BDBM134481 US8846656, 17-AP
BDBM204982 US9249085, I(ap)
US10106501, Example AP BDBM293080
US20230357139, Compound AP BDBM634024
albaspidin-AP BDBM50191691 CHEMBL439501
US10155732, Compound 3-AP Triapine BDBM50198892 3-AP 3-aminopyridine-2-carboxaldehyde thiosemicarbazone
BDBM292719 US10106501, Example AP-6
BDBM747347 US12325697, Compound AP-1
US10106501, Example AP-1 BDBM293081
US20240092799, Compound XXIX-AP BDBM660462
US9890185, Example 15(ap) BDBM381052
US9890185, Example 38(ap) BDBM381119
BDBM263254 US9708370, 65 CP-AP-5
BDBM728215 US20250092029, Compound AP-8-011
US11866416, Compound AP-I-240 BDBM644034
US20250092029, Compound AP-8-001 BDBM728213
US20250092029, Compound AP-8-012 BDBM728216
US20250092029, Compound AP-8-013 BDBM728212
US9708370, CP-AP-2 CHEMBL2163462 BDBM50394987
US9708370, CP-AP-3 CHEMBL2163461 BDBM50394988
BDBM50394989 US9708370, 62 CP-AP-4 CHEMBL2163460
Bms-986020 AP-3152 FREE ACID BDBM50581550
BDBM50260265 C-16-(S)-7-methylindolerapamycin AP-21967 CHEMBL525042
4-AP DALFAMPRIDINE BDBM10458 pyridin-4-amine CHEMBL284348 4-Aminopyridine 10
Brigatinib BDBM50185140 AP-26113 US11248003, Example Brigatinib US20230322822, Compound Brigatinib US20240409609, Compound Ref. 1
N-(7-((3r,5r,7r)-adamantan-1-ylmethoxy)-6-cyclopropyl-[1,2,4]triazolo[4,3-a]pyridin-3-0)-AP-methylsulfuric diamide) BDBM268549 US9550775, 73
3-{4-[(benzylethylamino)methyl]phenyl}-6,7-dimethoxychromen-2-one BDBM50218528 AP-2243 3-(4-((benzyl(ethyl)amino)methyl)phenyl)-6,7-dimethoxy-2H-chromen-2-one CHEMBL244416
AP-23464 BDBM50314074 3-(2-(2-cyclopentyl-6-(4-(dimethylphosphoryl)phenylamino)-9H-purin-9-yl)ethyl)phenol CHEMBL1089405 3-[2-(2-CYCLOPENTYL-6-{[4-(DIMETHYLPHOSPHORYL)PHENYL]AMINO}-9H-PURIN-9-YL)ETHYL]PHENOL 2,6,9-Trisubstitute purine, 6 (AP23464)
3-(2-(6-(cyclopropylamino)-9H-purin-9-yl)vinyl)-4-methyl-N-(4-(trifluoromethyl)pyridin-2-yl)benzamide AP-24226 (E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-methyl-N-(4-(trifluoromethyl)pyridin-2-yl)benzamide CHEMBL556874 BDBM50294010
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-methyl-N-(3-(4-methyl-1-imidazol-1-yl)-5-(trifluoromethyl)phenyl)benzamide CHEMBL539433 3-(2-(6-(cyclopropylamino)-9H-purin-9-yl)vinyl)-4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)benzamide (E)-3-(2-(6-(cyclopropylamino)-9H-purin-9-yl)vinyl)-4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)-phenyl)benzamide AP24163 BDBM50294011 3-{(E)-2-[6-(cyclopropylamino)-9H-purin-9-yl]ethenyl}-4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]benzamide AP-24163
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- ChEMBL_2376877 Antagonist activity at PAR2 (unknown origin)
- ChEMBL_2376876 Binding affinity to PAR2 (unknown origin) assessed as inhibition constant
- ChEMBL_2316142 Antagonist activity at PAR2 (unknown origin) expressed in human 1321N1 cells
- ChEMBL_984154 (CHEMBL2432082) Agonist activity at PAR2 (unknown origin) by cell-based assay
- ChEMBL_1458006 (CHEMBL3367180) Inhibition of AP-1 (unknown origin) binding to AP-1 oligonucleotide by gel shift assay
- ChEMBL_2459768 Inhibition of human recombinant CES2 activity assessed as fluorescence of the AP product using Benz-AP as substrate
- ChEMBL_449273 (CHEMBL899535) Agonist activity at PAR2 expressed in human HT29 cells assessed as intracellular calcium release
- ChEMBL_984152 (CHEMBL2432080) Agonist activity at PAR2 in human 16HBE14o- cells assessed as increase in MAPK level
- ChEMBL_984132 (CHEMBL2434653) Agonist activity at PAR2 in human platelets by platelet aggregation assay in presence of SFLLRN
- ChEMBL_984153 (CHEMBL2432081) Agonist activity at PAR2 in human 16HBE14o- cells assessed as increase in intracellular calcium level
- ChEMBL_1981506 (CHEMBL4614768) Inhibition of human AP protein factor D
- ChEMBL_28882 (CHEMBL645952) Ability to antagonise AP-1 transcriptional activity
- ChEMBL_2459782 Mixed type of inhibition at human recombinant CES2 activity assessed as fluorescence of the AP product using Benz-AP as substrate
- ChEMBL_2459783 Competitive type of inhibition at human recombinant CES2 activity assessed as fluorescence of the AP product using Benz-AP as substrate
- ChEMBL_674258 (CHEMBL1274355) Agonist activity at human PAR2 receptor in HEK293 cells assessed as induction of intracellular calcium release
- ChEMBL_984138 (CHEMBL2434659) Partial agonist activity at PAR2 in human SW620 cells assessed as increase in intracellular calcium release
- ChEMBL_984155 (CHEMBL2432083) Agonist activity at PAR2 in human HT-29 cells assessed as increase in intracellular calcium release
- ChEMBL_2277301 Inhibition of PAR2 expressed in human HT-29 cells incubated for 30 mins by FLIPR Tetra system method
- ChEMBL_674259 (CHEMBL1274356) Agonist activity at PAR2 receptor in human HT-29 cells assessed as induction of intracellular calcium release
- ChEMBL_674260 (CHEMBL1274357) Antagonist activity at PAR2 receptor in human HT-29 cells assessed as inhibition of intracellular calcium release
- ChEMBL_984143 (CHEMBL2432071) Antagonist activity at PAR2 in human HT-29 cells assessed as inhibition of agonist-induced calcium mobilization
- ChEMBL_2311808 Inhibition of recombinant AAK1 expressed in human HEK293F cells co-expressed with AP-2 assessed as reduction in AP-2 phosphorylation by western blotting
- ChEMBL_1458011 (CHEMBL3367185) Inhibition of AP-1 (unknown origin) by FRET assay
- ChEMBL_1458012 (CHEMBL3367186) Inhibition of TPA-induced AP-1 activation (unknown origin)
- ChEMBL_28877 (CHEMBL645947) AP-1 suppression activity using freshly prepared DMSO stock
- ChEMBL_305353 (CHEMBL832733) Inhibitory activity against transcription activator protein-1 (AP-1)
- ChEMBL_984140 (CHEMBL2432068) Antagonist activity at PAR2 in human HT-29 cells assessed as inhibition of trypsin-induced intracellular calcium release
- ChEMBL_984141 (CHEMBL2432069) Antagonist activity at PAR2 in human HCT15 cells assessed as inhibition of SLIGKV-NH2-induced intracellular calcium release
- ChEMBL_984142 (CHEMBL2432070) Antagonist activity at PAR2 in human HaCaT cells assessed as inhibition of SLIGKV-NH2-induced intracellular calcium release
- ChEMBL_984148 (CHEMBL2432076) Antagonist activity at rat PAR2 expressed in rat KNRK cells assessed as inhibition of trypsin-induced receptor activation
- ChEMBL_1549745 (CHEMBL3757301) Displacement of europium-labeled 2f-LIGRLO-NH2 from human PAR2 expressed in CHO cells after 1 to 2 hrs
- ChEMBL_984129 (CHEMBL2434650) Antagonist activity at PAR2 in human neutrophils assessed as inhibition of chemotactic migration of cells towards trypsin/SLIGRL-NH2
- ChEMBL_984135 (CHEMBL2434656) Agonist activity at human PAR2 expressed in African green monkey COS7 cells assessed as stimulation of inositol triphosphate production
- ChEMBL_984149 (CHEMBL2432077) Agonist activity at human PAR2 expressed in HEK293T cells assessed as increase in intracellular calcium level by bioluminescence assay
- ChEMBL_28878 (CHEMBL645948) AP-1 suppression activity using one month old DMSO stock
- ChEMBL_28879 (CHEMBL645949) AP-1 suppression activity using one week old DMSO stock
- ChEMBL_28880 (CHEMBL645950) AP-1 suppression activity using six months old DMSO stock
- ChEMBL_28881 (CHEMBL645951) AP-1 suppression activity using two months old DMSO stock
- ChEMBL_754808 (CHEMBL1805663) Transrepression activity of glucocorticoid receptor in human A549 cells expressing AP-1 assessed as inhibition of PMA-induced AP-1 activity by luciferase reporter gene assay
- ChEMBL_511265 (CHEMBL996091) Agonist activity at human PAR2 expressed in HEK293T cells assessed as effect on intracellular calcium mobilization by R-SAT assay
- ChEMBL_523563 (CHEMBL1001577) Antagonist activity at human recombinant PAR2 expressed in A549 cells assessed as inhibition of SLIGKV-NH2-induced intracellular calcium mobilization
- ChEMBL_984139 (CHEMBL2432067) Antagonist activity at PAR2 in human HT-29 cells assessed as inhibition of 2f-LIGRLO-NH2-induced intracellular calcium release
- ChEMBL_984147 (CHEMBL2432075) Displacement of [3H]2-furoyl-LIGRL-NH2 from human PAR2 expressed in human NCTC-2544 cells by scintillation counting analysis
- ChEMBL_1664557 (CHEMBL4014238) Agonist activity at human GPR34 expressed in HEK293 cells co-transfected with AP-TGFalpha/chimeric Galphaq/i1 assessed as induction of AP-TGFalpha release after 1 hr
- ChEMBL_1772742 (CHEMBL4229734) Inhibition of C3bi-AP binding to CR3 derived from human PMN
- ChEMBL_2237843 (CHEMBL5151739) Agonist activity at mouse GPR174 transfected in human HEK293FT cells co-transfected with AP-TGFalpha assessed as AP-TGFalpha release incubated for 90 mins by TGFalpha shedding assay
- ChEMBL_2045584 (CHEMBL4700283) Agonist activity at PAR2 in HUVEC cells assessed as stimulation of calcium mobilization by Fluo-4-AM dye-based FLIPR assay
- ChEMBL_736225 (CHEMBL1693369) Agonist activity at PAR2 in human 16HBE14o cells assessed as increase in intracellular calcium levels by in vitro physiological response assay
- ChEMBL_2237845 (CHEMBL5151741) Partial agonist activity at mouse GPR174 transfected in human HEK293FT cells co-transfected with AP-TGFalpha assessed as AP-TGFalpha release incubated for 90 mins by TGFalpha shedding assay
- ChEMBL_2350966 Agonist activity at N-terminal FLAG-tagged human LPS2/P2Y10 V159F mutant transfected in HEK293-A cells co-transfected with AP-TGFalpha assessed as AP-TGFalpha release by TGFalpha shedding assay
- ChEMBL_2350968 Agonist activity at N-terminal FLAG-tagged human LPS2/P2Y10 A163F mutant transfected in HEK293-A cells co-transfected with AP-TGFalpha assessed as AP-TGFalpha release by TGFalpha shedding assay
- ChEMBL_2045583 (CHEMBL4700282) Agonist activity at PAR2 in human EA.hy926 cells assessed as stimulation of calcium mobilization by Fluo-4-AM dye-based FLIPR assay
- ChEMBL_984136 (CHEMBL2434657) Antagonist activity at human PAR2 expressed in African green monkey COS7 cells assessed as inhibition of SLIGKV-NH2-induced inositol triphosphate production
- ChEMBL_984150 (CHEMBL2432078) Agonist activity at human PAR2 expressed in HEK293T cells assessed as stimulation of phosphatidylinositol hydrolysis after 45 mins by scintillation counting analysis
- ChEMBL_984151 (CHEMBL2432079) Agonist activity at human PAR2 expressed in HEK293T cells assessed as stimulation of cell proliferation after 5 days by beta-galactosidase assay
- ChEMBL_1664555 (CHEMBL4014236) Agonist activity at mouse GPR34 expressed in HEK293 cells co-transfected with AP-TGFalpha/chimeric Galphaq/i1 assessed as induction of ectodomain shedding of membrane bound AP-TGFalpha after 1 hr
- ChEMBL_984156 (CHEMBL2432084) Displacement of [3H]-2-furoyl-LIGRL-NH2 from human PAR2 expressed in human NCTC-2544 cells after 60 mins by scintillation counting analysis
- ChEMBL_1458004 (CHEMBL3367178) Inhibition of AP-1 bZIP domain DNA binding activity (unknown origin) by ELISA
- ChEMBL_1458013 (CHEMBL3367187) Inhibition of UVB-induced AP-1 activation (unknown origin) by cell-based assay
- ChEMBL_1624990 (CHEMBL3867402) Agonist activity at human PAR2 expressed in CHO cells assessed as induction of ERK1/2 phosphorylation incubated for 10 mins measured after 2 hrs
- ChEMBL_1985825 (CHEMBL4619231) Agonist activity at human PAR2 expressed in HEK293T cells assessed as induction of Galphaq-stimulated IP1 formation incubated for 2 hrs by FRET assay
- ChEMBL_984144 (CHEMBL2432072) Antagonist activity at human PAR2 expressed in human A549 cells coexpressing TACR1 assessed as inhibition of substance P-induced IL-8 production by ELISA
- ChEMBL_1458007 (CHEMBL3367181) Inhibition of AP-1-mediated collagenase production in IL1alpha-stimulated Lewis rat synovial cells
- ChEMBL_92286 (CHEMBL705832) Inhibition of AP-1 (activator protein-1) mediated transcriptional activation in Jurkat T-cells
- ChEMBL_984145 (CHEMBL2432073) Antagonist activity at human PAR2 expressed in human A549 cells assessed as inhibition of 2f-LIGRLO-NH2-induced NFkappaB activation by luciferase reporter gene assay
- ChEMBL_1458002 (CHEMBL3367176) Inhibition of AP-1-mediated transcriptional activation in HEK293 cells by luciferase reporter gene assay
- ChEMBL_2311807 Inhibition of recombinant AAK1 (unknown origin) expressed in baculovirus assessed as reduction in AP-2 phosphorylation
- ChEMBL_1624984 (CHEMBL3867396) Antagonist activity at PAR2 in human PC3 cells assessed as inhibition of endogenous trypsin-stimulated intracellular calcium release by Fluo-3 AM dye based FLIPR assay
- ChEMBL_1497755 (CHEMBL3583014) Antagonist activity against PAR2 in human HT29 cells assessed as inhibition of SLIGKV-induced Ca2+ responses pre-incubated for 30 mins before SLIGKV stimulation by FLIPR assay
- ChEMBL_1497756 (CHEMBL3583015) Antagonist activity against PAR2 in human HT29 cells assessed as inhibition of trypsin-induced Ca2+ responses pre-incubated for 30 mins before trypsin stimulation by FLIPR assay
- ChEMBL_1497757 (CHEMBL3583016) Antagonist activity against PAR2 in human HT29 cells assessed as inhibition of thrombin-induced Ca2+ responses pre-incubated for 30 mins before thrombin stimulation by FLIPR assay
- ChEMBL_1497758 (CHEMBL3583017) Antagonist activity against PAR2 in human HT29 cells assessed as inhibition of UTP-induced Ca2+ responses pre-incubated for 30 mins before UTP stimulation by FLIPR assay
- ChEMBL_1624986 (CHEMBL3867398) Antagonist activity at PAR2 in human HT-29 cells assessed as inhibition of endogenous trypsin-stimulated intracellular calcium release by Fluo-3 AM dye based FLIPR assay
- ChEMBL_1985827 (CHEMBL4619233) Agonist activity at PK1-tagged PAR2 (unknown origin) expressed in HEK293 cells assessed as induction of beta-arrestin recruitment incubated for 90 mins by Pathhunter chemiluminescent assay
- ChEMBL_1457997 (CHEMBL3367171) Inhibition of AP-1-mediated transcriptional activation in human Jurkat cells by luciferase reporter gene assay
- ChEMBL_162726 (CHEMBL765030) Inhibition of [14C]aminopyrine (AP) accumulation stimulated by dibutyryl cyclic AMP in isolated rabbit parietal cells
- ChEMBL_2073593 (CHEMBL4729127) Agonist activity at mouse GPR34 expressed in HEK293 cells co-transfected with AP-TGFalpha/Galphaq/i1 assessed as induction of ectodomain shedding of membrane bound AP-TGFalpha after 1 hr by p-NPP substrate based microplate reader analysis
- ChEMBL_2073596 (CHEMBL4729130) Agonist activity at mouse P2Y10 expressed in HEK293 cells co-transfected with AP-TGFalpha/Galphaq/i1 assessed as induction of ectodomain shedding of membrane bound AP-TGFalpha after 1 hr by p-NPP substrate based microplate reader analysis
- ChEMBL_2073600 (CHEMBL4729134) Agonist activity at human GPR34 expressed in HEK293 cells co-transfected with AP-TGFalpha/Galphaq/i1 assessed as induction of ectodomain shedding of membrane bound AP-TGFalpha after 1 hr by p-NPP substrate based microplate reader analysis
- ChEMBL_2237847 (CHEMBL5151743) Antagonist activity at mouse GPR174 transfected in human HEK293FT cells co-transfected with AP-TGFalpha assessed as inhibition of LysoPalloT-NH-C3-ph-m-O-C11-induced AP-TGFalpha release incubated for 90 mins by TGFalpha shedding assay
- ChEMBL_2237849 (CHEMBL5151745) Inverse agonist activity at mouse GPR174 transfected in human HEK293FT cells co-transfected with AP-TGFalpha assessed as inhibition of LysoPalloT-NH-C3-ph-m-O-C11-induced AP-TGFalpha release incubated for 90 mins by TGFalpha shedding assay
- ChEMBL_1549740 (CHEMBL3757296) Antagonist activity at PAR2 in human HT-29 cells assessed as inhibition of trypsin-induced ca2+ release preincubated for 15 mins measured after 1 hr by FLIPR assay
- ChEMBL_1651136 (CHEMBL4000391) Agonist activity at human PAR2 expressed in CHO cells assessed as induction of intracellular calcium release measured for 300 secs by Fluo-3 AM dye-based fluorescence assay
- ChEMBL_736221 (CHEMBL1693365) Agonist activity at PAR2 in human 16HBE14o cells assessed as increase in intracellular calcium levels measured every 15 second for 2 hrs by xCelligence real time cell analyzer
- ChEMBL_28876 (CHEMBL645946) Concentration required to inhibit activator protein-1(AP-1) mediated transcriptional activation in human Jurkat T-cells
- ChEMBL_2073602 (CHEMBL4729136) Agonist activity at zebrafish GPR34 type a expressed in HEK293 cells co-transfected with AP-TGFalpha/Galphaq/i1 assessed as induction of ectodomain shedding of membrane bound AP-TGFalpha after 1 hr by p-NPP substrate based microplate reader analysis
- ChEMBL_2073604 (CHEMBL4729138) Agonist activity at zebrafish GPR34 type b expressed in HEK293 cells co-transfected with AP-TGFalpha/Galphaq/i1 assessed as induction of ectodomain shedding of membrane bound AP-TGFalpha after 1 hr by p-NPP substrate based microplate reader analysis
- ChEMBL_1458005 (CHEMBL3367179) Inhibition of AP-1 (unknown origin) expressed in TPA-stimulated mouse NIH3T3 cells by luciferase reporter gene assay
- ChEMBL_1750384 (CHEMBL4185144) Inhibition of nuclease activity of APE1 in human glioblastoma cells assessed as reduction in AP site DNA cleavage
- ChEMBL_35369 (CHEMBL647950) Inhibitory potency against purified membrane bound rat brain Aminopeptidase M (AP-M) using [3H]Leu-enkephalin as substrate
- ChEMBL_2077653 (CHEMBL4733444) Inhibition of PAR1 in human whole blood assessed as inhibition of PAR1 AP (Ala-Phe (4fluro) Arg-Cha-homo Arg-Tyr-NH2)-induced platelet aggregation preincubated for 10 mins followed by PAR1 AP (Ala-Phe (4fluro) Arg-Cha-homo Arg-Tyr-NH2) stimulation
- ChEMBL_1549738 (CHEMBL3757294) Antagonist activity at PAR2 in human HT-29 cells assessed as inhibition of 2f-LIGRLO-NH2-induced ca2+ release preincubated for 15 mins measured after 1 hr by FLIPR assay
- ChEMBL_1459134 (CHEMBL3370895) Agonist activity at glucocorticoid receptor in human A549 cells assessed as AP-1 transrepression by luciferase reporter gene assay
- ChEMBL_164920 (CHEMBL770631) Antisecretory activity evaluated by the inhibition of 14C -AP uptake in isolated rabbit parietal cells stimulated by exogenous histamine
- ChEMBL_35346 (CHEMBL643875) Inhibitory potency against purified membrane bound rat brain Aminopeptidase B (AP-B) using L-lysine-beta napthylamide as substrate
- ChEMBL_1713912 (CHEMBL4123961) Antagonist activity at PAR2 in human HEK293 cells assessed as inhibition of trypsin-induced intracellular calcium flux pretreated for 15 mins followed by trypsin addition by Fluo-8 dye based fluorescence assay
- ChEMBL_2045587 (CHEMBL4700286) Agonist activity at PAR2 in human EA.hy926 cells assessed as stimulation of calcium mobilization treated with cRGD for 30 mins prior to compound addition by Fluo-4-AM dye-based FLIPR assay
- ChEMBL_1565175 (CHEMBL3782567) Inhibition of human GST tagged Tcf4/beta catenin interaction after 2 hrs using fluorescent AP Attophos substrate by ELISA analysis
- ChEMBL_1883558 (CHEMBL4385057) Inhibition of AP-1 (unknown origin) expressed in mouse JB6 Cl41 cells after 24 hrs by luciferase reporter gene assay
- ChEMBL_1713915 (CHEMBL4123964) Antagonist activity at PAR2 in human 1321N1 cells assessed as inhibition of trypsin-induced intracellular calcium flux pretreated for 60 mins followed by trypsin addition by Fluo-8 NW dye based FLIPR assay
- ChEMBL_1812465 (CHEMBL4311925) Antagonist activity at PAR2 in human EAhy926 cells assessed as inhibition of trypsin-induced intracellular calcium mobilization preincubated for 15 mins followed by trypsin addition by Fluo-4-AM dye based fluorescence assay
- Cellular Firefly Luciferase Assay (AP-1) Curcumin is a known inhibitor of the AP-1 activation cascade. Therefore, modification of the structure of curcumin could lead to analogs with enhanced activity. The library consisting of three series of curcumin analogs were used to examine the role of the enone functionality in aryl systems where the spacer is 7-carbons (as in curcumin), 5-carbons or 3-carbons in length. In addition, the importance of aryl ring substituents was assessed. The AP-1 activities of curcumin and analogs were determined by a cellular firefly luciferase assay. This assay utilized a commercially available cell line (Panomics 293-luc cellular assay) developed for screening inhibitors of AP-1. This cell line is stably transfected with a luciferase reporter controlled by an AP-1 dependent promoter. The cell is stimulated with phorbol ester which activates AP-1. AP-1 then binds to one of three promoter regions on the cells DNA leading to the production of a luciferase enzyme. Luciferin is added to the cell lysates and the luciferase enzyme catalyzes a cleavage of luciferin leading to the emission of light.
- ChEMBL_978866 (CHEMBL2423657) Inhibition of APE-1 (unknown origin) using double-stranded AP-site containing DNA as substrate after 30 mins by fluorescence assay
- ChEMBL_1624991 (CHEMBL3867403) Agonist activity at human PAR2 expressed in CHO cells assessed as inhibition of forskolin-induced cAMP accumulation incubated for 20 mins followed by forskolin addition for 10 mins measured after 1 hr by LANCE assay
- ChEMBL_1812457 (CHEMBL4311917) Antagonist activity at PAR2 in human EAhy926 cells assessed as inhibition of SLIGKV-NH2-induced intracellular calcium mobilization preincubated for 15 mins followed by SLIGKV-NH2 addition by Fluo-4-AM dye based fluorescence assay
- ChEBML_35366 Compound was evaluated for its inhibitory potency against purified membrane bound rat brain Aminopeptidase M (AP-M) using [3H]Leu-enkephalin as substrate
- ChEMBL_762451 (CHEMBL1816721) Inhibition of 12-lipoxygenase in human platelets assessed as reduction of PAR1-AP-induced 12 HETE production by LC-MS/MS analysis
- ChEMBL_816306 (CHEMBL2026982) Inhibition of human recombinant AP-N ectopeptidase activity using alanine-MCA as substrate preincubated for 10 mins with substrate by fluorimetric analysis
- ChEMBL_983187 (CHEMBL2428099) Transrepression of glucocorticoid receptor in human A549 cells assessed as inhibition of PMA-induced AP-1 activity by luciferase reporter gene assay
- ChEMBL_1713916 (CHEMBL4123965) Antagonist activity at PAR2 in human HEK293 cells assessed as inhibition of SLIGRL-induced intracellular calcium flux preincubated for 10 mins followed by SLIGRL-induction measured after 24 hrs by Fluo4-AM dye-based fluorescence assay
- ChEMBL_2045586 (CHEMBL4700285) Agonist activity at PAR2 in human EA.hy926 cells assessed as stimulation of calcium mobilization preincubated for 2 hrs with LPS followed by compound addition in absence of cRGD by Fluo-4-AM dye-based FLIPR assay
- ChEMBL_1435253 (CHEMBL3390699) Antagonist activity at PAR4 in PAR-4-AP-stimulated human platelets compound pretreated for 5 mins by fluorescent PAC1 integrin alpha2bb3 activation assay
- ChEMBL_1435254 (CHEMBL3390700) Antagonist activity at PAR1 in PAR-1-AP-stimulated human platelets compound pretreated for 5 mins by fluorescent PAC1 integrin alpha2bb3 activation assay
- ChEMBL_35363 (CHEMBL874110) Compound was evaluated for the inhibition of Aminopeptidase M (AP-M) and the inhibition constant was determined after preincubating the enzyme and inhibitor
- ChEMBL_982006 (CHEMBL2428210) Agonist activity at glucocorticoid receptor in human A549 cells assessed as inhibition of PMA-induced AP-1 activation by luciferase reporter gene assay
- ChEMBL_1624982 (CHEMBL3867394) Agonist activity at PAR2 in human HT-29 cells assessed as increase in intracellular calcium release incubated for 30 mins measured for 60 secs by Fluo-3 AM dye based FLIPR assay relative to 2f-LIGRLO-NH2
- ChEMBL_1812458 (CHEMBL4311918) Antagonist activity at PAR2 in human MDA-MB-231 cells assessed as inhibition of SLIGKV-NH2-induced intracellular calcium mobilization preincubated for 15 mins followed by SLIGKV-NH2 addition by Fluo-4-AM dye based fluorescence assay
- ChEMBL_2045585 (CHEMBL4700284) Agonist activity at PAR2 in human EA.hy926 cells assessed as stimulation of calcium mobilization preincubated for 2 hrs with LPS and 30 mins with cRGD followed by compound addition by Fluo-4-AM dye-based FLIPR assay
- ChEMBL_1290685 (CHEMBL3117488) Inhibition of transcription factor AP-1 binding to oligonucleotide containing TPA-responsive element in TPA-activated human HeLa cells after 1 hr by ELISA
- ChEMBL_164918 (CHEMBL872006) Antisecretory activity evaluated by the inhibition of 14C -AP uptake in isolated rabbit parietal cells stimulated by dibutyryl cyclic adenosine 3', 5' -monophosphate (dcAMP)
- ChEMBL_1750385 (CHEMBL4185145) Inhibition of APE1 in human HeLa cells assessed as reduction in AP site cleavage 5'-[gamma-32P]CTCGCAAGUGGGTACCGA-3' as substrate after 1 hr
- ChEMBL_2134238 (CHEMBL4843848) Antagonist activity at PAR4 in human platelet-rich plasma assessed as inhibition of PAR4 AP AYPGKF-NH2-induced platelet aggregation by photo-turbidimetry assay
- ChEMBL_2444013 Inhibition of rat NHE1 expressed in Chinese hamster ovary cells (AP-1) measured after 9 mins by BCECF-AM staining based NH4Cl pre-pulse method
- ChEMBL_35342 (CHEMBL646060) Compound was evaluated for its inhibitory potency against purified membrane bound rat brain Aminopeptidase B (AP-B) using L-lysine-beta napthylamide as substrate
- ChEMBL_1624983 (CHEMBL3867395) Antagonist activity at PAR2 in human PC3 cells assessed as inhibition of 2f-LIGRLO-NH2-stimulated intracellular calcium release preincubated for 30 mins followed by stimulation measured for 60 secs by Fluo-3 AM dye based FLIPR assay
- ChEMBL_2134237 (CHEMBL4843847) Antagonist activity at PAR4 in ICR mouse platelet-rich plasma assessed as inhibition of PAR4 AP AYPGKF-NH2-induced platelet aggregation by photo-turbidimetry assay
- ChEMBL_2437619 Agonist activity at mouse TLR7 expressed in HEK293 cells co-expressing NF-kappaB/AP-1 inducible SEAP reporter gene measured for 24 hrs by colorimetric analysis
- ChEMBL_458546 (CHEMBL942804) Antagonist activity at GnRH receptor expressed in AP Han Wistar rat primary pituitary cells assessed as inhibition of GnRH stimulated LH release after 24 hrs
- ChEMBL_741821 (CHEMBL1769966) Inhibition of TPA-induced AP-1 activation in cells expressing beta-lactamase after 18 hrs using FRET substrate by cell-based high-throughput screening assay
- ChEMBL_984146 (CHEMBL2432074) Antagonist activity at human PAR2 expressed in human NCTC-2544 cells assessed as inhibition of SLIGKV-NH2-induced NFkappaB activation preincubated for 15 to 30 mins followed by SLIGKV-NH2 addition measured after 6 hrs by luciferase reporter gene assay
- ChEMBL_1275793 (CHEMBL3091126) Competitive inhibition of human recombinant DPP4 using AP-pNA as substrate preincubated for 30 mins followed by substrate addition measured after 30 mins by spectrophotometric analysis
- ChEMBL_35358 (CHEMBL647940) Compound was evaluated for the inhibition of Aminopeptidase M (AP-M) from porcine kidney and the inhibition constant was determined after preincubating the enzyme and inhibitor
- ChEMBL_829669 (CHEMBL2061059) Inhibition of recombinant APE1 using [35P]-5'-AP-DNA as substrate incubated for 15 mins prior to substrate addition measured after 5 mins by PAGE analysis
- ChEMBL_1878589 (CHEMBL4379983) Inhibition of human DPP4 in pH 7.4 Tris buffer using AP-7-ATFMC as substrate preincubated for 15 mins followed by substrate addition by microplate reader analysis
- ChEMBL_651577 (CHEMBL1227960) Antagonist activity at human wild type ERbeta expressed in human HeLa cells co-expressing AP-1 assessed as inhibition of transactivation activity by luciferase reporter gene assay
- ChEMBL_946427 (CHEMBL2339607) Agonist activity at IgG1 Fc region fused-immobilized EphA2 ectodomain (unknown origin) assessed as inhibition of EphA2/ephrin-A5 AP interaction incubated for 3 hrs by ELISA
- ChEMBL_1624981 (CHEMBL3867393) Antagonist activity at PAR2 in human HT-29 cells assessed as inhibition of 2f-LIGRLO-NH2-stimulated intracellular calcium release preincubated for 30 mins followed by 2f-LIGRLO-NH2 stimulation measured for 60 sec by Fluo-3 AM dye based FLIPR assay
- ChEMBL_1771896 (CHEMBL4224008) Antagonist activity at PAR2 in human EAhy926 cells assessed as inhibition of SLIGKV-NH2-induced intracellular calcium mobilization pretreated for 15 mins followed by SLIGKV-NH2 addition measured at 5 secs interval for 250 secs by Fluo-4/AM dye based fluorescence assay
- ChEMBL_1278646 (CHEMBL3095634) Antagonist activity at human M3 receptor expressed in CHO cells assessed as inhibition of carbachol-induced response after 30 mins by AP-1-driven luciferase reporter gene assay
- ChEMBL_2185614 (CHEMBL5097696) Antagonist activity at R848 stimulated TRL7 in HEK-Blue hTLR7 cells assessed as inhibition of NF kappa B/Ap-1 activation measured after 20 hrs by SEAP reporter assay
- ChEMBL_2185615 (CHEMBL5097697) Antagonist activity at R848 stimulated TRL8 in HEK-Blue hTLR8 cells assessed as inhibition of NF kappa B/Ap-1 activation measured after 20 hrs by SEAP reporter assay
- ChEMBL_2185616 (CHEMBL5097698) Antagonist activity at R848 stimulated TRL9 in HEK-Blue hTLR9 cells assessed as inhibition of NF kappa B/Ap-1 activation measured after 20 hrs by SEAP reporter assay
- ChEMBL_2440613 Agonist activity at TLR7 in HEK-Blue hTLR7 cells expressing NF-kappaB/AP-1 inducible SEAP assessed as increase in SEAP activity incubated for 24 hrs by QUANTI-Blue assay
- ChEMBL_2440616 Agonist activity at TLR8 in HEK-Blue hTLR8 cells expressing NF-kappaB/AP-1 inducible SEAP assessed as increase in SEAP activity incubated for 24 hrs by QUANTI-Blue assay
- ChEMBL_651579 (CHEMBL1227962) Antagonist activity at human wild type ERbeta expressed in human HeLa cells co-expressing AP-1 assessed as inhibition of raloxifene-induced transactivation activity by luciferase reporter gene assay
- ChEMBL_759677 (CHEMBL1811510) Inhibition of TPA-induced AP-1 activity expressed in human HEK293T cells treated 1 hr before TPA challenge measured after 18 hrs by FRET based beta-lactamase reporter assay
- ChEMBL_2134240 (CHEMBL4843850) Antagonist activity at human PAR4 expressed in Ga15-HEK293 cells assessed as reduction in PAR4 AP AYPGKF-NH2-induced cytosolic calcium incubated for 24 hrs by FLIPR - calcium mobilization assay
- ChEMBL_2134241 (CHEMBL4843851) Antagonist activity at human PAR1 expressed in Ga15-HEK293 cells assessed as reduction in PAR1 AP SFFLRR-NH2-induced cytosolic calcium incubated for 24 hrs by FLIPR - calcium mobilization assay
- ChEMBL_35364 (CHEMBL647945) Compound was evaluated for the inhibition of Aminopeptidase M (AP-M) and the inhibition constant was determined after preincubating the enzyme and inhibitor Value in the parentheses is IC50 (uM).
- ChEMBL_63593 (CHEMBL675240) Inhibition of the heparin binding epidermal growth factor (HB-EGF) release from fibrosarcoma HT1080 transfectants expressing alkaline phosphate (AP) tagged HB-EGF stimulated by 12-O-tetradecanoylphorbol 13-acetate(TPA).
- ChEMBL_726917 (CHEMBL1686706) Transrepression activity at GR expressed in IL-1beta- and TNFalpha-stimulated HepG2 cells assessed as inhibition of NFKB- or AP-1 mediated E-selectin transcription by luciferase reporter gene assay
- ChEMBL_625244 (CHEMBL1106187) Inhibition of AP-1 expressed in HEK293 cells assessed as PMA-stimulated transcriptional activation treated 24 hrs before PMA challenge measured after 20 to 24 hrs by luciferase reporter gene assay
- ChEMBL_2056279 (CHEMBL4711280) Inhibition of DPP4 in human Caco-2 cells using AP-AFC as substrate preincubated with enzyme for 10 mins followed by substrate addition and measured after 10 mins by fluorescence based analysis
- ChEMBL_2278366 Modulation of glucocorticoid receptor (unknown origin) transfected in human HeLa cells assessed as transrepression by measuring inhibition of PMA-induced AP-1 signalling incubated for 18 hrs by dual-luciferase reporter based assay
- ChEMBL_1680907 (CHEMBL4031184) Transrepression of glucocorticoid receptor in PMA-stimulated human ChaGoK1 cells expressing TRE-LacZ construct assessed as inhibition of AP-1 mediated TRE-LacZ activity after 24 hrs by beta-galactosidase reporter gene assay
- ChEMBL_2025658 (CHEMBL4679471) Inhibition of human TLR9 expressed in HEK-Blue cells assessed as inhibition of ODN2006-induced NF-kappaB and AP-1 activation measured after 20 hrs by quanti-blue based SEAP reporter gene assay
- ChEMBL_2025659 (CHEMBL4679472) Inhibition of human TLR8 expressed in HEK-Blue cells assessed as inhibition of R848-induced NF-kappaB and AP-1 activation measured after 20 hrs by quanti-blue based SEAP reporter gene assay
- ChEMBL_2025660 (CHEMBL4679473) Inhibition of human TLR7 expressed in HEK-Blue cells assessed as inhibition of R848-induced NF-kappaB and AP-1 activation measured after 20 hrs by quanti-blue based SEAP reporter gene assay
- ChEMBL_728111 (CHEMBL1687053) Agonist activity at GR expressed in IL-1beta- and TNFalpha-stimulated HepG2 cells assessed as inhibition of NFKB- or AP-1 mediated E-selectin transcription by luciferase reporter gene assay relative to Dexamethasone
- ChEBML_1658795 Agonist activity at human TLR7 expressed in HEK293 cells co-expressing secreted alkaline phosphatase assessed as induction of NF-kappaB/AP-1 promoter activity by measuring increase in secreted alkaline phosphatase level by spectrophotometric method
- ChEBML_1658796 Agonist activity at human TLR8 expressed in HEK293 cells co-expressing secreted alkaline phosphatase assessed as induction of NF-kappaB/AP-1 promoter activity by measuring increase in secreted alkaline phosphatase level by spectrophotometric method
- ChEMBL_1658795 (CHEMBL4008407) Agonist activity at human TLR7 expressed in HEK293 cells co-expressing secreted alkaline phosphatase assessed as induction of NF-kappaB/AP-1 promoter activity by measuring increase in secreted alkaline phosphatase level by spectrophotometric method
- ChEMBL_1658796 (CHEMBL4008408) Agonist activity at human TLR8 expressed in HEK293 cells co-expressing secreted alkaline phosphatase assessed as induction of NF-kappaB/AP-1 promoter activity by measuring increase in secreted alkaline phosphatase level by spectrophotometric method
- ChEMBL_1911350 (CHEMBL4413796) Inhibition of full length human APE1 assessed as reduction in endonuclease activity by measuring DNA cleavage of abasic sites using 6-FAM labelled two annealed oligonucleotides as AP site mimic substrate by fluorescence-based assay
- ChEMBL_767399 (CHEMBL1825511) Inhibition of hypoxia-induced HIF1 activation in human LN229 cells expressing HRE-AP reporter gene preincubated for 1 hr under normoxia condition followed by 24 hrs incubation under hypoxia condition by HRE-luciferase reporter assay
- ChEMBL_1663630 (CHEMBL4013311) Inhibition of complement factor D in human whole blood assessed as decrease in zymosan-induced AP activation mediated soluble MAC complex formation preincubated for 15 mins followed by zymosan addition measured after 40 mins by ELISA
- ChEMBL_2127307 (CHEMBL4836652) Inhibition of human TLR7 expressed in HEK293-Blue cells assessed as inhibition of R848-induced NF-kappaB/AP-1 activation measured after 20 hrs using quanti-blue as substrate by SEAP reporter gene based spectrophotometry assay
- ChEMBL_2127308 (CHEMBL4836653) Inhibition of human TLR8 expressed in HEK293-Blue cells assessed as inhibition of R848-induced NF-kappaB/AP-1 activation measured after 20 hrs using quanti-blue as substrate by SEAP reporter gene based spectrophotometry assay
- ChEMBL_2127309 (CHEMBL4836654) Inhibition of human TLR9 expressed in HEK293-Blue cells assessed as inhibition of R848-induced NF-kappaB/AP-1 activation measured after 20 hrs using quanti-blue as substrate by SEAP reporter gene based spectrophotometry assay
- ChEMBL_1989194 (CHEMBL4622741) Antagonist activity at human TLR7 overexpressed in HEK-Blue cells assessed as inhibition of gardiquimod-induced NFkappaB and AP-1 activation preincubated for 30 mins followed by gardiquimod stimulation measured after 22 hrs by quanti-blue SEAP assay
- ChEMBL_1989195 (CHEMBL4622742) Antagonist activity at human TLR9 overexpressed in HEK-Blue cells assessed as inhibition of ODN2006-induced NFkappaB and AP-1 activation preincubated for 30 mins followed by ODN2006 stimulation measured after 22 hrs by quanti-blue SEAP assay
- ChEMBL_1989198 (CHEMBL4622745) Antagonist activity at human TLR8 overexpressed in HEK-Blue cells assessed as inhibition of R848-induced NFkappaB and AP-1 activation preincubated for 30 mins followed by R848 stimulation measured after 22 hrs by quanti-blue SEAP assay
- ChEMBL_2185611 (CHEMBL5097693) Antagonist activity at R848 stimulated TRL7 overexpressed in HEK293 cells assessed as inhibition of NF kappa B/Ap-1 activation preincubated with compound for 30 mins followed by R848 stimulation measured after 20 hrs by SEAP reporter assay
- ChEMBL_2222656 (CHEMBL5135990) Antagonist activity at gardiquimod stimulated human TRL7 overexpressed in HEK293 cells assessed as inhibition of NF kappa B/Ap-1 activation preincubated with compound for 30 mins followed by gardiquimod stimulation measured after 22 hrs by SEAP reporter assay
- ChEMBL_2222657 (CHEMBL5135991) Antagonist activity at R848 stimulated human TRL8 overexpressed in HEK293 cells assessed as inhibition of NF kappa B/Ap-1 activation preincubated with compound for 30 mins followed by R848 stimulation measured after 22 hrs by SEAP reporter assay
- ChEMBL_2222658 (CHEMBL5135992) Antagonist activity at ODN2006 stimulated human TRL9 overexpressed in HEK293 cells assessed as inhibition of NF kappa B/Ap-1 activation preincubated with compound for 30 mins followed by ODN2006 stimulation measured after 22 hrs by SEAP reporter assay
- ChEMBL_1989196 (CHEMBL4622743) Antagonist activity at human TLR3 overexpressed in HEK-Blue cells assessed as inhibition of Poly IC-induced NFkappaB and AP-1 activation preincubated for 30 mins followed by Poly IC stimulation measured after 22 hrs by quanti-blue SEAP assay
- ChEMBL_1989197 (CHEMBL4622744) Antagonist activity at human TLR4 overexpressed in HEK-Blue cells assessed as inhibition of LPD-RK induced NFkappaB and AP-1 activation preincubated for 30 mins followed by LPD-RK stimulation measured after 22 hrs by quanti-blue SEAP assay
- ChEMBL_2222660 (CHEMBL5135994) Antagonist activity at Poly C stimulated human TRL3 overexpressed in HEK293 cells assessed as inhibition of NF kappa B/Ap-1 activation preincubated with compound for 30 mins followed by Poly C stimulation measured after 22 hrs by SEAP reporter assay
- ChEMBL_2222661 (CHEMBL5135995) Antagonist activity at LPD-RK stimulated human TRL4 overexpressed in HEK293 cells assessed as inhibition of NF kappa B/Ap-1 activation preincubated with compound for 30 mins followed by LPD-RK stimulation measured after 22 hrs by SEAP reporter assay
- Activities on KLK6 (PAR2) In a typical test, 100 μl of reaction medium contains the buffer, 1 μl of recombinant enzyme, 1 μl of substrate (PAR2) and 1 μl of the compound tested at 10 μM (in the control, 1 μl of DMSO). In each well, the inhibitor is deposited in the presence of the enzyme-buffer mixture for preincubation for 15 minutes at 37° C. The enzymatic reaction is then triggered by adding the buffer-substrate mixture (100 μM). The release of the 7-amino-4-methylcoumarin fluorescent group (AMC, λex=360 nm, λem=460 nm) following the hydrolysis of the substrate catalyzed by the enzyme is monitored for 30 minutes at 37° C. using a FLUOstar spectrofluorimeter.
- ChEMBL_1731440 (CHEMBL4146976) Antagonist activity at human GnRH receptor expressed in HEK293 cells co-expressing pGL4-NFATpromoter AP-1-luc assessed as inhibition of GnRH-induced NFAT promoter activation preincubated for 1 hr followed by GnRH addition measured after 6 hrs by luciferase reporter gene assay
- ChEMBL_1731445 (CHEMBL4146981) Antagonist activity at rat GnRH receptor expressed in HEK293 cells co-expressing pGL4-NFATpromoter AP-1-luc assessed as inhibition of GnRH-induced NFAT promoter activation preincubated for 1 hr followed by GnRH addition measured after 6 hrs by luciferase reporter gene assay
- ChEMBL_751649 (CHEMBL1787847) Inhibition of catalytic activity of recombinant human AP-N transfected in human HEK-293 cells assessed as cleavage of substrate L-leucine-p-nitroanilide to L-leucine and p-nitroaniline measured every 5 mins for 10 to 50 mins by spectrophotometric analysis
- ChEMBL_1750400 (CHEMBL4185160) Inhibition of His-tagged human APE1 expressed in Escherichia coli M15 assessed as reduction in cleavage of 26 mer substrate to 13 mer products pre-incubated for 30 mins before substrate addition for 15 mins using 5' end [32P] AP site containing 26 mer oligonucleotide by polyacrylamide gel electrophoresis
- TLR9 Antagonist Assay HEK-Blue-hTLR9 cells were obtained from InvivoGen Inc. and used to determine test compound antagonism of human TLR9 (hTLR9) driven responses. HEK-Blue-hTLR9 cells are designed for studying the stimulation of human TLR9 by monitoring the activation of NF-kB. As described by the manufacturer, HEK-Blue-hTLR9 cells were obtained by co-transfection of the hTLR9 gene and an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK293 cells. The SEAP reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-kB and AP-1 binding sites. Stimulation with a TLR9 ligand activates NF-kB and AP-1 which induces the production of SEAP. Levels of SEAP can be easily determined with QUANTI-Blue a detection medium that turns purple/blue in the presence of alkaline phosphatase.
- Rapid-Fire Mass Spectrometry (RFMS) Assay A rapid-fire mass spectrometry (RFMS) assay was used to assess RNR enzyme activity using a 384 well plate and a robotic platform. The plate layout included two validated reference compounds (Triapine (3-AP) and Hydroxyurea (HU)): A dose response in duplicate; top concentration: 5 uM (3-AP) and 250 uM (HU), semi-log dilutions. Spike wells in triplicate randomly spotted at four concentrations: 250 uM, 100 uM, 30 uM and 2 uM for HU 5 uM, 2 uM, 0.6 uM and 0.04 uM for 3-AP First, the multidrop pipes were saturated for 30 minutes with enzymatic solution. Then 30 uL of Stop solution was distributed in column 24. Next, 15 uL of enzyme was distributed in column 1 to 24. Next, a pre-incubation step of 15 minutes at room temperature occurred, followed by distribution of 15 uL of substrate solution (column 1 to 24). Next, the plate was incubated for 45 minutes at 37 C. 30 uL of Stop solution was distributed to columns 1 to 23. The final parameters for the enzyme reactions were: Incubation: 37 C., 45 min [CDP]: 5 uM; [ATP]: 1 mM; [NADPH]: No [RNR]final: 50 nM with 1:1 (RNR1:RNR2) ratio Final volume: 30 uL Stop solution: 6% HCOOH containing 2 uM of 15 The compounds were screened at concentrations from 10 nM to 30 uM.
- Biological Assay HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
- Inhibition Reporter Assay HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
- RNR Enzyme Activity Assay A rapid-fire mass spectrometry (RF/MS) assay was used to assess RNR enzyme activity using a 384 well plate and a robotic platform.[0402]The plate layout included two validated reference compounds (Triapine (3-AP) and Hydroxyurea (HU)):A dose response in duplicate; top concentration: 5 μM (3-AP) and 250 μM (HU), semi-logdilutions.Spike wells in triplicate randomly spotted at four concentrations:250 μM, 100 μM, 30 μM and 2 μM for HU5 μM, 2 μM, 0.6 μM and 0.04 μM for 3-APFirst, the multidrop pipes were saturated for 30 minutes with enzymatic solution. Then 30 μL of Stop solution was distributed in column 24. Next, 15 μL of enzyme was distributed in column 1 to 24. Next, a pre-incubation step of 15 minutes at room temperature occurred, followed by distribution of 15 μL of substrate solution (column 1 to 24). Next, the plate was incubated for 45 minutes at 37° C. 30 μL of Stop solution was distributed to columns 1 to 23.The final parameters for the enzyme reactions were:Incubation: 37° C., 45 min[CDP]: 5 μM; [ATP]: 1 mM; [NADPH]: No[RNR]final: 50 nM with 1:1 (RNR1:RNR2) ratioFinal volume: 30 μLStop solution: 6% HCOOH containing 2 μM of 15
- IP-One HTRF Assay Inositol monophosphate (IP1) production were measured in 1321N1 cells stably expressing human PAR2 using an IP-One HTRF assay kit (Cisbio). 80 nl of compound in 100% DMSO were added in white small-volume 384-well plates using an Echo 555 (Labcyte) and were incubate for 30 min at 37° C. with 4 μl of cells (15,000 cells per well) in stimulation buffer (HBSS with 20 mM HEPES, pH 7.4). IP1 production was initiated by addition of 4 μl of 140 μM SLIGRL-NH2 (SEQ ID NO.: 4) in stimulation buffer supplemented with 100 mM LiCl. After 60 min at 37° C. cells were lysed and IP1 concentrations were detected according to the manufacturers protocol (Cisbio). Data were normalized to IP1 concentrations using a IP1 standard curve according to the manufacturers protocol (Cisbio).
- TLR7/8/9 Inhibition Reporter Assays HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κc Band AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Bue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
- TLR7/8/9 Inhibition Reporter Assays HEK-Blue -cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.