Query String: TOLBUTAMIDE
1-Butyl-3-tosylurea N-[(butylamino)carbonyl]-4-methylbenzenesulfonamide Orinase (TN) BDBM50027886 N-(4-Methylphenylsulfonyl)-N'-butylurea N-Butyl-N'-(p-tolylsulfonyl)urea 1-Butyl-3-(p-tolylsulfonyl)urea 1-Butyl-3-(p-methylphenylsulfonyl)urea CHEMBL782 N-Butyl-N'-(4-methylphenylsulfonyl)urea N-n-Butyl-N'-tosylurea TOLBUTAMIDE
- ChEMBL_948336 (CHEMBL2341170) Inhibition of CYP2C9 (unknown origin)-mediated tolbutamide hydroxylation to hydroxy tolbutamide
- ChEMBL_563300 (CHEMBL981057) Inhibition of CYP2C9 assessed as Tolbutamide hydroxylation
- ChEMBL_690429 (CHEMBL1635166) Inhibition of CYP2C9 using Tolbutamide as probe
- ChEMBL_1868902 (CHEMBL4369968) Inhibition of human CYP2C9 using tolbutamide as substrate
- ChEMBL_1898405 (CHEMBL4400440) Inhibition of CYP2C9 (unknown origin) using tolbutamide as substrate
- ChEMBL_1903825 (CHEMBL4406047) Inhibition of CYP2C9 (unknown origin) using tolbutamide as substrate
- ChEMBL_1972823 (CHEMBL4605641) Inhibition of CYP2C9 (unknown origin) using tolbutamide as substrate
- ChEMBL_2265486 Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate
- ChEBML_1683156 Inhibition of CYP2C9 in human liver microsomes using tolbutamide or diclofenac as substrates
- ChEMBL_2103952 (CHEMBL4812455) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate
- ChEMBL_2151596 (CHEMBL5036058) Inhibition of CYP2C9 in human liver microsome using tolbutamide as substrate
- ChEMBL_2234461 (CHEMBL5148233) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate
- ChEMBL_813723 (CHEMBL2019981) Inhibition of CYP2C9 in human liver microsomes co-incubated with tolbutamide
- ChEMBL_875800 (CHEMBL2186746) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate
- ChEMBL_1362981 (CHEMBL3292549) Inhibition of CYP2C9 in human liver microsomes assessed as tolbutamide conversion to 4-hydroxy tolbutamide preincubated for 10 mins followed by NADPH addition measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1656440 (CHEMBL4005910) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate in presence of NADPH
- ChEMBL_1661022 (CHEMBL4010634) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate by HPLC analysis
- ChEMBL_52553 (CHEMBL666252) Inhibitory potential of human cytochrome P450 2C9 as Tolbutamide methylhydroxylation (100 uM)
- ChEMBL_738489 (CHEMBL1743390) Mechanism based inhibition of human cytochrome P450 2C19 evaluated using Tolbutamide
- ChEMBL_788301 (CHEMBL1919564) Inhibition of CYP2C9-mediated Tolbutamide methlyhydroxylation in human liver microsomes by LCMS analysis
- ChEMBL_813038 (CHEMBL2020550) Inhibition of human CYP2C9 using tolbutamide as substrate by LC/MS/MS analysis
- ChEMBL_2272996 Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate by LC-MS/MS analysis
- ChEMBL_874593 (CHEMBL2186636) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 10 mins
- ChEMBL_1781440 (CHEMBL4252957) Inhibition of CYP2C9 in human pooled human liver microsomes using tolbutamide as substrate assessed as tolbutamide 4-methylhydroxylation preincubated for 5 mins followed by NADPH addition measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1485704 (CHEMBL3541096) Inhibition of CYP2C9 in human liver microsomes using tolbutamide substrate by LC-MS/MS method
- ChEMBL_1576883 (CHEMBL3806592) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate by LC-MS/MS analysis
- ChEMBL_1653833 (CHEMBL4003199) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate by HPLC-MS/MS method
- ChEMBL_1655300 (CHEMBL4004666) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 5 to 15 mins
- ChEMBL_2149009 (CHEMBL5033407) Inhibition of human CYP2C9 using tolbutamide as substrate in presence of NADPH incubated for 15 to 45 mins
- ChEMBL_2156138 (CHEMBL5040798) Inhibition of CYP2C9 in human liver microsomes using Tolbutamide as substrate by LC-MS/MS analysis
- ChEMBL_690426 (CHEMBL1635163) Inhibition of CYP2C9 in human liver microsomes assessed as Tolbutamide 4'-hydroxylation after 60 mins
- ChEMBL_959659 (CHEMBL2382852) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate by LC-MS/MS analysis
- ChEMBL_1279430 (CHEMBL3096586) Inhibition of human CYP2C9 assessed as tolbutamide hydroxylation after 20 mins by LC-MS/MS analysis
- ChEMBL_1635604 (CHEMBL3878502) Inhibition of CYP2C9 in human liver microsomes assessed as tolbutamide 4-methylhydroxylation by LC/MS/MS analysis
- ChEMBL_2205589 (CHEMBL5118297) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate and measured by LC-MS/MS analysis
- ChEMBL_850904 (CHEMBL2156887) Inhibition of human CYP2C9 using tolbutamide as substrate after 15 mins by LC/MS/MS analysis
- ChEMBL_956255 (CHEMBL2379432) Inhibition of human CYP2C9 assessed as tolbutamide hydroxylation after 20 mins by LC/MS/MS analysis
- ChEBML_1682567 Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 30 mins by LC-MS/MS analysis
- ChEMBL_1584744 (CHEMBL3821695) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 20 mins by LC-MS analysis
- ChEMBL_1833052 (CHEMBL4333060) Inhibition of CYP2C9 in human liver microsomes assessed as reduction in tolbutamide 4-hydroxylation by tandem mass spectrometry analysis
- ChEMBL_2081965 (CHEMBL4737756) Inhibition of CYP2C9 in human liver microsomes using tolbutamide 4-hydroxylation as substrate by LC-MS/MS analysis
- ChEMBL_2082010 (CHEMBL4737801) Inhibition of CYP2C19 in human liver microsomes using tolbutamide 4-hydroxylation as substrate by LC-MS/MS analysis
- ChEMBL_2160241 (CHEMBL5044991) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 15 to 45 mins in presence of NADPH
- ChEMBL_434567 (CHEMBL914214) Activity at Kir6.2/SUR1 KATP channels expressed in HEK293 cells assessed as repolarization of tolbutamide-induced membrane depolarization
- ChEMBL_790067 (CHEMBL1925457) Inhibition of human B-lymphoblastoid cell microsomal CYP2C8 assessed as Tolbutamide hydroxylation preincubated for 5 mins with substrate
- ChEMBL_813724 (CHEMBL2019982) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 30 mins prior to substrate addition
- ChEMBL_1366894 (CHEMBL3297535) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 15 mins by LC/MS/MS analysis
- ChEMBL_1803977 (CHEMBL4276269) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 10 to 30 mins by LC/MS analysis
- ChEMBL_1823315 (CHEMBL4323079) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 15 mins in presence of NADPH by fluorescence method
- ChEMBL_1921505 (CHEMBL4424350) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 15 mins by LC-MS/MS analysis
- ChEMBL_1933018 (CHEMBL4478670) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 10 mins by LC/MS/MS analysis
- ChEMBL_2030246 (CHEMBL4684404) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 30 mins by LC-MS/MS analysis
- ChEMBL_2187308 (CHEMBL5099390) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 60 mins in presence of NADPH by HPLC analysis
- ChEMBL_690377 (CHEMBL1634994) Inhibition of CYP2C9 in human liver microsomes assessed as Tolbutamide 4-methylhydroxylation after 60 mins by Dixon plot analysis
- ChEMBL_790068 (CHEMBL1925458) Inhibition of human B-lymphoblastoid cell microsomal CYP2C9 (Arg) assessed as Tolbutamide hydroxylation preincubated for 5 mins with substrate
- ChEMBL_800479 (CHEMBL1948599) Inhibition of CYP2C9 in human liver microsomes using Tolbutamide as substrate after 60 mins by LC-MS/MS analysis
- ChEMBL_986816 (CHEMBL2438457) Inhibition of CYP2C9 in human liver microsomes assessed as tolbutamide hydroxylation after 20 mins by LC/MS/MS analysis
- ChEMBL_1713505 (CHEMBL4123554) Inhibition of CYP2C9 in human liver microsomes assessed as tolbutamide methylhydroxylation after 4 to 40 mins in presence of NADPH by LCMS analysis
- ChEMBL_1904190 (CHEMBL4406412) Inhibition of CYP2C9 in human pooled liver microsomes using tolbutamide as substrate after 10 mins by UPLC-MS/MS analysis
- ChEMBL_2069795 (CHEMBL4725048) Inhibition of human liver microsome CYP2C9 using tolbutamide as substrate incubated for 20 mins by LC-MS/MS with HPLC analysis
- ChEMBL_2073305 (CHEMBL4728839) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1660773 (CHEMBL4010385) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 60 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1760218 (CHEMBL4195226) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate after 15 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_1823224 (CHEMBL4322988) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 15 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_1904559 (CHEMBL4406781) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 40 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1992479 (CHEMBL4626214) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 20 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_2075860 (CHEMBL4731394) Inhibition of CYP2C9 in human pooled liver microsomes using tolbutamide as substrate measured up to 20 mins by UHPLC-MS/MS analysis
- ChEMBL_2103387 (CHEMBL4811890) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 15 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_2280220 Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate measured after 15 mins in the presence of NADPH by LC/MS/MS analysis
- ChEMBL_1859909 (CHEMBL4360765) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate measured after 10 mins in presence of NADPH by UHPLC-MS/MS analysis
- ChEMBL_2046445 (CHEMBL4701144) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate measured after 15 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2101712 (CHEMBL4810108) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate preincubated for 5 mins followed by NADPH addition by LC-MS/MS analysis
- ChEMBL_2277114 Inhibition of CYP2C9 (unknown origin) using tolbutamide as substrate incubated for 10 to 20 mins in presence of NADPH-generating system by LC-MS/MS analysis
- ChEMBL_306750 (CHEMBL830895) Inhibitory activity against cytochrome P450 determined using human liver microsome upon 15 min incubation with probes substrates Tolbutamide (2C9)
- ChEMBL_1972620 (CHEMBL4605438) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate measured for 10 to 30 mins in presence of NADPH regenerating system by UPLC-MS analysis
- ChEMBL_872613 (CHEMBL2183772) Inhibition of CYP2C9 in human liver microsomes assessed as decrease in formation of 4-hydroxytolbutamide from tolbutamide substrate after 60 mins by LC-MS/MS
- ChEMBL_1502034 (CHEMBL3588593) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as susbtrate incubated for 30 mins prior to substrate addition for 10 mins by LC-MS/MS analysis
- ChEMBL_1817312 (CHEMBL4316972) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate preincubated for 10 mins followed by NADPH addition and measured for 15 mins by LC-MS/MS analysis
- ChEMBL_2273350 Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate preincubated for 10 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1349492 (CHEMBL3270811) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 5 mins prior to substrate addition measured after 10 mins by LC/MS/MS analysis
- ChEMBL_1902874 (CHEMBL4405096) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate preincubated for 10 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
- ChEMBL_2014366 (CHEMBL4667944) Inhibition of recombinant human CYP2C9 expressed in baculovirus infected insect cells using tolbutamide as substrate incubated for 30 mins in presence of NADPH-regenerating system by fluorescence based assay
- ChEMBL_2212607 (CHEMBL5125556) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate preincubated for 5 mins followed by NADPH addition measured after 20 mins by LC-MS/MS analysis
- ChEMBL_2235522 (CHEMBL5149294) Inhibition of CYP2C9 in human liver microsome using tolbutamide as substrate preincubated for 10 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1750151 (CHEMBL4184911) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate preincubated for 10 mins with substrate followed by NADPH addition measured after 15 mins by LC/MS/MS analysis
- ChEMBL_2025120 (CHEMBL4678933) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate preincubated for 5 mins followed by NADPH generating system addition and measured after 120 mins by LC-MS/MS
- ChEMBL_2248035 (CHEMBL5162245) Inhibition of CYP2C9 in human liver microsomes tolbutamide as cocktail probe substrate preincubated for 5 mins followed by NADPH addition and measured after 30 mins by HPLC-MS/MS analysis
- ChEMBL_1805641 (CHEMBL4305000) Inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate preincubated for 5 mins followed by NADPH-generating system addition and measured after 20 mins by LC-MS/MS analysis
- ChEMBL_1805642 (CHEMBL4305001) Inhibition of CYP2D6 in human liver microsomes using tolbutamide as substrate preincubated for 5 mins followed by NADPH-generating system addition and measured after 20 mins by LC-MS/MS analysis
- ChEMBL_1904185 (CHEMBL4406407) Inhibition of CYP2C9 in human pooled liver microsomes using tolbutamide as substrate preincubated with NADPH for 30 mins followed by substrate addition and measured after 10 mins by UPLC-MS/MS analysis
- ChEMBL_959656 (CHEMBL2382849) Time-dependent inhibition of CYP2C9 in human liver microsomes using tolbutamide as substrate incubated for 30 mins prior to substrate addition measured after 30 mins by LC-MS/MS analysis
- ChEMBL_2064946 (CHEMBL4720199) Inhibition of human liver microsome CYP2C9 using tolbutamide as substrate incubated for 5 mins followed by NADPH addition and further incubated for 10 mins in shaking water bath by LC-MS/MS analysis
- Inhibition Assay Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration=0.25%) are incubated with human liver microsomes (1 mg/mL) and NADPH (1 mM) in the presence of the probe substrate tolbutamide (120 μM) for 60 min at 37° C. The selective CYP2C9 inhibitor, sulphaphenazole, is screened alongside the test compounds as a positive control.
- CYP450 Enzyme Inhibition Test Experimental Method: 4-hydroxydiclofenac (the substrate for CYP450 and 2C9 enzymes) and different doses of compounds were added into human liver microsome (Xenotech, LLC), then NADPH (Reduce dcoenzyme II, Chem-impex international, Inc.) was added, mixed, and then incubated in 37° C. water bath, at the terminal time, the stop solution (200 ng/mL tolbutamide and 200 ng/mL labetalol dissolved in acetonitrile) was added to stop the reaction, methanol or ethanol was used to precipitate proteins, the concentration of the metabolites of substrates was determined by using LC-MS/MS to obtain the IC50 values of compounds on CYP450 and 2C9 enzyme.
- Inhibition Assay Each five kinds of substrates, human hepatic microsome, and a test drug in 50 mmol/L Hepes buffer as a reaction solution was added to a 96-well plate as the composition ad described above, NADPH, as a coenzyme was added to initiate metabolism reactions as markers and, after the incubation at 37° C. for 15 minutes, a methanol/acetonitrile=1/1 (V/V) solution was added to stop the reaction. After the centrifugation at 3000 rpm for 15 minutes, resorufin (CYP1A2 metabolite) in the supernatant was quantified by a fluorescent multilabel counter and tolbutamide hydroxide (CYP2C9 metabolite), mephenytoin 4′ hydroxide (CYP2C19 metabolite), dextrorphan (CYP2D6 metabolite), and terfenadine alcohol (CYP3A4 metabolite) were quantified by LC/MS/MS.
- CYP Activity Assay Inhibition of hepatic cytochromes P450 was assessed in human liver microsomes using a substrate-specific approach of monitoring metabolites formed by each specific CYP enzyme. Metabolic reactions included phenacetin-O-deethylation (1A2), tolbutamide methylhydroxylation (2C9), S-mephenytoin 4'-hydroxylation (2C19), dextrome-thorphan-O-deethylation (2D6), and testosterone-6-β-hydroxylation (3A4). Each substrate was added at a concentration less than or equal to the Km for themetabolic pathway, and microsomal protein concentrations and assay incubation times were optimized for each reaction to ensure linear metabolite formation based on initial rates (Table 1). Microsomes were suspended in phosphate buffer and incubated at 37 °C in the presence of probe substrates. Reactions were initiated by the addition of an NADPH-regenerating buffer system and were quenched at the appropriate times using acetonitrile. Samples were centrifuged and concentrations of metabolites assessed by LC/MS.
- Inhibition Assay Compounds were assessed for inhibition (IC50, n=2) of CYP2C8, CYP2C9 and CYP3A4 in pooled human liver microsomes using selective probe substrates at their previously determined Km values (CYP2C8: paclitaxel, 4 μM; CYP2C9: diclofenac, 5 μM; CYP3A4: midazolam, 0.5 μM). Incubations contained 0.1 mg/mL human liver microsomes, 3 mM MgCl2, probe substrate and various concentrations of inhibitor (12-point IC50 curve) in 100 mM potassium phosphate buffer (pH 7.4). Concentrations of organic solvents were kept to <1% (v/v). All incubations were pre-incubated at 37° C. for 5 minutes prior to addition of 1 mM NADPH (final concentration). Incubations were stopped after 5 (CYP3A4) or 15 minutes (CYP2C8 and CYP2C9) with one volume (v/v) of ice-cold acetonitrile containing 0.1 μM tolbutamide as an internal standard. All samples were vortexed and centrifuged prior to LC-MS/MS analysis.
- CYP1A2 Inhibition Assay CYP1A2 Inhibition in Human Liver Microsomes. Pooled human liver microsomes were incubated with individual CYP, CYP1A2, isozyme-specific marker substrate (Phenacetin) in the presence of test compound at various concentrations (0.05, 0.15, 0.5, 1.5, 5, 15, 50 uM). The specific marker metabolites are measured with LC/MS/MS. The remaining enzymatic activities and inhibitory potency IC50 are determined. Procedure: Microsomes are removed out of the −80° C. freezer to thaw on ice and 20 μL of the substrates solution added to the corresponding wells. Then 20 μL PB was added to blank wells and 2 μL of the test compounds and positive control working solution added to the corresponding wells. 2 μL of solvent was added to No Inhibitor wells and Blank wells. Then 158 μL of the HLM working solution was added to all wells of the incubation plate. The plate was pre-warmed for about 10 min using a 37° C. water bath. Then 20 μL of the NADPH cofactor solution was added to all incubation wells, mixed and incubated for 10 minutes at 37° C. water bath. The reaction is terminated by adding 400 μL cold stop solution (200 ng/mL Tolbutamide and 200 ng/mL Labetalol in ACN). The samples were centrifuged at 4000 rpm for 20 minutes to precipitate protein. Finally 200 μL of the supernatant was transferred to 100 μL HPLC water, shaken for 10 min and analyzed by LC/MS/MS.
- Cytochrome P450 Isoenzyme Inhibitory Activity Test The inhibitory activities of test compound against different isoforms of human cytochrome P450 isoenzymes were determined. Experimental Operation. The test compound, standard inhibitor (100×final concentration) and mixed substrate working solution were prepared; the microsome frozen in −80° C. refrigerator was taken out and thawed. 2 μL of the compound to be tested and standard inhibitor solution were added to the corresponding wells, and at the same time, 2 μL of the corresponding solvent was added to the non-inhibitor control wells (NIC) and the blank control wells; secondly, 20 μL of mixed substrate solution was added to the corresponding wells except the blank wells (20 μL of Pb was added to the blank wells); human liver microsome solution was prepared (the solution was put back in the refrigerator immediately after using and marking the date), and then 158 μL of human liver microsome solution was added to all wells; the sample plate was put in a 37° C. water bath for pre-incubation, and then a coenzyme factor (NADPH) solution was prepared; after 10 minutes, 20 μL of NADPH solution was added to all wells, the sample plate was shaken well, and incubated in a 37° C. water bath for 10 minutes; at the corresponding time point, 400 μL of cold acetonitrile solution (internal standard is 200 ng/mL tolbutamide and labetalol) was added to terminate the reaction; after the plates were evenly mixed, the mixture was centrifuged at 4000 rpm for 20 minutes to precipitate protein; 200 μL of supernatant was added into 100 μL of water, shaken well and detected by LC/MS/MS.
- Inhibition Against Cytochrome P450 Isoenzymes Table 9: The inhibition of the test compound against different subtypes of the human cytochrome P450 isoenzyme was determined. The test compound, a standard inhibitor (100×final concentration) and a mixed substrate working solution were prepared; the microsomes frozen in a refrigerator at −80° C. were taken out and thawed. 2 μL of a solution of the test compound and the standard inhibitor was added to corresponding wells, and 2 μL of a corresponding solvent was added to a non-inhibitor control (NIC) well and a blank control (Blank) well; then, 20 μL of a solution of mixed substrate was added to corresponding wells except for the Blank well (adding 20 μL of PB to the Blank well); a human liver microsome solution (labeled with the date after use and immediately putting back to a refrigerator) was prepared and then added to all wells at 158 μL per well; the sample plate was put into a 37° C. water bath for pre-incubation, and then a coenzyme factor (NADPH) solution was prepared; after 10 min, the NADPH solution was added to all the wells at 20 μL per well, and the sample plate was shaken to mix well and then incubated in a 37° C. water bath for 10 min; at corresponding time points, 400 μL of cold acetonitrile solution (internal standard: 200 ng/mL tolbutamide and labetalol) was added to stop the reaction; after being mixed well, the mixture in the sample plate was centrifuged at 4,000 rpm for 20 min, and proteins were precipitated; 200 μL of supernatant was collected and added into 100 μL of water, and the mixture was mixed well and then analyzed by LC/MS/MS.
- GCS Enzymatic Assay This assay was modified based on the study by Larsen et al. (J. Lipid Res. 2011, 53, 282). Madin-Darby canine kidney (MDCK) cell lysate was prepared using M-PER Mammalian Protein Extraction Reagent (Thermal Scientific) in the presence of a protease inhibitor cocktail (Roche). Protein concentration was determined using BCA assay kit (Pierce). Sixty micrograms of MDCK cell lysate was incubated with various concentrations of a compound described herein from 0.001 μM-10 μM, respectively, or as indicated in Table 2, in 100 mM Tris buffer (pH 7.5) containing 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 2 mM NAD, 100 μM UDP-glucose, 10 μM C6-NBD-Ceramide (Matreya LLC, Pleasant Gap, Pa.), 35 μM dioleoylphosphatidylcholine and 5 μM sulfatide (Sigma) in a final reaction volume of 100 μL at 37° C. for 1 hour. 0.1% DMSO was used as mock treatment or control. The reaction was terminated by adding 100 μL acetonitrile solution and subjected to LC/MS analysis.The quantitative analysis of NBD-Ceramide and glucosylceramide was performed on a Shimadzu ultra-fast liquid chromatography (Shimadzu, Japan) coupled with API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Concord, Ontario, Canada). Sample separation was conducted on a Waters Xbridge BEH130 C18, 100 mm×4.6 mm i.d, 3.5 μm (Milford, Mass., USA). The mobile phase consisted of water and acetonitrile supplemented with 0.1% formic acid (v/v). The flow rate was 1.0 mL/min. The initial mobile phase was 20% acetonitrile and was ramped in a linear fashion to 50% acetonitrile in 0.4 min. From 0.4 to 1.5 min, the gradient was ramped to 98% acetonitrile, and then was held at 100% until 8.0 min. Acetonitrile was reset to 20% in 1.5 min, and maintained until 10.0 min. The total run time was 10.0 min. The MS/MS detection was performed in ESI positive mode. The mass transition of NBD-Ceramide was m/z 576.36→558.40 under the collision energy of 15 V, and the mass transition of glucosylceramide was m/z 738.35-558.40 under 21V collision energy. The cell lysate was diluted with equal volume of acetonitrile. Aliquots of 50 μL diluted samples were added to 1.5 mL tubes, and 100 μL of acetonitrile containing internal standard (100 ng/mL tolbutamide) were added for protein precipitation. The mixture were vortexed and then centrifuged at 13000 rpm for 10 min. 70 μL of supernatant were mixed with 140 μL of H2O and the final solution were injected for LC/MS/MS analysis and IC50's and/or percent inhibitions calculated.