The absorbance was measured for 30 minutes on a SpectraMax reader (Molecular Devices) after ATP was added. The concentrations provided are based on the final mixture volume of 40 μL (referred to as the final concentration).
- Biological Assay Assay A, a 384-well format on a Corning 3653 assay plate is used, and monitored by UV-vis spectroscopy in continuous mode at rt. Compounds were prepared in DMSO as 4 mM stocks, diluted using an 11-point half-log scheme on a Biomek FX (Beckman Coulter), and incubated at rt for 30 minutes with the reaction mixture containing 50 mM HEPES, pH 7.4, 140 mM KCl, 3.5 mM MgCl2, 0.8 mM fructose, 2 mM TCEP, 0.8 mM PEP, 0.7 mM NADH, 0.01% Triton X-100, 30 U/mL pyruvate kinase-lactate dehydrogenase, and 10 nM purified KHK-C. The compound concentration in each well ranged from 1 nM to 100 uM. The reaction was initiated with the addition of 0.2 mM ATP. The absorbance was measured for 30 minutes on a SpectraMax reader (Molecular Devices) after ATP was added. The concentrations provided are based on the final mixture volume of 40 uL (referred to as the final concentration).
- Cell Based Assay ATR (Ataxia Telangiectasia+Rad3-related kinase) is a PI3-kinase-related kinase which phosphorylates multiple substrates on serine or threonine residues in response to DNA damage or replication blocks. Chk1, a downstream protein kinase of ATR, plays a key role in DNA damage checkpoint control. Activation of Chk1 involves phosphorylation of Ser317 and Ser345 (the latter regarded as the preferential target for phosphorylation/activation by ATR).This was a cell based assay to measure inhibition of ATR kinase, by measuring a decrease in phosphorylation of Chk1 (Ser 345) in HT29 cells, following treatment with compound and the UV mimetic 4NQO (Sigma #N8141). HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 6000 cells/well in 40 ul EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compounds in 100% DMSO were added to assay plates by acoustic dispensing.
- FRET Assay For the FRET measurements, the Cerulean-Runt (Runx1 41-190) and Venus-CBFbeta (1-141) fusion proteins were dialyzed into FRET assay buffer. Protein concentrations were determined by UV absorption at 433 nm for Cerulean-Runt and 513 nm for Venus-CBFbeta. Equal molar concentrations of the two proteins were mixed together, diluted to 150 nM with FRET assay buffer containing 0.1% BSA, and incubated for 1 hr. A PHERAstar microplate reader (BMG Labtech, Durham, NC, USA) was used to measure fluorescence (excitation at 433 nm and emission measured at 474 and 525 nm). For IC50 determinations, the ratios of the fluorescence intensities at 525 nm and 474 nm were plotted versus the log of compound concentration, and the resulting curve was fit to a sigmoidal curve using Origin 7.0 (MicroCal, Northampton, MA, USA). Mean values of IC50 originating from two independent measurements performed in duplicate together with the standard deviations are reported.
- Functional Peptidyl Prolyl Isomerase (PPlase) Spectrophotometric Assay Cyclophilins are enzymes that catalyse the cis-trans isomerisation of peptide bonds to the amino acid proline. This event is important in many biological processes, including protein folding and signal transduction. Cyclosporins and the compounds of the present invention inhibit this catalytic activity. The method to measure this activity and its inhibition is similar to that described by Jankowski (Analytical Biochemistry, 252, 2, 299-307 (1997)). The assay measures the cis to trans isomerisation of a peptide substrate catalysed by a PPlase enzyme using a UV/Vis spectrophotometer. This assay is a manual single cuvette-based assay. A first order rate equation is fitted to the absorbance data to obtain a rate constant. The catalytic rate is calculated from the enzymatic rate minus the background rate. The Ki (the concentration required to produce half maximum inhibition) for an inhibitor is obtained from the rate constant plotted against the inhibitor concentration.
- IC50-Activity Assay (AKR1B10) The IC50-activity assays were carried out on the basis of the quantification of the NADPH consumption that takes place when the enzyme catalyzes the conversion of the aldehyde substrate into the alcohol product. The assays were performed at 25 °C in a 100 mM sodium phosphate buffer (pH 7.0), with the protein amount reaching the Vmax, and 0.2 mM NADPH. The final reaction volume was 600 μL per reaction. All compounds were dissolved in dimethyl sulfoxide (DMSO), and the corresponding solution was added to the cell to be assayed in a final concentration of 2% (v/v) DMSO and incubated for 5 min at 25 °C prior to the addition of the substrate. The reaction was initiated by the addition of 60 mM glyceraldehyde and 0.2 mM pyridine-3-aldehyde, and the decrease in opticaldensity at 340 nm was monitored for at least 3 min at 25 °C in a UV−vis spectrophotometer (Cary 400 Bio (Varian)).
- IC50-Activity Assay (AR) The IC50-activity assays were carried out on the basis of the quantification of the NADPH consumption that takes place when the enzyme catalyzes the conversion of the aldehyde substrate into the alcohol product. The assays were performed at 25 °C in a 100 mM sodium phosphate buffer (pH 7.0), with the protein amount reaching the Vmax, and 0.2 mM NADPH. The final reaction volume was 600 μL per reaction. All compounds were dissolved in dimethyl sulfoxide (DMSO), and the corresponding solution was added to the cell to be assayed in a final concentration of 2% (v/v) DMSO and incubated for 5 min at 25 °C prior to the addition of the substrate. The reaction was initiated by the addition of 1 mM glyceraldehyde, and the decrease in optical density at 340 nm was monitored for at least 3 min at 25 °C in a UV−vis spectrophotometer (Cary 400 Bio (Varian)).
- Inhibition Assay Compounds were added in 5 uL volume to wells in UV transparent 96-well plates ( area well size). The final compound concentrations tested ranged from 0.5 nM to 10 uM). Assays were performed in duplicate. CaMKIIδ is added to at a final concentration of 16 nM to a mixture containing 100 mM Tris (pH 7.5), 150 mM KCl, 0.27 mM EGTA, 1.3 mM PEP, 0.2 mg/ml AC3, 6.9% (v/v) PK/LDH mixture (Sigma P0294), 0.38 mM NADH and kept on ice. 72 uL of the enzyme mixture was added to the wells containing compounds and the plate was shaken briefly and kept on ice. The assay was initiated by adding 23 uL of a mixture containing 100 mM Tris (pH 7.5), 150 mM KCl, 1.7 mM CaCl2, 48 mM MgCl2, 0.35 mM ATP and 6.7 ug/mL calmodulin. The rate of ADP released was measured as the rate of absorbance decrease at 340 nM at 25° C.
- Inhibition Assay Compounds were tested against mPTPB using the p-nitrophenyl phosphate (pNPP) assay system in a Cary 100 UV-Vis spectrophotometer by monitoring the increase in absorbance of product formed, p-nitrophenol (pNP) at 405 nm. The reaction was started by the addition of mPTPB into a final volume of 200 μL master mix containing pH 7.0 3,3-Dimethyl glutarate buffer (50.0 mM, 1 mM EDTA, 0.15M NaCl), 3.0 mM pNPP. IC50 values for each compound were determined by varying the concentration of the inhibitor at fixed concentrations of the enzyme and by fitting the dose-response data into the four-parameter logistic curve (eq 1) model of GraphPad prism 7.02, as follows. A I /A 0 =IC 50/(IC 50+[I]).The inhibition assay for these PTPs were performed under the same conditions as mPTPB except using a different pNPP concentration corresponding to the Km of the FTP studied.
- LOX Activity Assay Enzyme activity was monitored by UV analysis. 0.05 cm^3 of enzyme solution was added to a cuvette containing 2 cm^3 linoleic acid solution and the appropriate amounts of buffer and inhibitor solutions in thermostatic water bath at 25°C. There was no pre-incubation time of the enzyme with inhibitor solution. The activity of the enzyme was determined by monitoring the increase in the absorption caused by the oxidation of linoleic acid at 234 nm and 25°C (ε = 25 000 M^−1 cm^−1). One standard solution of complexes in DMSO (10^-2 M) was used for theinhibition activity experiments (three sets). In this case, the substrate concentration was kept constant (0.3 mM), while the amounts of buffer and inhibitor solutions varied according to the inhibitor final concentration needed(0.5-30 μM or 0.15-9 μL from standard solutions). The total experiment volume was 3 cm^3.
- Topoisomerase I Relaxation Assay Supercoiled pHOT1 DNA (0.5 μg) was incubated with four units of human topoisomerase I in relaxation buffer (10mM Tris-HCl (pH 7.8), 1mM EDTA, 0.15M NaCl, 0.1% BSA, 0.1 spermidine, 5% glycerol), in the presence of varying concentrations of the test compounds. Reactions were carried out at 37 °Cfor 1 h and then terminated by the addition of sodium dodecyl sulfate (SDS) to 0.25% and proteinase K to 50 μg/ml. The reaction mixture was subjected to electrophoresis through a 0.8% agarose gel containing 0.5 mg/ml ethidium bromide in TAE buffer (40mM Tris-borate and 1mM EDTA). The gels were stained with ethidium bromide and photographed under UV light. For the quantitative determination of topoisomerase concentration activity, photographic negatives were scanned and the area representing supercoiled DNA, migrating as a single band at the bottom of the gel was measured using UVI-KS4000i gel documentation and analysis system (SyngenBiotech, Sacramento, CA).
- Biological Assay Reaction buffers used for the lipoxygenase assay were as follows: 25 mM HEPES (pH 7.5), with 0.01% Triton X-100. 1 mM 12-HPETE or 1 mM 15-HPETE stock solution was prepared in 25 mM HEPES with 0.01% Triton X-100.The standard curve of 12-HPETE was made by serial dilution of 0, 6.25, 12.5, 25, 50, 75 and 100 μM, and the final reaction volume in 96 Well Clear Flat Bottom UV-Transparent Microplate is 100 μL. The absorbance of the 12-HPETE at each concentration was measured at 234 nm.The standard curve of 15-HPETE was made by serial dilution of 0, 3.125, 6.25, 12.5, 25, 50 and 100 μM, and the final reaction volume in 96 Well Clear Flat Bottom UV-Transparent Microplate is 100 μL. The absorbance of the 12-HPETE at each concentration was measured at 234 nm.[0453]IC50 values were obtained by determining the % inhibition at various inhibitor concentrations. The final concentrations of the control inhibitors and test compounds in the IC50 determination assay are 0, 0.03, 0.1, 0.3, 1, 3, 10 and 20 μM. The final concentration of DMSO in the assay is 0.05%.12-LOX IC50 determination. The 12-LOX enzyme and AA were diluted in the HEPES buffer to 120 nM and 200 μM, respectively. Pre-incubation 50 μL of indicated test compounds and 25 μL 120 nM enzyme at room temperature for 5 min. The reaction is started by adding 25 μL 200 μM AA. After a short spin (1000 rpm, 15 s), incubate the reaction system for 5 min. The blank control was set by adding 25 μL HEPES to 50 μL of control inhibitor compound (MHL355) and 25 μL 200 μM AA. The final concentrations of 12-LOX enzyme and AA were 30 nM and 50 μM, respectively. The absorbance of the 12-HPETE at 234 nm was measured.
- In Vitro Urease Inhibition Assay The assay mixture, containing 50 μl (2 mg/ml) of enzyme and 100 μl of different concentration of the tested agents, was added to 850 μl of 25 mM urea and preincubated for 0.5 h in water bath at 37 °C. The urease reaction was stopped after 30 min incubation by the following procedure. After pre-incubation, 500 μl of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 500 μl of alkali reagent (1% w/v NaOH and 0.075% active chloride NaOCl) were added to100 μl of incubation mixture and kept at 37 °C for 30 min. The absorbance (A) was measured at 625 nm by using following equation A = kbc, where c is the concentration of solution (mol/L), b the Length of the UV cell [Golbabaei et al., Daru., 21(1):2; Akhtar et al., Chem. Biol. Drug Des., 84:92-98]. All experiments were performed in triplicate in a final volume of 1 ml, and AHA was used as a standard urease inhibitor.
- Inhibition Assay To gain insight into the selectivity of the synthetic compounds for inhibition of other CYPs, we examined the major CYPs present in human liver. Prior to these studies we showed that the nicotine analogs were quite metabolically stable in the presence of mouse or rat or human liver microsomes (i.e., T1/2>60 mins). That the nicotine analogs showed low or no inhibitory activity against other CYPs suggests that the inhibitors examined selectively inhibited CYP2A6. A typical incubation mixture (final volume 0.25 mL) contained 50 mM Tris or KPhos buffer (pH 7.5), 0.5 mM NADP+, 2.0 mM G6P, 1 U of G6P dehydrogenase and 0.6 mg DETAPAC and the inhibitor was added last to minimize interaction with the protein. After mixing on ice, the reaction was initiated by the addition of substrate and incubated at 37 ° C. with shaking in air. Organic extracts were analyzed by fluorescence (for fluorometric substrates) or injected onto a Hitachi L-7100 system equipped with a Hitachi L-7400 UV detector.
- Kinetic Inhibition Assay Purified CtBP2 in 50% glycerol was added to 150 μM NADH and variable amounts of MTOB and HIPP in buffer containing 25 mM HEPES, pH 7.1, 25 mM potassium chloride, and 1mM DTT. The final concentration of CtBP was 40 μg/mL (986 nM) per reaction. HIPP was dissolved in DMSO (1% total volume). Final MTOB concentrations were the following: 36, 24, 12, 8, 4, 2, 1, and 0 μM for 0 μM HIPP; 64, 48, 24, 12, 8, 4, 2, and 0uM for 500nM HIPP; 80, 64, 48, 24, 12, 8, 4, and 0 μM for 1 μM HIPP; 96, 80, 64, 48, 36, 24, 12, and 0 μM for 2 μM HIPP. Reaction components wereadded to 96-well UV-Star Microplates (Greiner Bio-One). Upon addition of CtBP,reactions were mixed vigorously and immediately read by a Synergy H1 microplatereader (BioTek). Absorbance was recorded at A=340nm every 7 seconds for 7 minutes at 25°C to measure CtBP dehydrogenase function (NADH, but not NAD+ absorbs light at 340nm).
- MraY Assay Park's nucleotide-N-C6-dansyl (2 mM stock solution, 1.88 μL), MgCl2 (0.5 M, 5 μL), KCl (2 M, 5 μL), Triton X-100 (0.5%, 5.63 μL), Tris buffer (pH 8.0, 50 mM), neryl phosphate (0.1 M, 2.25 μL), and inhibitor molecule (0-100 μg/mL in Tris buffer) were placed in a 500 μL Eppendorf tube. To a stirred reaction mixture, P-60 (10 μL) was added (total volume of reaction mixture: 50 μL adjust with Tris buffer). The reaction mixture was incubated for 2 h at room temperature (26 oC) and quenched with CHCl3 (100 μL). Two phases were mixed via vortex and centrifuged at 25,000 xg for 10 min. The upper aqueous phase was assayed via reverse-phase HPLC. The water phase (10 μL) was injected into HPLC (solvent: CH3CN/0.05 M aq. NH4HCO3=25:75; UV: 350 nm; flow rate: 0.5 mL/min; column: Kinetex 5 μm C8, 100 Å, 150×4.60 mm), and the area of the peak for lipid I-neryl derivative was quantified to obtain the IC50 value.
- TR-FRET Assay Table 1: Briefly, 20 μL aliquots of a 1.5× stock of ASK1, STK Substrate 3 and Assay Buffer were distributed in the wells of a white 384 well Optiplate, prior to addition of 0.3 μL of a 100× compound stock in DMSO, or DMSO alone in Blank and 100% Activity Controls. After a 10 minute pre-incubation, ASK1 protein kinase activity was initiated by addition of 10 μL of 300 μM ATP. After 5 hours, the kinase activity was quenched by addition of 30 μL of CisBio Kit 62ST3PEJ components: 0.5 μM of Streptavidin XL665; and 100×STK Antibody-Eu Cryptate; in Detection Buffer containing sufficient EDTA to chelate Mg2+ in the assay buffer. The plates were read after 65 minutes in an Envision Plate reader, with the following components and settings: Top Mirror, Lance Delfia Dual (662); UV Ex Filter, 320 nm (111); Emission Filter for donor, 615 nm (203); Emission Filter for Acceptor; 665 nM (205); and 100 usec delay, with 665/615 ratio output.
- ThermoFluor Assay ThermoFluor experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
- WecA Assay DP-Glucosamine-C6-FITC (2 mM stock solution, 0.56 μL), MgCl2 (0.5 M, 4 μL), β-mercaptoethanol (50 mM, 5 μL), CHAPS (5%, 11.25 μL), Tris buffer (pH 8.0, 50 mM), undecaprenyl phosphate (4 mM, 1.4 μL), and inhibitor molecule (0-100 μg/mL in Tris buffer) were place in a 500 μL Eppendorf tube. To a stirred reaction mixture, P-60 (10 μL) was added (total volume of reaction mixture: 50 μL adjust with Tris buffer). The reaction mixture was incubated for 4 h at 37° C. and quenched with n-butanol (150 μL). Two phases were mixed via vortex and centrifuged at 10,000 xg for 3 min. The upper organic phase was assayed via reverse-phase HPLC. The organic phase (30 μL) was injected into HPLC (solvent: gradient elution of 85:15 to 95:5 MeOH/0.05 M aq. NH4HCO3; UV: 485 nm; flow rate: 0.5 ml/min; column: Kinetex 5 μm C8, 100 Å, 150×4.60 mm), and the area of the peak for C55-P—P-glucosamine-C6-FITC was quantified to obtain the IC50 value.
- Xanthine Oxidase (XO) Assay Xanthine oxidase (XO) assay of 2-(benzylamino)-4-methyl-1,3-thiazole-5-carboxylic acid derivatives 5a-p was evaluated using Bovine milk XO (grade 1, ammonium sulphate suspension, purchased by Sigma Aldrich). UV visible spectrophotometer (EI 2371) was used for measuring uric acid formation at 293 nm at 25 °C under aerobic condition. The reaction mixture containing 1 mL xanthine (0.15 mM), 2.5 mL potassium phosphate buffer (50 mM, pH 7.4) and 0.5 mL of XO solution (0.405 U/mL) was incubated for 5 min at 25 °C. Inhibition of XO activity by various inhibitors was measured by following the decrease in the uric acid formation at 293 nM at 25 °C. The blank was prepared without enzyme solution. Febuxostat was used as positive control. The enzyme was preincubated for 5 min, with test compounds ranging from 6.25 to 100 µM (six concentrations in triplicate) dissolved in DMSO (1% v/v) (38,39), and the reaction started by the addition of xanthine. DMSO (1% v/v) did not interfere with the enzyme ac
- 3β-HSD1 Inhibition Assay DHEA and NAD+ were enzymatically converted to androstenedione and NADH, respectively. Final NADH concentrations were spectroscopically quantified using a commercially available Amplite™ Colorimetric NADH Assay Kit which utilizes an NADH probe with an absorbance wavelength of 460 nm. Enzymatic assays with a final volume of 25 μL were ran in a 384-well, Greiner Bio-One UV-STAR® assay plate in 50 mM TRIS pH 8.0 assay buffer. Compound IC50 values were experimentally determined in duplicate using a 10-point concentration response curve (CRC) with a top concentration of 50 μM and a bottom concentration of 25 pM. Compound stocks at 10 mM in DMSO were serial diluted 5-fold in DMSO to generate an ECHO® compatible source plate. 125 nL of DMSO, Trilostane, or serial diluted compounds were then acoustically transferred into an empty UV-STAR® assay plate to give a final assay concentration of 0.5% v/v DMSO. The stamp volume and dilution scheme can be adjusted to accommodate compound potency. The assay can tolerate up to a 5% v/v DMSO final assay concentration. 15 μL of 1 μg purified 3β-HSD1 and 50 μM DHEA (final assay volume concentration) was dispensed per well into the stamped plate, centrifuged for 1 minute at 1000 RPM, covered, and allowed to incubate for 15 minutes at room temp. Enzymatic reactions were initiated upon the addition of 10 μL of NAD+ diluted in assay buffer to give a final assay volume concentration of 2 mM. The assay plate was centrifuged for 1 minute at 1000 RPM, covered, and incubated at room temperature for 1 hour. 25 μL Amplite™ Colorimetric NADH Assay Kit detection solution (prepared as recommended) was added to all reaction wells, the plate centrifuged for 1 minute at 1000 RPM, covered, and incubated for 15 minutes. Final absorbance values (λ=460 nm) are spectroscopically determined on BioTek® Cytation5 or Synergy4 plate readers. The background signal was determined by averaging 32 fully inhibited reactions (Trilostane treated) and subtracted across the plate. From the background subtracted values, the non-inhibited enzyme signal (DMSO treated) was averaged from 32 control reactions.
- Lipoxygenase UV-Vis Assay The inhibitor compounds were screened initially using one concentration point on a Perkin-Elmer Lambda 40 UV-Vis spectrometer. The percent inhibition was determined by comparing the enzyme rates of the control (DMSO solvent) and the inhibitor sample by following the formation of the conjugated diene product at 234 nm (ε=25,000 M−1 cm−1). The reactions were initiated by adding either of 40 nM 12-LOX, 40 nM 12/15-LOX, 0.5 μM 15-LOX-2 or 200 nM 5-LOX (ammonium sulfate suspension) to a cuvette with a 2 mL reaction buffer constantly stirred using a magnetic stir bar at room temperature (22° C.). It should be noted that LOX isozymes are often expressed in the inactive demetallated form, so it is best to utilize activity to determine the optimal LOX concentration for the assay (optimal rate of approximately 0.001 abs/sec at 10 μM AA). Reaction buffers used for various lipoxygenase were as follows-25 mM HEPES (pH 7.3), 0.3 mM CaCl2, 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X-100, 10 μM AA for the crude, ammonium sulfate precipitated 5-LOX; 25 mM Hepes (pH 8), 0.01% Triton X-100, 10 μM AA for 12-LOX and 25 mM Hepes buffer (pH 7.5), 0.01% Triton X-100, 10 μM AA for 12/15-LOX and 15-LOX-2. The substrate concentration was quantitatively determined by allowing the enzymatic reaction to go to completion in the presence of 15-LOX-2. For the inhibitors that showed more than 50% inhibition at the one point screens (25 μM inhibitor), IC50 values were obtained by determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor concentration (Approximate range: 0.1 to 25 μM inhibitor), followed by a hyperbolic saturation curve fit (assuming total enzyme concentration [E]<
- In Vitro Enzymatic Activity Assay The activity of recombinant PHGDH was measured by monitoring the reduced nicotinamideadenine dinucleotide (NADH) to nicotinamideadenine dinucleotide (NAD+) change in fluorescence emission at 456 nm. PHGDH (final concentration of 30 ng/ L) was first pre-incubated with enzyme samples in the assay buffer (25 mM HEPES, pH 7.1, 400 mM KCl, 5 M phosphopyridoxa (PLP), 0.5 mM ±-ketoglutarate, 150 M NADH, PSAT1) for 10 min in 96-well plate, then 10 L of DMSO (control) or a small molecule of DMSO solution was added, shaken at 550 rpm for 5 minutes at 25 ° C. and balanced for 5 minutes. In the in vivo testing system of enzyme, each compound was dissolved in DMSO at a final concentration of 5% (v/v), which did not affect the assay signal. The reaction was started by adding L-phospho-O-serine (Pser) solution. The UV-visible microplate reader was used to monitor the change of NADH consumption at 456 nm with time. Protein activity was assessed by using an initial rate of reaction within 30 s, at which time NADH consumption was linear over time.
- Lipoxygenase UV-Vis-based IC50 Assay The full IC50 experiments were done with at least five different inhibitor concentrations. All reaction mixtures were 2 mL in volume and constantly stirred using a magnetic stir bar at room temperature (23° C.) with the appropriate amount of LOX isozyme [h5-LOX (˜200 nM); h12-LOX (50 nM); h15-LOX-1 (60 nM); h15-LOX-2 (200 nM)]. The protein concentrations are the total protein concentration; active protein concentration can be significantly less due to incomplete metalation. Reactions with h12-LOX were carried out in 25 mM HEPES (pH 8.0) 0.01% Triton X-100 and 10 μM AA. Reactions with the crude, ammonium sulfate precipitated h5-LOX were carried out in 25 mM HEPES (pH 7.3), 0.3 mM CaCl2), 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X100 and 10 μM AA. Reactions with h15-LOX-1 and h15-LOX-2 were carried out in 25 mM HEPES buffer (pH 7.5), 0.01% Triton X-100 and 10 μM AA. The concentration of AA was quantitated by allowing the enzymatic reaction to proceed to completion in the presence of soybean 15-LOX-1 (s15-LOX-1).
- MraY Assay Park's nucleotide-N -C6-dansyl (2 mM stock solution, 1.88 μL), MgCl2 (0.5 M, 5 L), KCI (2 M, 5 μL), Triton X-100 (0.5%, 5.63 μL), Tris buffer (pH 8.0, 50 mM), neryl phosphate (0.1 M, 2.25 μL), and inhibitor molecue (0-100 μg/mL in Tris buffer) were placed in a 500 μL Eppendorf tube. To a stirred reaction mixture, P-60 (10 μL) was added (total volume of reaction mixture: 50 μL adjust with Tris buffer). The reaction mixture was incubated for 2 h at room temperature (26 oC) and quenched with CHCl3 (100 μL). Two phases were mixed via vortex and centrifuged at 25,000 xg for 10 min. The upper aqueous phase was assayed via reverse-phase HPLC. The water phase (10 μL) was injected into HPLC (solvent: CH3CN/0.05 M aq. NH4HCO3=25:75; UV: 350 nm; flow rate: 0.5 mL/min; column: Kinetex 5 μm C8, 100 Å, 150 ×4.60 mm), and the area of the peak for lipid I-neryl derivative was quantified to obtain the IC50 value.
- ThermoFluor Assay 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt 60 μM 1,8-ANS 100 mM Hepes, pH 7.0 10 mM NaCl 2.5 mM GSH 0.002% Tween-20
- Urease Inhibition Assay The inhibition studies of soybean urease were initiated with boric acid and boronic acids (butylboronic acid, 4-bromophenylboronic acid, and phenylboronic acid). Also, heavy metal ions (HgCl2, AgCl, and Cu(CH3COO)2) and sodium salts of mineral acids (NaF, NaCl, NaNO3, and Na2SO4) were investigated for their inhibitory effects. Stock solutions of inhibitors, except for 4-bromophenylboronicacid, were prepared in 0.05 M Tris-acetate buffer, pH 7.0, and were suitably diluted for experiments, whereas a stock solution of 4-bromophenylboronic acid was prepared in absolute ethanol and subsequently diluted in respective buffer. The activity assay was carried out at standard conditions as described earlier in the presence of varying concentrations of inhibitors. The yellow-orange colored solution was measured at 405 nm on a Unicam UV-2 spectrophotometer. The amount of NH3 liberated in the reaction mixture was estimated by calibrating Nessler's reagent with standard NH4Cl solution. One enzyme unit is defined as the amount of urease required to liberate 1 μmol of ammonia per minute under our test conditions (0.1 M urea, 0.05 M Trisacetatebuffer, pH 7.0, 37°C).
- Enolase Enzymatic Assays Enolase activity was measured by two different methods: a fluorometric NADH-linked assay or adirect spectrophotometric assay via formation of PEP. In the fluorescent assay,enolase activity was measured via NADH oxidation in a pyruvate kinase-dehydrogenase coupled assay. The assay is conducted in 10 mM KCl, 5 mM MgSO4, and 100 mM triethanolamine at pH 7.4, with 400 uM NADH and 2 mM ADP. 2-PGA, pyruvate kinase (PK) and lactate dehydrogenase(LDH) are provided in excess, with conversion of 2-PGA to PEP by enolase being rate limiting. PEP (with ADP) is substrate of PK; pyruvate formed by thisreaction is linked to NADH oxidation by LDH. Enolase activity is determinedby fluorescence measurement of oxidation of NADH by excitation at 340 nmand emission at 460 nm. The substrate concentration was 5 mM 2-PGA unlessotherwise indicated. Fluorescence was measured using an Omega FluorescencePlate Reader (BMG Labtech). Alternatively, enolase activity was measured directly by the appearance of PEP from 2-PGA via absorption at 240 nm. The assay medium was the same, except that all the auxiliary reagents (PK or LDH, NADH and ADP) are omitted. Both assays were conducted in a 96-well plate format, with the direct assay performed in UV-transmissible plates.
- Esterase Activity Assay CA activity was assayed according to method of Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229] described previously by Innocenti et al. [Innocenti et al., Bioorg. Med. Chem., 18:2159-2164; Innocenti et al., Bioorg. Med. Chem. Lett., 20:5050-5053] CA activity was determined by following thechange in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion over a period of 3 min at 25°C using a spectrophotometer (CHEBIOS UV-VIS). The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL, 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. The inhibitory effects of ampicillin sulfate, ceftriaxone, ceftizoxime, ranitidine were examined. All compounds were tested in triplicate at each concentration used. Different inhibitor concentrations were used. HCA-I enzyme activities were measured for ceftriaxone (0.003-0.033 mM), ceftizoxime (0.017-0.096 mM), ranitidine (0.026-0.053 mM) at cuvette concentrations and HCAII enzyme activities were measured for ampicillin sulfate (0.038-0.095 mM), ceftriaxone (0.003-0.021 mM), ceftizoxime (0.026-0.061 mM) and ranitidine (0.032-0.058 mM) at cuvette concentrations.
- ThermoFluor® Assay ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20.
- WecA Assay UDP-Glucosamine-C6-FITC (2 mM stock solution, 0.56 L), MgCl2 (0.5 M, 4 μL), β-mercaptoethanol (50 mM, 5 μL), CHAPS (5%, 11.25 μL), Tris buffer (pH 8.0, 50 mM), undecaprenyl phosphate (4 mM, 1.4 μL), and inhibitor molecule (0-100 μg/mL in Tris buffer) were place in a 500 μL Eppendorf tube. To a stirred reaction mixture, P-60 (10 μL) was added (total volume of reaction mixture: 50 μL adjust with Tris buffer). The reaction mixture was incubated for 4 h at 37° C. and quenched with n-butanol (150 μL). Two phases were mixed via vortex and centrifuged at 10,000 xg for 3 min. The upper organic phase was assayed via reverse-phase HPLC. The organic phase (30 μL) was injected into HPLC (solvent: gradient elution of 85:15 to 95:5 MeOH/0.05 M aq. NH4HCO3; UV: 485 nm; flow rate: 0.5 ml/ min; column: Kinetex 5 μm C8, 100 Å, 150 ×4.60 mm), and the area of the peak for C55-P-P-glucosamine-C6-FITC was quantified to obtain the IC50 value. The IC50 values were calculated from plots of the percentage product inhibition versus the inhibitor concentration.
- Esterase Activity Assay Esterase activity assay was performed based on the method of by Verpoorte et al. [Verpoorte et al., J. Biol. Chem., 242:4221-4229] as described in previous studies [Innocenti et al., Bioorg. Med. Chem., 18:2159-2164; Akıncıoğlu et al., Arch. Pharm., 347:68-76; Akıncıoğlu et al., Bioorg. Med. Chem., 21:1379-1385]. The differences in absorbances due to the hCA I and II isoenzyme activities converting 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion at 348 nm were observed in 3 min duration at room temperature using a spectrophotometer (Shimadzu, UVmini-1240 UV-VIS) [Çetinkaya et al., Arch. Pharm., 347:354-359; Aksu et al., Bioorg. Med. Chem., 21:2925-2931]. The reactants were 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1 mL 3 mM NPA, 0.5 mL H2O and 0.1 mL enzyme solution making a total volume of 3 mL. A cuvette containing all reactants without the enzyme solution was used as blank. For the determination of Ki values three different bromophenols concentrations with NPA as the substrate at five different concentrations were used for making of Lineweaver-Burk curves [Lineweaver et al., J. Am. Chem. Soc., 56:656-666] as previously described [Atasever et al., Food Chem., 136:864-870; Aydin et al., Int. J. Food Propert., 18:2735-2745].
- EGFR Inhibitory Activity With regard to the setting of the conditions for a method for measuring the inhibitory activity of a compound against EGFR kinase activity in vitro, it is described in the consumable reagent supplies price list for LabChip (registered trademark) series of PerkinElmer, Inc. that FL-PEPTIDE 22 corresponds to a substrate peptide for the measurement of EGFR kinase activity. Therefore, a biotinated peptide (biotin-EEPLYWSFPAKKK) was produced by referring to the amino acid sequence of the peptide. The purified recombinant human EGFR protein used in the test was purchased from Carna Biosciences, Inc.With regard to the measurement of the inhibitory activity of the compounds, firstly, the compounds of the present invention were diluted stepwise with dimethyl sulfoxide (DMSO). Subsequently, EGFR protein, a substrate peptide (final concentration was 250 nM), magnesium chloride (final concentration was 10 mM), manganese chloride (final concentration was 10 mM), ATP (final concentration was 1.5 μM), and a DMSO solution of the compound of the present invention (final concentration of DMSO was 2.5%) were added to a buffer solution for kinase reaction (20 mM HEPES (pH 7.5), 2 mM dithiotheitol, 0.01% Triton X-100), and after the solution was incubated for 120 minutes at 25° C., a kinase reaction was carried out. The reaction was terminated by adding EDTA thereto in order to obtain a final concentration of 24 mM. Subsequently, a detection liquid containing Eu-labeled anti-phosphorylated tyrosine antibody PT66 (PerkinElmer, Inc.) and SURELIGHT APC-SA (PerkinElmer, Inc.) was added thereto, and the system was left to stand for 2 hours or longer at room temperature. Finally, the amount of fluorescence upon irradiation of excitation light having a wavelength of 337 nm was measured at two wavelengths of 620 nm and 665 nm, using a PHERAstar FS (BMG Labtech GmbH). The amount of phosphorylation reaction was determined from the ratio of the amounts of fluorescence at the two wavelengths, and the compound concentration at which the phosphorylation reaction could be suppressed in 50% was defined as the IC50 value (nM).
- Measurement of EGFR Inhibitory Activity With regard to the setting of the conditions for a method for measuring the inhibitory activity of a compound against EGFR kinase activity in vitro, it is described in the consumable reagent supplies price list for LabChip (registered trademark) series of PerkinElmer, Inc. that FL-PEPTIDE 22 corresponds to a substrate peptide for the measurement of EGFR kinase activity. Therefore, a biotinated peptide (biotin-EEPLYWSFPAKKK) was produced by referring to the amino acid sequence of the peptide. The purified recombinant human EGFR protein used in the test was purchased from Carna Biosciences, Inc.With regard to the measurement of the inhibitory activity of the compounds, firstly, monofumarate of Compound A was diluted stepwise with dimethyl sulfoxide (DMSO). Subsequently, EGFR protein, a substrate peptide (final concentration was 250 nM), magnesium chloride (final concentration was 10 mM), manganese chloride (final concentration was 10 mM), ATP (final concentration was 1.5 μM), and a DMSO solution of the test compounds (final concentration of DMSO was 2.5%) were added to a buffer solution for kinase reaction (20 mM HEPES (pH 7.5), 2 mM dithiotheitol, 0.01% Triton X-100), and after the solution was incubated for 120 minutes at 25° C., a kinase reaction was carried out. The reaction was terminated by adding EDTA thereto so as to obtain a final concentration of 24 mM. Subsequently, a detection liquid containing Eu-labeled anti-phosphotyrosine antibody PT66 (PerkinElmer, Inc.) and SURELIGHT APC-SA (PerkinElmer, Inc.) was added thereto, and the system was left to stand for 2 hours or longer at room temperature. Finally, the amount of fluorescence upon irradiation with excitation light having a wavelength of 337 nm was measured at two wavelengths of 620 nm and 665 nm, with a PHERAstar FS (BMG Labtech GmbH). The amount of phosphorylation reaction was determined from the ratio of the amounts of fluorescence at the two wavelengths, and the compound concentration at which the phosphorylation reaction could be suppressed by 50% was defined as the IC50 value (nM).
- Biochemical Assay For the SAR (structure-activity relationship) and compound screening, LanthaScreen TR-FRET (Time-Resolved fluorescence energy transfer) assay was employed using the phospho-tyrosine specific Terbium (Tb)-labeled antibody with a fluorescein labeled poly-GT (glutamate-tyrosine) as a substrate. Upon excitation at 340 nm by UV, the energy from Tb donor of the antibody is transferred to the fluorescein of the phosphorylated poly GT substrate, and fluorescein emits light at 520 nm. The ratio between the intensity of primary emission at 495 nm and that of secondary emission at 520 nm was used to quantify the level of kinase activity. The recombinant proteins of human c-MER and AXL catalytic domains, Fluorescein-labeled poly-GT substrate, Tb-labeled anti-phosphorylated tyrosine antibodies, the kinase assay buffer, and 0.5M EDTA solution were purchased (Life technologies, USA). The TR-FRET assays were carried out in the white low volume 384-well plate (Corning, USA). To measure the compound mediated inhibition of kinase activity, the recombinant kinases were pre-incubated with test compounds for 20 minutes prior to the addition of 200 nM fluorescein labeled poly-GT substrates and 10 uM ATP, and then the reaction was carried out for 1 hour at room temperature. 10 mM EDTA was added to terminate the enzyme reaction, and the level of phosphorylation of poly-GT substrate was determined following 30 min incubation with 2 nM Tb-labeled antibody. The fluorescence intensity was measured with Envision plate reader (PerkinElmer, USA).
- Enzyme Activity Assay An enzyme activity assay for BLVRB was conducted. Since BLVRB catalyzes the NAD(P)H dependent readuction of FMN, changes in the NAD(P)H concentration in the presence of FMN can be used to measure enzyme activity in the absence (control) and presence of drug candidates. The IC50 (the concentration of the drug candidate at which BLVRB activity to convert NADPH to NADP+ in the presence of FMN was reduced to 50%) was used as the selection criterion for the screenings of more potent inhibitory molecules with lower IC50 value. Native form of BLVRB (without NADP+) was used for enzyme activity assay. Assay was performed by monitoring the rate of oxidation of NADPH at 340 nm with Gemini EM Microplate Reader. All assays were operated at 25° C. in 100 mM Phosphate-buffered saline, 0.01% of triton X-100, 100 μM FMN, 100 μM NADPH, 1 μM BLVRB and variable concentration of compounds of the present disclosure. The control reactions were carried out in the absence of compounds of the present disclosure to compare the effectiveness of the drug. FMN and NADPH were freshly constructed with 10 mM stock daily, calculated by UV/Vis spectrometer. In each case, FMN was added to initiate the reaction and measured for 30 minutes. The concentration of the compounds of the present disclosure was sequentially decreased in the order of 100 μM, 25 μM, and 5 μM.
- Enzyme Inhibition Assay (IC50) and Fluorescence Quench Assay (KD) Inhibitors were assayed in a 96-well plate format. Typically, protease was preincubated with concentrations of test compounds at 37 deg C for 30 min. The reaction was initiated by the addition of substrate Bz-nKRR-AMC. Reaction progress was monitored continuously by following the increase in fluorescence on a Tecan Safire2 plate reader. IC50 values of inhibitors were derived by fitting the calculated initial velocities to a nonlinear regression curve using GraphPad Prism software. Each point of the IC50 curve was measured in duplicate during a single experiment. Compounds were tested for binding (KD) using fluorescence quench assay. Two protein solutions were prepared (2-5 uM protein with and without 40 uM compound) and were mixed in a microplate to obtain 12 different compound concentrations ranging from 0 to 40 uM. Then each dilution comprising 90 ul was transferred to a UV-Star 96-well microplate. After 1 h incubation at room temperature, fluorescence was measured at 25 deg C on a Tecan Safire2 with ex = 280 nm and em = 340 nm. At the end of the measurements, 11 uM bovine pancreatic trypsin inhibitor (BPTI) was added to the wells containing 40 uM compound and the fluorescence was measured again. Binding curves were analyzed according to the two-state model describing the formation of a 1:1 complex. Kd values were obtained from curve-fit of binding isotherms.
- IDO1 Enzymatic Assay The compounds described in this disclosure were dissolved in 10% DMSO to predefined concentration. All of the reactions were conducted at room temperature in this disclosure. Human recombinant IDO1 (BPS Bioscience) was added so that its concentration was 200 nm in the reaction buffer solution (200 μL). The solution also contained 500 nM tryptophan, ascorbic acid (20 mM), methylene blue (10 μM), 126ydroxyl peroxidase (10 g/mL) and sodium phosphate buffer solution Ph 6.5 (100 mM). 10 μL test compound solution was added into the above reaction solution (200 μL), so that the final concentration of DMSO in all reaction wells was 0.5%. % concentration was volume %. After 80 min incubation of the reaction solution, the UV absorbance was measured using TECAN Infinite M1000 plate reader. For the negative control (blank), 10 μl of the assay buffer was added instead of the IDO1All reagents (excluding human recombinant IDO1) were purchased from Sigma Aldrich, MO, USA.The percent activity in the presence of each compound was calculated according to the following equation: % activity=[(A−Ab)/(At−Ab)]×100, where A=the absorption signal in the presence of the compound, Ab=the absorption signal of blank, At=the absorption signal in the absence of the compound. The percent inhibition was calculated according to the following equation: % inhibition=100−% activity. The IC50 was calculated using non-linear regression in Graph Pad Prism 4.
- Phosphotyrosine (PY) ELISA Cells used were tumor cell lines naturally expressing high levels of tyrosine kinases. Expression levels at the RNA level were derived from the NCI Developmental Therapeutics Program (NCI-DTP) web site public molecular target information (http://www.dtp.nci.nih.gov/mtargets/mt_index.html). Briefly, cells at 60-75% confluence are placed in serum-free medium for 18 h to reduce the background of phosphorylation. Cells were always >98% viable by Trypan blue exclusion. Cells are then pre-treated for 60 min with various concentrations of test compound, followed by growth factor. The reaction was stopped and cells permeabilized by quickly removing the media from the cells and adding ice-cold Tris-buffered saline (TBS) containing 0.05% triton X-100, protease inhibitor cocktail and tyrosine phosphatase inhibitor cocktail. The TBS solution is then removed and cells fixed to the plate by 30 min at 60 deg C and further incubation in 70% ethanol for an additional 30 min. Cells are further exposed to block (TBS with 1% BSA) for 1 h, washed, and then a horseradish peroxidase (HRP)-conjugated phosphotyrosine antibody added overnight. The antibody was removed; cells are washed again in TBS, exposed to an enhanced luminol ELISA substrate (Pierce Chemical, Rockford, IL, USA) and light emission measured using an UV Products (Upland, CA, USA) BioChemi digital darkroom. Data were graphed as a percent of cells receiving growth factor alone and IC50 values estimated from 2-3 separate experiments (n=8-24) using Prism 5.0.
- In-vitro IDO1 Enzyme (Indoleamine 2,3-dioxygenase) Assay Human indoleamine 2,3-dioxygenasel (hIDO1) catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N-formylkynurenine which can be converted to kynurenine (KYN) by deformylation. hIDO1 with an N-terminal His tag, expressed and purified from E. coli cells was procured from either Enzo LifeScienccs, NY, USA). Unless otherwise stated, all materials were procured from Sigma Aldrich, Mo., USA.The assay monitoring the conversion of L-tryptophan to KYN was carried out as follows. hIDO1 (10 nM) was incubated with tryptophan (30 μM) in the presence of ascorbic acid (20 mM), methylene blue (10 μM) and catalase (100 μg/mL) in potassium phosphate buffer (50 mM; pH 6.5) at 37C for 30 min. The reaction was terminated with 30% trichloroacetic acid (TCA) and further incubated at 65C for 15 min to fully convert N-formylkynurenine to KYN. The reaction mixture was then centrifuged to remove sediments and the KYN in the supernatant was estimated by UV-visible absorption spectroscopy at 360 nm using a Waters HPLC system fitted with a C-18 column or by LC/MS/MS. See, Sono, 1980, J. Biol. Chem., 255:1339-1345, which is herein incorporated by reference.Percent inhibition at each concentration of test compounds was determined by estimating the decrease in KYN with reference to the reaction control with 1% DMSO vehicle. Data were analyzed using nonlinear regression to generate IC50 values using Graph Pad Prism 5.
- KHK-C Assay A A 384-well format on a Corning 3653 assay plate is used, and monitored by UV-vis spectroscopy in continuous mode at rt. Compounds were prepared in DMSO as 4 mM stocks, diluted using an 11-point half-log scheme on a Biomek FX (Beckman Coulter), and incubated at rt for 30 minutes with the reaction mixture containing 50 mM HEPES, pH 7.4, 140 mM KCl, 3.5 mM MgCl2, 0.8 mM fructose, 2 mM TCEP, 0.8 mM PEP, 0.7 mM NADH, 0.01% Triton X-100, 30 U/mL pyruvate kinase-lactate dehydrogenase, and 10 nM purified KHK-C. The compound concentration in each well ranged from 1 nM to 100 μM. The reaction was initiated with the addition of 0.2 mM ATP. The absorbance was measured for 30 minutes on a SpectraMax reader (Molecular Devices) after ATP was added. The concentrations provided are based on the final mixture volume of 40 μL (referred to as the final concentration). Controls: N8-(cyclopropylmethyl)-N4-(2-(methylthio)phenyl)-2-(piperazin-1-yl)pyrimido[5,4-d]pyrimidine-4,8-diamine at 2 μM final concentration was used as high percent effect (HPE) control, and 2.5% DMSO which was present in all reaction wells was used as zero percent effect (ZPE) control. Reaction rates were obtained for 300-1800 seconds time window in units of 1000*AU/min (absorbance unit per minute), and average values for ZPE and HPE controls from 16 wells each were calculated, AveZPE and AveHPE, respectively.
- TR-FRET Kinase Activity Biochemical Assay Table 2: For the SAR (structure-activity relationship) and compound screening, LanthaScreen™ TR-FRET (Time-Resolved fluorescence energy transfer) assay was employed using the phospho-tyrosine specific Terbium (Tb)-labelded antibody with a fluorescein labeled poly-GT (glutamate-tyrosine) as a substrate. Upon excitation at 340 nm by UV, the energy from Tb donor of the antibody is transferred to the fluorescein of the phosphorylated poly GT substrate, and fluorescein emits light at 520 nm. The ratio between the intensity of primary emission at 495 nm and that of secondary emission at 520 nm was used to quantify the level of kinase activity. The recombinant proteins of human c-MER and AXL catalytic domains, Fluorescein-labeled poly-GT substrate, Tb-labeled anti-phosphorylated tyrosine antibodies, the kinase assay buffer, and 0.5M EDTA solution were purchased (Life technologies, USA). The TR-FRET assays were carried out in the white low volume 384-well plate (Corning, USA). To measure the compound mediated inhibition of kinase activity, the recombinant kinases were pre-incubated with test compounds for 20 minutes prior to the addition of 200 nM fluorescein labeled poly-GT substrates and 10 uM ATP, and then the reaction was carried out for 1 hour at room temperature. 10 mM EDTA was added to terminate the enzyme reaction, and the level of phosphorylation of poly-GT substrate was determined following 30 min incubation with 2 nM Tb-labeled antibody. The fluorescence intensity was measured with Envision™ plate reader (PerkinElmer, USA).
- ThermoFluor® Assay Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
- FRET Assay An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
- FRET Assay Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat#Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
- Fluorescence Direct Binding Assay Determination of the affinities of compounds to protein containing one or more tryptophan is measurable by monitoring the fluorescence emission in direct mode. The measurements depending on the protein available amounts are performed either manually in a cuvette on ISS-PC1 photon counting spectrofluorometer or automatically in well plates on a fluorescence plate reader device. Fluorescence titrations are performed at 20° C. in the chosen binding assay buffer by using a defined constant protein concentration against ligand concentration variations. Small aliquots of known ligand concentration solubilized in DMSO were added and the fluorescence, excited at 280 nm, was recorded at 340 nm. The fluorescence intensity was corrected for protein dilution and for the filter effect (Birdsall et al.72). The corrected fluorescence intensity was plotted against the ligand concentration and fitted using a four-parameter sigmoidal function, from which the equilibrium dissociation constant Kd was computed using the law of mass action assuming a 1:1 protein-ligand complex (Eftink, 199773).Process1) Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content2) Titration measurements of the protein against ligand by at least 12 titration steps to obtain a good s-curve fit3) Repeat the same titration measurements with the ligand alone to enable correction4) Check the stability of the protein once by titration against DMSO alone5) Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of an UV-spectrophotometer6) Use Excel template for the correction of the measured raw data7) Use GraphPad Prism software for the quadratic binding fit and the KD evaluation.
- In-vitro IDO2 Enzyme (Indoleamine 2,3-dioxygenase2) Assay Human indoleamine 2,3-dioxygenase-2 (hIDO2) catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N-formylkynurenine which can be converted to kynurenine (KYN) by deformylation. hIDO2 with an C-terminal His tag (Sino Biological Inc (China)) was expressed in E. coli cells and the protein was purified using standard methods well known in the art. Unless otherwise stated, all materials were procured from Sigma Aldrich, Mo., USA.The assay monitoring the conversion of L-tryptophan to KYN was carried out as follows. hIDO2 (160 nM) was incubated with tryptophan (5000 04) in the presence of ascorbic acid (20 mM), methylene blue (10 04) and catalase (100 μg/mL) in potassium phosphate buffer (100 mM; pH 7.5) at 37 degrees C. for 30 min. The reaction was terminated with 30% trichloroacetic acid (TCA) and further incubated at 65 degrees C. for 15 min to fully convert N-formylkynurenine to KYN. The reaction mixture was then centrifuged to remove sediments, and the KYN in the supernatant was measured by UV-visible absorption spectroscopy at 360 nm using a Waters HPLC system fitted with a C-18 column or by LC/MS/MS (C. J. D Austin et. Al, Amino Acid 2009, 565-578).Percent inhibition at each concentration of test compounds was determined by determining the decrease in KYN with reference to the reaction control with 1% DMSO vehicle. Data were analyzed using nonlinear regression to generate IC50 values using Graph Pad Prism 5. Compounds 2 and 184 were tested as described above.
- MERS-CoV nLUC in Calu-3 At 48 hours prior to infection, Calu-3 2B4 cells were plated in a 96-well black-walled clear bottom plate at 5x104 cells/well. A 10 mM stock of NHC was serially diluted in 100% DMSO in 3-fold increments to obtain a ten-point dilution series. MERS-nLUC was diluted in DMEM supplemented with 10% FBS, and 1% Antibiotic-Antimycotic to achieve a multiplicity of infection (MOI) of 0.08. Cells were infected and concurrently treated with NHC in triplicate per drug dilution for 1hr, after which viral inoculum was aspirated, cultures were rinsed once and fresh medium containing drug or vehicle was added. At 48 hours post infection, nanoluciferase expression as a surrogate for virus replication was quantitated on a Spectramax plate reader (Molecular Devices) according to the manufacturer s instructions (Promega, NanoGlo). For the 100% inhibition control, diluted MERS-nLUC was exposed to short-wave UV light (UVP, LLC) for 6 min to inhibit the ability of the virus to replicate. For the 0% inhibition control, cells were infected in the presence of vehicle only. DMSO was kept constant in all conditions at 0.05%. Values from triplicate wells per condition were averaged and compared to controls to generate a percent inhibition value for each drug dilution. The IC50 value was defined as the concentration at which there was a 50% decrease in luciferase expression. Data were analyzed using GraphPad Prism 8.0. The IC50 values were calculated by non-linear regression analysis using the dose-response (variable slope) equation (four parameter logistic equation): Y = Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)).
- fluorescence resonance energy transfer (FRET)-based CoV-2 3CLpro inhibition assay SARS-CoV-2 3CLpro expression and purification is based on a published procedure and our modified protocol is found in the supplementary file. A highly sensitive FRET based protease assay was developed to identify inhibitors of 3CL proteases based on a published protocol. The peptide substrate (Dabcyl)KTSAVLQSGFRKM(Glu)(EDANS) was synthesized by Genscript (USA). The test compounds were 3-fold serially diluted in 100% DMSO to 15 concentrations, starting at 3.33 mM. 1.5 μl of the serially diluted compounds were transferred to a black 384 well assay plate (Cat. 781900, Greiner). 23.5 μl of 2.13X concentration of SARS-CoV-2 Chis-3CLpro enzyme prepared in assay buffer was added to the compounds and incubated for 30 mins at 25 ◦C. 25 μl of 2X concentration of peptide substrate was added to the assay plate and incubated at 37 ◦C for 1.5 h. The final assay contained 12.5 nM of enzyme, 6 μM substrate and 3% DMSO in assay buffer containing 50 mM HEPES at pH 7.5, 100 mM NaCl, and 0.01% Triton X-100 and 1 mM DTT. The starting test compound concentration started at 100 μM. The FRET signal was measured using an excitation wavelength of 340 nm (UV[TRF] 340/60 nm, Barcode 101), emission wavelength of 490 nm (DSPPsion 486/10 filter, Barcode 220) and Lance/DELFIA D400 single mirror (Barcode 412) on Envision plate reader (2104 EnVision Multilabel Plate Readers, Perkin Elmer). The dose response curves were fitted with a variable slope using GraphPad Prism software (GraphPad, USA) to determine a compound s IC50.
- Fluorescence Direct Binding Asaay Determination of the affinities of compounds to protein containing one or more tryptophan is measurable by monitoring the fluorescence emission in direct mode. The measurements, depending on the protein available amounts, were performed either manually in a cuvette on ISS-PCI photon counting spectrofluorometer or automatically in well plates on a fluorescence plate reader device. Fluorescence titrations were performed at 20° C. in the chosen binding assay buffer by using a defined constant protein concentration against ligand concentration variations. Small aliquots of known ligand concentration solubilized in DMSO were added and the fluorescence, excited at 280 nm, was recorded at 340 nm. The fluorescence intensity was corrected for protein dilution and for the filter effect (Birdsall, B., King, R. W., Wheeler, M. R., Lewis, C. A. Jr, Goode, S. R., Dunlap, R. B. & Roberts, G. C. (1983). Anal. Biochem.132, 353-361). The corrected fluorescence intensity was plotted against the ligand concentration and fitted using a four-parameter sigmoidal function from which the equilibrium dissociation constant Kd was computed using the law of mass action assuming a 1:1 protein ligand complex (Eftink, Methods Enzymol. 1997; 278:221-57). The process includes:Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content. Titration measurements of the protein against ligand by at least 12 titration steps to obtain an s-curve fit. Repeat the same titration measurements with the ligand alone to enable correction. Check the stability of the protein once by titration against DMSO alone. Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of an UV-spectrophotometer. Use Excel template for the correction of the measured raw data. Use GraphPad Prism software for the quadratic binding fit and the KD evaluation.
- ThermoFluor Assay For the RORγt construct used in the ThermoFluor assay, numbering for the nucleotide sequences was based on the reference sequence for human RORγt, transcript variant 2, NCBI Accession: NM_001001523.1 (SEQ ID NO:1). Nucleotides 850-1635 (SEQ ID NO:2) coding for the wild type human RORγt ligand binding domain (RORγt LBD) were cloned into the pHIS1 vector, a modified pET E. coli expression vector (Accelagen, San Diego), containing an in-frame N-terminal His-tag and a TurboTEV protease cleavage site (ENLYFQG, SEQ ID NO:3) upstream of the cloned insert sequence. The amino acid sequence for the RORγt construct used in the Thermofluor assay is shown as SEQ ID NO:4. ThermoFluor experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
- DAAO Enzymatic Assay The DAAO enzymatic activity assay was modified according to the report of Oguri et al (Oguri, S., Screening of d-amino acid oxidase inhibitor by a new multi-assay method. Food chemistry 2007, 100 (2), 616). The DAAO activity was measured by using substrate D-alanine reaction produced hydrogen peroxide (H2O2) to further react with 3-(4-hydroxyphenyl) propionic acid (HPPA). The HPPA were oxidized by H2O2 and peroxidase to become the fluorogenic dimer which was measured to represent the activity of DAAO. The substrate of DAAO was prepared in 50 mM D-alanine (dissolved in 0.2 M Tris-HCl buffer, pH 8.3). A 100 ul of D-alanine solution was mixed with 4 ul (in 100%) dimethyl sulfoxide, DMSO) of different concentrations of drugs ranging from 31.36 nM, 94.08 nM, 0.28 uM, 0.85 uM, 2.54 uM, 7.62 uM, 22.86 uM, 68.59 uM, 0.21 mM, 0.62 mM, 1.85 mM, 5.56 mM, 16.67 mM, and 50.00 mM with a final DMSO concentrations of 0.167% in each reaction concentration. A 10 ul of D-alanine and drug mixture was incubated with 220 ul of Reaction Master Mix in black 96 well plate at 37° C. for 5 min. The Reaction Master Mix contained 110 ul of 5 U/mL porcine kidney DAAO (Sigma-Aldrich, USA) solution (dissolved with 0.2 M Tris-HCl buffer, pH 8.3), 1.1 mL of 15 U/mL peroxidase solution (dissolved with 0.2 M Tris-HCl buffer, pH 8.3), 1.1 mL of 20 mM HPPA solution (dissolved with 0.2 M Tris-HCl buffer, pH 8.3), and 2.2 ml of 2 M Tris-HCl buffer (pH 8.3) for 110 reaction assays.Fluorescence intensity (Fs) was measured at 405 nm by irradiation excitation at 320 nm. The higher is the DAAO enzymatic activity, the higher is the fluorescence intensity. The fluorometric inhibition indicator (Fi) was obtained from the following equation: Fi=(Fs−FDrug)/(FDMSO). Where the fluorescent drug blank (FDrug) were measured in the drug mixture solution (using 0.2 M Tris HCl buffer, pH 8.3, without D-alanine). A DMSO blank (FDMSO) was measured under a 100% DMSO solution. Although, in the assay for D-amino acid oxidase, FAD was generally included in the reaction mixture because this co-factor is easily dissociated from the holoenzyme, the present method was performed without FAD. The inhibitory effect of DAAO inhibitors was compared by using inhibitory concentrations which reduce 50% of DAAO activity (IC50). The IC50 value was calculated by GraphPad Prism, version 5 software (GraphPad Software, Inc., La Jolla, Calif.) (GraphPad Prism 5, GraphPad software Inc: California, USA) through nonlinear regression model.
- 5α-Reductase Activity Assay The reaction mixture contained a final volume of 1 mL: 1 mM dithiothreitol, sodium phosphate buffer 40 mM, at pH 6.5 for human prostate, 2 mM NADPH, 2 nM [1,2,6,7-3H]T [Cabeza et al., Steroids 60:630-5]. The reaction in duplicate was started when it was added to the enzymatic fraction (500 μg protein in a volume of 80 μL) incubated at 37°C for 60 min [Hirosumi et al., J. Steroid Biochem. Mol. Biol., 52:357-63] and stopped by mixing with 1 mL of dichloromethane; this was considered as the end point. Incubation without tissue was used as a control. The mixture (incubation medium/dichloromethane) was agitated on a vortex for 1 min and the dichloromethane phase was separated and placed in another tube. This procedure was repeated four more times. The dichloromethane extract was evaporated to dryness under a nitrogen stream and suspended in 50 mL of methanol that was spotted on high-performance thin layer chromatography (HPTLC) Keiselgel 60 F254 plates. T and DHT were used as carriers, and were applied in different lanes on both lateral sides of the plates (T, T + DHT, and DHT). The plates were developed in chloroform-acetone, 9:1, and were air-dried; the chromatography was repeated twice more. The steroid carriers were detected using phosphomolybdic acid reagent (DHT) and with an ultraviolet (UV) lamp (254 nm) (T). After the plates were segmented into pieces of 1 cm each, these were cut off and the strips soaked in 5 mL of Ultima Gold (Packard). The radioactivity was determined in a scintillation counter (Packard Tri-Carb 2100 TR). The radioactivity content in the segment corresponding to T and DHT carriers was identified. Radioactivity with identical chromatographic behavior to the DHT standard was considered as the DHT transformation. Control incubations, chromatography separations, and identifications were carried out in the same manner as described above, except that the tubes did not contain tissue.
- HTRF Kinase Activity Biochemical Assay Table 3: For the SAR (structure-activity relationship) and compound screening, HTRF (Homogeneous Time Resolved Fluorescence) kinase activity assay was employed for all MER, AXL and TYRO3 kinases using Cisbio HTRF® KinEASE™-TK kit (Cisbio, USA). The kit includes biotin-labeled TK substrate, streptavidin-XL665, Eu3+-cryptate-labeled TK antibody and HTRF® Detection buffer. There are two main steps in the kinase assay: kinase reaction and detection of phosphorylated substrate. The reaction was carried out in white low volume 384-well plate (Corning, USA) with 25 nL compound in dimethyl sulfoxide in each well. To measure the compound mediated inhibition of kinase activity, 2.5 uL the recombinant kinases were pre-incubated with test compounds for 30 minutes in the kinase reaction buffer (20 mM HEPES pH7.4, 2 mM MnCl2, 10 mM MgCl2, 100 uM Na3VO4, 0.0075% Triton X 100, 0.005% BSA and 1 mM DTT) prior to the addition of 2.5 uL of 1 uM biotin-labeled TK substrates and 10 uM ATP. Then the reaction was stopped after 1 hour incubation at room temperature by adding 5 uL of HTRF® Detection buffer which also contains 0.375 nM Eu3+-cryptate-labeled TK antibody and 0.062 uM streptavidin-XL665(SA-XL665) to allow for detection of the phosphorylated peptide product. After 1 h incubation at room temperature, the fluorescence intensity was measured with Envision™ plate reader (PerkinElmer, USA). Upon excitation at 340 nm by UV, the energy from Eu3+ donor of the antibody is transferred to the FRET acceptor XL665, and XL655 emits light at 665 nm. The level of kinase activity was quantified by the HTRF ratio that calculated from the intensity of emission at 665 nm and emission at 620 nm (fluorescence intensity @ 665 nm/fluorescence intensity @ 620 nm×10,000). The recombinant protein of human MER (528-end) was purchased from Carnabio, Japan. The recombinant human AXL (473-end) and TYRO3 (455-end) were purchased from SignalChem, Canada.
- KHK Assay A A 384-well format on a Corning 3653 assay plate is used, and monitored by UV-vis spectroscopy in continuous mode at rt. Compounds were prepared in DMSO as 4 mM stocks, diluted using an 11-point half-log scheme on a Biomek FX (Beckman Coulter), and incubated at rt for 30 minutes with the reaction mixture containing 50 mM HEPES, pH 7.4, 140 mM KCl, 3.5 mM MgCl2, 0.8 mM fructose, 2 mM TCEP, 0.8 mM PEP, 0.7 mM NADH, 0.01% Triton X-100, 30 U/mL pyruvate kinase-lactate dehydrogenase, and 10 nM purified KHK-C. The compound concentration in each well ranged from 1 nM to 100 μM. The reaction was initiated with the addition of 0.2 mM ATP. The absorbance was measured for 30 minutes on a SpectraMax reader (Molecular Devices) after ATP was added. The concentrations provided are based on the final mixture volume of 40 μL (referred to as the final concentration).Controls: N8-(cyclopropylmethyl)-N4-(2-(methylthio)phenyl)-2-(piperazin-1-yl)pyrimido[5,4-d]pyrimidine-4,8-diamine at 2 μM final concentration was used as high percent effect (HPE) control, and 2.5% DMSO which was present in all reaction wells was used as zero percent effect (ZPE) control. Reaction rates were obtained for 300-1800 seconds time window in units of 1000*AU/min (absorbance unit per minute), and average values for ZPE and HPE controls from 16 wells each were calculated, AveZPE and AveHPE, respectively.Percent inhibition (% inhibition) was calculated for each well using this equation:100 - 100 × ( Compound absorbance rate value - Ave HPE ) ( Ave ZPE - Ave HPE )The % inhibition was then plotted against the log of compound concentration using GraphPad Prism, and the data was fit to the equation log [compound] vs. response variable slope using nonlinear regression analysis to give IC50 values. For each compound tested, the IC50 provided is the average based on at least two separate assays conducted on separate days.
- ThermoFluor® Assay ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >60D cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1x=46 mL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
- GOAT activity assay GOAT activity was assessed using a time-resolved fluorescence energy transfer (TR-FRET) assay in a 384-well format. His-tag human GOAT enzyme was in the form of a cell membrane preparation from sf9 cells infected with hGOAT-V5-His baculovirus. Varying concentrations of test compound with final DMSO concentration kept to 0.5% were added to membrane solution. Human GOAT membrane activity was established in a buffer having final concentration 0.25 mg/mL in 50 mM MOPS, pH7.5; 50 mM KCl; 0.1 mg/mL BSA; 50 μM CHAPS; and 2 mM EDTA. Substrate solution consisting of biotinylated ghrelin peptide (final concentration 100 nM), octanoyl coA (final concentration 2 μM) and palmitoyl CoA (final concentration 50 μM) was added to initiate the reaction. Plates were sealed, centrifuged for 1 minute at 2000 rpm, then incubated at 30° C. for 80 minutes with gentle shaking on an Eppendorf mix plate. Reaction termination and detection mix consisting of chicken anti-active ghrelin antibody (final concentration of 10 nM), Europium W1024-labeled streptavidin (final concentration of 4 nM), GOAT anti-chicken Dylight (final concentration of 12.5 nM), and GS[DAP-oc]-FL-amide inhibitor (final concentration of 1 μM) was added before further incubation for 40 minutes at 30° C. The plate was then read on an Envision in HTRF mode with excitation filter UV (TRF) 340 and first emission filter of APC 665 and a second emission filter of Europium 615. HTRF readings were acquired as per instrument defined LANCE-DELFIA protocol with a delay and window times of 50 μs for both; number of sequential windows: 1; time between flashes: 2000 μs between each of 100 flashes and 10 flashes for the second detector. The HTRF ratio was calculated directly by the instrument as the ratio of 665 window/615 window. Percent inhibition was calculated as 100−(100×(U−NC)/(PC−NC)) where U was the unknown value HTRF ratio (test compound value), NC was the negative control (100% inhibition value generated from a potent inhibitor), and PC was the positive control (100% activity generated from 0.5% DMSO vehicle). IC50 values were generated in GraphPad Prism (Version 4.03) using non-linear regression curve fit and sigmoidal dose response variable slope analysis.
- In Vitro Plasma Kallikrein Inhibition Assay The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
- In Vitro Fluorescence-Based Recombinant Tau Binding Assay (A) Expression and Purification of Human Tau: 1 mM IPTG (Sangon Biotech, Cat. No A100487) was used to induce production of tau by bacteria (BL21, Invitrogen, Cat. No C600003) transformed with a full-length 2N4R tau expression plasmid. After 3 hours, cell pellet was resuspended in lysis buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgSO4, pH 6.8) and lysed by sonication followed by centrifugation (15,000 rpm, 15 min, 4° C.). The supernatant was then placed in a boiling water bath for 20 min, followed by centrifugation (15,000 rpm, 15 min, 4° C.). Supernatant was loaded onto a Q-Sepharose Fast Flow column (GE healthcare, Cat. No 17-0510-01), the flow-through fraction was loaded onto an SP-Sepharose Fast Flow column (GE healthcare, Cat. No 17-0729-01), and tau protein was eluted with elution buffer (100 mM PIPES, 1 M NaCl, 1 mM EGTA, 1 mM MgSO4, 0.2 M NaCl, pH 6.8). Collected fractions of tau-containing eluates were pooled, concentrated and dialyzed against HEPES buffer (25 mM HEPES, 0.1 mM EDTA, 0.5 mM DTT, 100 mM NaCl, pH 7.2) and stored at −80° C. in small aliquots until use. Protein concentration was determined by UV absorption.(B) Preparation of Heparin-Induced Aggregated Tau (aTau):>2 μM of tau prepared in 30 mM Tris-HCl, pH 7.5 buffer was incubated in tube with 15 μM heparin (Sigma, Cat. No H3149) at 37° C. for 24 hours. (C) Compound Fluorescent Spectra Scanning Assay: Compounds were dissolved in 100% DMSO, and 40 nM aTau was incubated with 10 μM of each compound in 2% DMSO in 96 well plate (Corning, Cat. No 3573) at 37° C. for 1 hour. Emission and excitation of the compound was scanned by microplate spectrometer (EnSpire 2300, PerkinElmer).(D) In Vitro Fluorometric aTau Binding Assays: 2 μM aTau was diluted to 0.04 μM with 30 mM Tris-HCl (pH 6.8), and then incubated with serially diluted compound (three-fold serial dilutions, from 10 to 0.00017 μM) in a 96-well plate (Corning, Cat. No 3573) at 37° C. for 1 hour. Fluorescence intensity (excitation/emission=370/500 nm) of APN-0729 was read by microplate spectrometer (EnSpire 2300, PerkinElmer). Compound Kd values were calculated using the following equation: Y=B max *X/(Kd+X), where X is the concentration of compound; Y is the fluorescence signal of (compound+aTau)−(compound+DMSO); and B max is the maximum signal.
- Protease-Free PPIase Assay The protease-free PPIase assay measures the rate of cis to trans conversion of a peptide substrate catalyzed by the enzyme cyclophilin A. Addition of a cyclophilin A inhibitor (e.g., a test compound) slows the catalyzed rate and a Ki value is obtained. A Ki value of less than 10 nM demonstrates that the test compound is a potent inhibitor of cyclophilin A.MaterialsAssay Buffer:35 mM HEPES pH 7.8, filtered through a 0.2 μm filter. 50 μM DTT was added prior to use each day and then the buffer was stored on ice.Enzyme:Human recombinant cyclophilin A (Cyp A) (Sigma C3805) enzyme was diluted to 1 μM with enzyme dilution buffer (20 mM HEPES pH 7.8, 40% glycerol, 50 μM DTT and 1 μM BSA) and stored at −20° C.Substrate:Succinimide-Ala-Ala-Pro-Phe-p-nitroanilide (SUC-AAPF-pNA) (from Bachem AG, L-1400), 20 mg/ml prepared in 0.5 M LiCl in trifluoroethanol.MethodAll readings were taken with an Agilent 8453 Spectrophotometer which includes of a cuvette holder, stirrer and chiller to maintain a stirred cuvette temperature of 10.0±0.1° C. The temperature is monitored by the use of a temperature probe. To prevent UV degradation of test compounds, the light below 290 nm was blocked using a glass slide in the light path. 1.5 ml of the assay buffer was put into a 3 ml quartz cuvette and cooled to 10.0±0.1° C. while stirring (vigorous but not so fast as to produce cavitation). The inhibitor was diluted in 100% DMSO, and then added to the assay to a maximum final concentration of 0.5% DMSO in the assay. A blank spectrum was obtained, then 3 μL of enzyme was added (2 nM final concentration) and then 3 μL substrate (60 μM final concentration) added. The absorbance was measured at 330 nm for 300 s or 500 s for blank runs (NOTE: the substrate must be added in one quick injection and the measurements started immediately to minimize mixing errors).A first order rate equation was fitted to the absorbance data, for each concentration of inhibitor, to obtain the rate constant (the first 10 to 15 seconds were excluded as mixing causes errors in this portion of curve). The catalytic rate was calculated from the enzymatic rate constant minus the background rate constant. An exponential curve was generated using the catalytic rate constants versus the inhibitor concentration to obtain the Ki value for the inhibitor. The Ki value is indicative of the binding affinity between the test compound and cyclophilin A.
- RORgammat ThermoFluor Assay For the RORγt construct used in the ThermoFluor assay, numbering for the nucleotide sequences was based on the reference sequence for human RORγt, transcript variant 2, NCBI Accession: NM_001001523.1 (SEQ ID NO:1). Nucleotides 850-1635 (SEQ ID NO:2) coding for the wild type human RORγt ligand binding domain (RORγt LBD) were cloned into the pHIS1 vector, a modified pET E. coli expression vector (Accelagen, San Diego), containing an in-frame N-terminal His-tag and a TurboTEV protease cleavage site (ENLYFQG, SEQ ID NO:3) upstream of the cloned insert sequence. The amino acid sequence for the RORγt construct used in the Thermofluor assay is shown as SEQ ID NO:4.ThermoFluor experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows:0.065 mg/mL RORγt60 M 1,8-ANS100 mM Hepes, pH 7.010 mM NaCl2.5 mM GSH0.002% Tween-20Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1x=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
- PDE4 Inhibition Assay The assay buffer was prepared by diluting 5× supplied Tween-based buffer in 1:5 in water to make 1× buffer. Add desired additive to buffer (DTT or MnCl2). In a separate 96-well polypropylene plate, compound dilutions were prepared in assay buffer. Separate microcentrifuge tubes were prepared of PDE4B and PDE4D according to assay template in assay buffer. The tubes were kept on ice. The enzyme concentration shown on the template were diluted by 1:4. FAM-cAMP substrate solution was prepared according to assay template. 5 μl compound was transferred from polypropylene plate into black 384-well plate. This plate was centrifuged briefly to make sure all 5 μl is on the bottom. Up to 80 μl of prepared PDE4B enzyme solution was transferred into alternate wells on row N of 384-well plate starting from cell N1. Up to 80 μl of prepared PDE4D enzyme solution was transferred into alternate wells on row 'O' of 384-well plate, starting from cell O2. cAMP substrate solution was transferred into bottom row of separate 96-well plate. 5 μl of enzyme solution from the "reservoir" row (N or O) was transferred to each of the wells containing compounds, per layout map. Next, 10 μl of cAMP substrate was transferred to these wells. The order of substrate-first or enzyme-first can be switched depending on what is optimal. The final cAMP concentration was 100 nM in the reaction. 20 μl of assay buffer was pipetted into 4 separate wells these are the blanks. The plate was sealed with an aluminum strip and incubated at 30° C. for 90 minutes. A TR-FRET solution was prepared. 4 ml of 1× IMAP Buffer A was added to 6 ml of IMAP Buffer B. 25 μl (1/800 of 20 ml) of binding beads was added to this and mix by inverting. 60 μl of this mixture was pipetted into 2 of the wells containing the "blank" assay buffer. Next, 49.7 μl (1/400 of remaining volume) of Tb donor solution was added to the remaining TR-FRET solution and mixed by inverting. 60 μl of this solution was pipetted into remaining 2 blank assay buffer-containing wells. The TR-FRET solution was poured into a pipette boat and a multichannel pipette was used to drop 60 μl of solution into all assay wells. The wells were covered with a foil strip and incubated for at least 3 hours or overnight protected from light (e.g. in a drawer) at room temperature. The plate was read on the Envision Reader: Emission 1: 520/Emission 2: 486/Exc: 340. Mirror: Umbelliferone (UV).
- TR-FRET-Based Assay The assay buffer was prepared by diluting 5× supplied Tween-based buffer in 1:5 in water to make 1× buffer. Add desired additive to buffer (DTT or MnCl2). In a separate 96-well polypropylene plate, compound dilutions were prepared in assay buffer. Separate microcentrifuge tubes were prepared of PDE4B and PDE4D according to assay template−in assay buffer. The tubes were kept on ice. The enzyme concentration shown on the template were diluted by 1:4. FAM-cAMP substrate solution was prepared according to assay template. 5 μl compound was transferred from polypropylene plate into black 384-well plate. This plate was centrifuged briefly to make sure all 5 μl is on the bottom. Up to 80 μl of prepared PDE4B enzyme solution was transferred into alternate wells on row 'N' of 384-well plate starting from cell N1. Up to 80 μl of prepared PDE4D enzyme solution was transferred into alternate wells on row 'O' of 384-well plate, starting from cell O2. cAMP substrate solution was transferred into bottom row of separate 96-well plate. 5 μl of enzyme solution from the "reservoir" row (N or O) was transferred to each of the wells containing compounds, per layout map. Next, 10 μl of cAMP substrate was transferred to these wells. The order of substrate-first or enzyme-first can be switched depending on what is optimal. The final cAMP concentration was 100 nM in the reaction. 20 μl of assay buffer was pipetted into 4 separate wells−these are the blanks. The plate was sealed with an aluminum strip and incubated at 30° C. for 90 minutes. A TR-FRET solution was prepared. 4 ml of 1×IMAP Buffer A was added to 6 ml of IMAP Buffer B. 25 μl ( 1/800 of 20 ml) of binding beads was added to this and mix by inverting. 60 μl of this mixture was pipetted into 2 of the wells containing the "blank" assay buffer. Next, 49.7 μl ( 1/400 of remaining volume) of Tb donor solution was added to the remaining TR-FRET solution and mixed by inverting. 60 μl of this solution was pipetted into remaining 2 "blank" assay buffer-containing wells. The TR-FRET solution was poured into a pipette boat and a multichannel pipette was used to drop 60 μl of solution into all assay wells. The wells were covered with a foil strip and incubated for at least 3 hours or overnight protected from light (e.g. in a drawer) at room temperature. The plate was read on the Envision Reader: Emission 1: 520/Emission 2: 486/Exc: 340. Mirror: Umbelliferone (UV).