lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1(C)COCCN1C(=O)c1ccc(C#Cc2cccc(F)c2)cn1
InChIKey: ZFKBTPPHOPDBMS-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Binding Assay: For binding experiments, cDNA encoding human mGlu 5a receptor was transiently transfected into EBNA cells using a procedure described by Schlaeger and Christensen [Cytotechnology 15:1-13 (1998)]. Cell membrane homogenates were stored at -80° C. until the day of assay where upon they were thawed and resuspended and polytronised in 15 mM Tris-HCl, 120 mM NaCl, 100 mM KCl, 25 mM CaCl2, 25 mM MgCl2 binding buffer at pH 7.4 to a final assay concentration of 20 ug protein/well.Saturation isotherms were determined by addition of twelve [3H]MPEP concentrations (0.04-100 nM) to these membranes (in a total volume of 200 ul) for 1 h at 4° C. Competition experiments were performed with a fixed concentration of [3H]MPEP (2 nM) and IC50 values of test compounds evaluated using 11 concentrations (0.3-10,000 nM). Incubations were performed for 1 h at 4° C.At the end of the incubation, membranes were filtered onto unifilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.1% PEI in wash buffer, Packard BioScience, Meriden, Conn.) with a Filtermate 96 harvester (Packard BioScience) and washed 3 times with cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 uM MPEP. The radioactivity on the filter was counted (3 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 ul of microscint 40 (Canberra Packard S. A., Zurich, Switzerland) and shaking for 20 min.
Measurement: Ki = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 338.38, ALogP 2.87, HBA 3, HBD 0, PSA 42.43, QED 0.75
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3ccc(O)cc3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: LDYHWJYPYDCMNK-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 488.55, ALogP 4.75, HBA 8, HBD 2, PSA 111.85, QED 0.37
Compound (SMILES): c1ccc(-c2cc(Nc3nccc(OC4CCNCC4)n3)ccc2N2CCOCC2)cc1
InChIKey: GQSHHVMPXKNCSM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 46.9 nM (pChEMBL 7.329999923706055)
Descriptors: MW 431.54, ALogP 3.85, HBA 7, HBD 2, PSA 71.54, QED 0.61
Compound (SMILES): CN1C(=O)N(c2ccc(C#Cc3cccc(F)c3)cn2)[C@@H]2CCC[C@@H]21
InChIKey: CXSRRWDESYDWAM-ZWKOTPCHSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Intracellular Ca2+ Mobilization Assay: A monoclonal HEK-293 cell line stably transfected with a cDNA encoding for the human mGlu5a receptor was generated; for the work with mGlu5 Positive Allosteric Modulators (PAMs), a cell line with low receptor expression levels and low constitutive receptor activity was selected to allow the differentiation of agonistic versus PAM activity. Cells were cultured according to standard protocols (Freshney, 2000) in Dulbecco's Modified Eagle Medium with high glucose supplemented with 1 mM glutamine, 10% (vol/vol) heat-inactivated bovine calf serum, Penicillin/Streptomycin, 50 μg/ml hygromycin and 15 μg/ml blasticidin (all cell culture reagents and antibiotics from Invitrogen, Basel, Switzerland). bout 24 hrs before an experiment, 5×104 cells/well were seeded in poly-D-lysine coated, black/clear-bottomed 96-well plates. The cells were loaded with 2.5 μM Fluo-4AM in loading buffer (1×HBSS, 20 mM HEPES) for 1 hr at 37 °C. and washed five times with loading buffer. The cells were transferred into a Functional Drug Screening System 7000 (Hamamatsu, Paris, France), and 11 half logarithmic serial dilutions of test compound at 37 °C. were added and the cells were incubated for 10-30 min. with on-line recording of fluorescence. Following this pre-incubation step, the agonist L-glutamate was added to the cells at a concentration corresponding to EC20 (typically around 80 μM) with on-line recording of fluorescence; in order to account for day-to-day variations in the responsiveness of cells, the EC20 of glutamate was determined immediately ahead of each experiment by recording of a full dose-response curve of glutamate. Responses were measured as peak increase in fluorescence minus basal (i.e. fluorescence without addition of L-glutamate), normalized to the maximal stimulatory effect obtained with saturating concentrations of L-glutamate.
Measurement: EC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 335.38, ALogP 3.41, HBA 2, HBD 0, PSA 36.44, QED 0.75
Compound (SMILES): O=C(O)Cn1nnc(-c2cnc(N3CCC4(C=Cc5ccccc54)CC3)s2)n1
InChIKey: PEXPKNONLMRNCB-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Enzyme Activity Assay: The potency of compounds of formula I against the stearoyl-CoA desaturase was determined by measuring the conversion of radiolabeled stearoyl-CoA to oleoyl-CoA using rat liver microsome or human SCD1 following previously published procedures with some modifications (Joshi, et al., J. Lipid Res., 18: 32-36 (1977); Talamo, et al., Anal. Biochem, 29: 300-304 (1969)). Liver microsome was prepared from male Wistar or Sprague Dawley rats on a high carbohydrate diet for 3 days (LabDiet #5803, Purina). The livers were homogenized (1:10 w/v) in a buffer containing 250 mM sucrose, 1 mM EDTA, 5 mM DTT and 50 mM Tris-HCl (pH 7.5). After a 100,000xg centrifugation for 60 min, the liver microsome pellet was suspended in a buffer containing 100 mM sodium phosphate, 20% glycerol, 2 mM DTT, and stored at -78° C. Human SCD1 desaturase system was reconstituted using human SCD1 from a baculovirus/Sf9 expression system, cytochrome B5 and cytochrome B5 reductase.
Measurement: IC50 = 244 nM (pChEMBL 6.610000133514404)
Descriptors: MW 394.46, ALogP 2.45, HBA 8, HBD 1, PSA 97.03, QED 0.73
Compound (SMILES): N=C(N)NCCC[C@@H]1N[C@H](CNC(=O)/C=C/c2ccc(Cl)cc2Cl)CCN(CC(c2ccccc2)c2ccccc2)C1=O
InChIKey: BQDYRGULYZIWNJ-FMDWORLUSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 621.61, ALogP 4.78, HBA 4, HBD 5, PSA 123.34, QED 0.09
Compound (SMILES): FC(F)(F)c1ccc2nc(C3C4Oc5ccc(Oc6ccncc6)cc5C43)[nH]c2c1
InChIKey: MQIILRMBQJPTPO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 518 nM (pChEMBL 6.289999961853027)
Descriptors: MW 409.37, ALogP 5.41, HBA 4, HBD 1, PSA 60.03, QED 0.49
Compound (SMILES): Cl.Oc1ccc(OC[C@@H]2CNCC[C@H]2c2ccc(F)cc2)cc1O
InChIKey: GIPUDSSHAJOHQK-LINSIKMZSA-N
Target: Beta-adrenergic receptor kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25098
Assay: Inhibition of human GRK2 S670A mutant assessed as decrease in phosphorylation of light-activated bovine rod outer segments preincubated for 30 mins followed by ATP addition
Measurement: IC50 = 63100 nM (pChEMBL 4.199999809265137)
Descriptors: MW 317.36, ALogP 3.01, HBA 4, HBD 3, PSA 61.72, QED 0.76
Compound (SMILES): C[C@]12CC[C@H]3[C@@H](C=CC4=C(O)C(=O)CC[C@@]43C)C1=CC=C2c1cccnc1
InChIKey: OVJPNGCRJYHHBM-SWZCZGJTSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels at 1 nM after 90 mins by ELISA relative to control
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 359.47, ALogP 5.19, HBA 3, HBD 1, PSA 50.19, QED 0.75
Compound (SMILES): CN(C)c1ccc(C(O)(c2ccc3nc(Cl)c(Cc4ccc(-n5cccn5)cc4)c(Cl)c3c2)c2cncn2C)cc1
InChIKey: XJIYGWJODWNNBE-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: 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.
Measurement: Kd = 440 nM (pChEMBL 6.360000133514404)
Descriptors: MW 583.52, ALogP 6.4, HBA 7, HBD 1, PSA 72, QED 0.22
Compound (SMILES): CN1CCN(C2CCN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)CC2)CC1=O
InChIKey: KCJZFSGHEZZMNI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.41 nM (pChEMBL 9.390000343322754)
Descriptors: MW 642.07, ALogP 2.86, HBA 10, HBD 1, PSA 130.2, QED 0.31
Compound (SMILES): N#Cc1cncc(S(=O)(=O)c2ccc(CNC(=O)c3cc4ccncc4o3)nc2)c1
InChIKey: WLEPCUNXLYYUGI-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT Enzymatic Reaction. The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 pM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 217 nM (pChEMBL 6.659999847412109)
Descriptors: MW 419.42, ALogP 2.25, HBA 8, HBD 1, PSA 138.84, QED 0.52
Compound (SMILES): CCC(CC)(COc1ccc(-c2ccc(-c3ncc(C(F)(F)F)[nH]3)nc2)cn1)C(=O)O
InChIKey: XZFVKWGKDMBZSN-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: DGAT1 Inhibitory Assay: As a buffer to be used in the enzymatic reaction of DGAT1, 100 mM Tris-HCl (pH 7.4), 200 mM Sucrose, 20 mM MgCl2, 0.125% Bovine Serum Albumin (BSA) were used. To the buffer were added Test compound with a predetermined concentration as well as 15 μM dioleoylglycerol, 5 μM [14C]-palmitoyl-CoA, 100 μg protein/nth DGAT1 highly expressed-expresSF+® microsome, 0.75% acetone, and 1% dimethylsulfoxide, and triglyceride (TG) synthetic reaction was carried out at 30 °C. for 20 minutes with a volume of 100 μL. 90 μL of the reaction solution was added to 810 μL of methanol to stop the reaction. The reaction solution was added to Oasie® μElution plate (available from Waters Corporation), and eluted with 150 μL of a mixed solution of acetonitrile: isopropanol (=2:3). To elute was added 150 μL of MicroScinti™-40 (available from PerkinElmer Inc.), and after thoroughly stirring the mixture, a [14C]-TG amount formed by the reaction was quantitated by measuring the same using TopCount™-NXT (available from PerkinElmer Inc.).
Measurement: IC50 = 0.76 nM (pChEMBL 9.119999885559082)
Descriptors: MW 434.42, ALogP 4.82, HBA 5, HBD 2, PSA 100.99, QED 0.53
Compound (SMILES): O=C(CN1CCN(C(c2ccccc2)c2ccccc2)CC1)C1c2ccccc2-c2ccccc21
InChIKey: WFYLVPRSRGXJDR-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Inhibition of human alpha1G T-type calcium channel expressed in HEK293 cells assessed as current dependent channel blocking at -110 mV holding potential with pre-pulse at -100 mV for 1 sec prior to test pulse at -40 mV for 50 ms by whole cell patch clamp assay
Measurement: IC50 = 404 nM (pChEMBL 6.389999866485596)
Descriptors: MW 458.61, ALogP 5.78, HBA 3, HBD 0, PSA 23.55, QED 0.37
Compound (SMILES): CS(=O)(=O)Nc1ccc(S(=O)(=O)N(CCC2CC2)c2sc3ccccc3c2Cl)cc1
InChIKey: MDQYFXBLALCVBY-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 485.05, ALogP 4.92, HBA 5, HBD 1, PSA 83.55, QED 0.49
Compound (SMILES): Cn1c(SCCCN2CC[C@]3(C[C@@H]3c3ccc(C(F)(F)F)cc3)C2)nnc1-c1ccc(=O)n(C)c1
InChIKey: DRMNJQDZXRGJRN-YKSBVNFPSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]-spiperone from human dopamine D3 receptor expressed in CHO-K1 cell membranes after 90 mins by liquid scintillation counting
Measurement: Ki = 1.738 nM (pChEMBL 8.760000228881836)
Descriptors: MW 503.59, ALogP 4.56, HBA 7, HBD 0, PSA 55.95, QED 0.35
Compound (SMILES): CCCCOC(=O)Nc1ccc2c(c1)C(C)(c1ccccc1)CC(C)(C)N2C(C)=O
InChIKey: OFWGBZCYRKFABH-UHFFFAOYSA-N
Target: Thyrotropin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P16473
Assay: Antagonist Activity Assay: Antagonist activity of three test compounds against human TSI at the human TSH receptor was tested in Chinese Hamster Ovary (CHO) cells (cultured 16 h in the absence of serum prior to the assay) stably expressing the human TSH receptor and a CRE-driven firefly luciferase reporter gene. TSI was partially purified from the serum of a GD patient by filtration over a 0.45 mm filter, protein G-Sepharose column chromatography, dialysis against phosphate-buffered saline and subsequent concentrating on a 10K Amicon filter. It was confirmed that the TSI preparation does not activate luciferase activity in control CHO cells lacking the human TSHR. The cAMP phosphodiesterase inhibitor rolipram was included in the assay medium (10 μM) to augment TSHR-induced CRE-luciferase synthesis, which was quantified using a luminescence counter. The cells were incubated with compound A, B or C (0.316 nM-10 μM) together with 3.16 mg/ml TSI (or bovine TSH at a equieffective concentration of 18 nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 408.54, ALogP 5.88, HBA 3, HBD 1, PSA 58.64, QED 0.63
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2ccccc2)C(=O)N[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: UCIXIQRCAGGOJY-AJQTZOPKSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 578.71, ALogP 3.93, HBA 5, HBD 4, PSA 122.55, QED 0.18
Compound (SMILES): COc1ccc2cc(CN3CCN(C(=O)Nc4ccccc4)CC3)ccc2c1
InChIKey: VOODDNMALWNZME-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture was prepared from 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA; Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 200 nM. The reaction mixture was incubated at rt for 1 hour (h). During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 375.47, ALogP 4.2, HBA 3, HBD 1, PSA 44.81, QED 0.74
Compound (SMILES): CC(C)n1cc(-c2cc(-c3ccc4c(c3)CCN4C)cnc2N)nn1
InChIKey: OTCKNJDQPALGIV-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Kinase Activity Assay: The kinase reaction buffer consists of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT. In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase is incubated with the test compound (typically at 0 to 30 μM) for 30 minutes before the kinase reaction is initiated by addition of 1.3 mM ATP and 0.4 μM fluorescein-LRRKtide. The reaction mixture (20 μl total volume) is incubated for 2 hours at 30 °C. before the reaction is terminated by addition of 10 mM EDTA and 1 nM terbium-labelled anti-phospho-LRRKtide antibody (final volume 20 μl). The mixture is further incubated for 30 minutes at RT. TR-FRET is measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 μs) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 μs. The measurement is repeated 10 times for fluorescein and 10 times for terbium emission with a 2000 μs time window between repeats. TR-FRET measurements are performed on a Biomek Synergy plate. The TR-FRET signal is calculated as the emission-ratio at 520 nm over 495 nm.
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 334.43, ALogP 3.16, HBA 6, HBD 1, PSA 72.86, QED 0.8
Compound (SMILES): C[C@H](Nc1ncnc2[nH]nnc12)c1nc2c(F)cccc2c(=O)n1-c1cnc(N)cn1
InChIKey: VLMXVJDEMLNUBP-QMMMGPOBSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The activities were measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site. Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (75-500 uM), 2 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 uM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) and Compound Y ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window). The results were normalized based on positive (1 uM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ and γ were calculated from the fit of the dose-response curves to a four-parameter equation.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 419.38, ALogP 1.13, HBA 11, HBD 3, PSA 166.07, QED 0.38
Compound (SMILES): COc1cc(CN(C)C(C)c2ccc3c(c2)CCO3)ccc1OCCN1C(=O)CN(C)C1=O
InChIKey: NJVQAWSOVXFONW-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 202 nM (pChEMBL 6.699999809265137)
Descriptors: MW 453.54, ALogP 3.1, HBA 6, HBD 0, PSA 71.55, QED 0.54
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: UXBSQZKSGDJREX-GOYLXPRVSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 2457.87
Compound (SMILES): CON=C1CCC(c2cc(NC(=O)c3cnn4cccnc34)n(-c3ccc(C)cc3)n2)CC1
InChIKey: VJTJGOGWYCCWQQ-MIXAMLLLSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 443.51, ALogP 4.14, HBA 8, HBD 1, PSA 98.7, QED 0.47
Compound (SMILES): C[C@H]1CNC[C@H](c2ccc(NC(=O)c3cccc(Cl)c3)cc2)O1
InChIKey: VHLRQDIZVICDNS-YVEFUNNKSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 115.9 nM (pChEMBL 6.940000057220459)
Descriptors: MW 330.82, ALogP 3.64, HBA 3, HBD 2, PSA 50.36, QED 0.9
Compound (SMILES): NC(=O)c1ccc2[nH]c(-c3ccc(OC[C@H]4CC[C@@H](O)CC4)cc3)nc2c1
InChIKey: LYTQPKFVJFPASC-AKAXFMLLSA-N
Target: Serine/threonine-protein kinase Chk2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96017
Assay: Inhibition of human CDS1 using N-terminal biotin labeled SGLYRSPSMPENLNRPR as substrate in presence of [gamma-33P]-ATP after 3 hrs by scintillation counting method
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 365.43, ALogP 3.26, HBA 4, HBD 3, PSA 101.23, QED 0.65
Compound (SMILES): CCCC1(C(O)c2cccc([C@H]3CCC(=O)[C@@H]3CCCCCCC(=O)O)c2)CCC1
InChIKey: HOCLEBHKVQABNF-QQMHWKEJSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Agonist activity at human recombinant EP2 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 = 3407 nM (pChEMBL 5.46999979019165)
Descriptors: MW 414.59, ALogP 6.18, HBA 3, HBD 2, PSA 74.6, QED 0.4
Compound (SMILES): NC(=O)c1cc(-c2ccc(F)cc2)c2ccc([C@@H](O)Cc3ccc(Cl)nc3)cc2n1
InChIKey: CWRYRTIWJWUWEF-NRFANRHFSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr-cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity. Then 2,500 nM glutamate is added and the antagonist response is monitored. The maximum calcium response at each concentration of compound for agonist or antagonist were plotted as dose responses and the curves were fitted with a four parameter logistic equation giving IC50 and Hill coefficient using the iterative non linear curve fitting software ADA (Merck & Co).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 421.86, ALogP 4.46, HBA 4, HBD 2, PSA 89.1, QED 0.47
Compound (SMILES): N#C/C(=C\C1CC1)C(=O)N1CCC[C@H]1Cn1nc(-c2ccc(Oc3c(F)cccc3F)cc2F)c2c(N)ncnc21
InChIKey: WBPYSPBQYMHIEC-RLGUWMIQSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzymatic Activity Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of Btk kinase activity of a compound of Formula (IA), (I′) or (I). Serial dilutions of test compounds were incubated with human recombinant Btk (2 nM), ATP (40 μM) and a phosphoacceptor peptide substrate FAM-GEEPLYWSFPAKKK-NH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper Desktop Profiler (Caliper LabChip 3000). Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 559.55, ALogP 5.14, HBA 8, HBD 1, PSA 122.95, QED 0.25
Compound (SMILES): O=S(=O)(Nc1ccc(-c2nc3cc(NS(=O)(=O)c4ccccc4F)ccc3o2)cc1)c1ccccc1F
InChIKey: IZGYHBGXECNFGB-UHFFFAOYSA-N
Target: GroEL/GroES (PROTEIN COMPLEX), Escherichia coli
UniProt: Q7BGE6
Assay: Inhibition of Escherichia coli GroEL/GroES expressed in Escherichia coli DH5alpha/BL21 (DE3) assessed as inhibition of denatured MDH refolding preincubated for 10 mins followed by ATP addition measured after 20 to 40 mins by fluorescence based assay
Measurement: IC50 = 22000 nM (pChEMBL 4.659999847412109)
Descriptors: MW 541.56, ALogP 5.37, HBA 6, HBD 2, PSA 118.37, QED 0.29
Compound (SMILES): Fc1ccc(Nc2nnc(-c3ccc4[nH]cnc4c3)o2)cc1
InChIKey: UFUXCXWKPAKGOW-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Fluorometric Assay: All measurements were performed with a BioAssay Reader HTS-7000Plus for microplates (Perkin Elmer) at 30 C. QC activity was evaluated fluorometrically using H-Gln-beta NA. The samples consisted of 0.2 mM fluorogenic substrate, 0.25 U pyroglutamyl aminopeptidase (Unizyme, Horsholm, Denmark) in 0.2 M Tris/HCl, pH 8.0 containing 20 mM EDTA and an appropriately diluted aliquot of QC in a final volume of 250 ul. Excitation/emission wavelengths were 320/410 nm. The assay reactions were initiated by addition of glutaminyl cyclase. QC activity was determined from a standard curve of beta -naphthylamine under assay conditions. One unit is defined as the amount of QC catalyzing the formation of 1 umol pGlu-beta NA from H-Gln-beta NA per minute under the described conditions.In a second fluorometric assay, QC was activity determined using H-Gln-AMC as substrate. Reactions were carried out at 30 C. utilizing the NOVOStar reader for microplates (BMG labtechnologies).
Measurement: Ki = 6840 nM (pChEMBL 5.170000076293945)
Descriptors: MW 295.28, ALogP 3.5, HBA 5, HBD 2, PSA 79.63, QED 0.6
Compound (SMILES): C[C@H](Nc1ncc(F)c(N2C(=O)OC[C@@H]2CF)n1)c1cn(-c2ccc(F)cc2)cn1
InChIKey: GXKZHLYWKDTOMF-FZMZJTMJSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 418.38, ALogP 3.41, HBA 7, HBD 1, PSA 85.17, QED 0.66
Compound (SMILES): FC(F)(F)Oc1cccc(/C=C/c2n[nH]c3cc(-c4ccncc4)ccc23)c1
InChIKey: JPQSUYRVRQRMPH-QPJJXVBHSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Inhibition of full length human C-terminal 6His-tagged CHK1 expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using Syntide2 as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 232 nM (pChEMBL 6.630000114440918)
Descriptors: MW 381.36, ALogP 5.69, HBA 3, HBD 1, PSA 50.8, QED 0.49
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCCCN(C5CCC5)C5CC5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: VOSHORWBODTEPW-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 921.56, ALogP 9.64, HBA 11, HBD 3, PSA 166.04, QED 0.05
Compound (SMILES): CC(C)Nc1c(-c2cccc(Cl)c2)nc2ccc(Cl)cn12
InChIKey: TWBKIWUMAUJIEJ-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Dose Response Assay: Harvested cells with 0.025% trypsin/EDTA. Resuspended cells in growth media without selection antibiotics. Measured cell density and diluted to 2.4×10^5 cells/ml in media containing 1 μg/ml Doxycycline Plate 25 μl/well into 384 well black/clear tissue culture-treated plates. Incubated overnight at 37° C. Growth media was removed from the cell plates (20 μl) and 20 μl of the Replacement Buffer was added followed by addition of 25 μl of diluted dye. All three steps were performed using a Plate Washer BioTek 407. The plates were then incubated for 30' at RT. After incubation, both the cell and compound plates were brought to the FLIPR and 20 μl of the diluted compounds/antagonist/bk were transferred to the cell plates by the FLIPR. Plates were then incubated for 30' at room temperature. After 30' incubation, plates were returned to the FLIPR and 20 ul of 4.5× Cinnamaldehyde was added to the cell plates. During the compound addition as well as agonist addition, fluorescence readings were taken simultaneously from all 384 wells of the cell plate every 1.5 seconds. Five readings were taken to establish a stable baseline, then 20 μl of sample was rapidly (30 μl/sec) and simultaneously added to each well of the cell plate. The fluorescence was continuously monitored before, during and after sample/agonist addition for a total elapsed time of 100 seconds (compound addition) and 120 seconds (agonist addition).
Measurement: IC50 = 195 nM (pChEMBL 6.710000038146973)
Descriptors: MW 320.22, ALogP 5.13, HBA 3, HBD 1, PSA 29.33, QED 0.72
Compound (SMILES): NC(=O)[C@@H](CCC(F)(F)F)[C@@H](CCC(F)(F)F)C(=O)N[C@H]1N=C(c2ccccc2)c2cccc(C3CC3)c2NC1=O
InChIKey: UGHSMELYSLKUPL-XLNZFTOWSA-N
Target: Neurogenic locus notch homolog protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P46531
Assay: Biological Assay: The pharmacological properties of the compounds of this invention may be confirmed by a number of biological assays.
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 582.55, ALogP 5.2, HBA 4, HBD 3, PSA 113.65, QED 0.33
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)C(=O)N(c3ccc(F)cc3)CO4)cc12
InChIKey: MFARMFXLOUWZSG-UHFFFAOYSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Non-nucleoside inhibition of NS5B polymerase in HCV genotype 1a by Taqman probe based RT-PCR analysis
Measurement: EC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 618.62, ALogP 5.19, HBA 7, HBD 1, PSA 122.05, QED 0.28
Compound (SMILES): C[C@H](Nc1ncc(F)c(N2C(=O)OC[C@@H]2[C@H](C)F)n1)c1cn(-c2cc(F)cc(F)c2)cn1
InChIKey: SHUACSXYCKAFCG-PSVSLLHZSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 29.7 nM (pChEMBL 7.53000020980835)
Descriptors: MW 450.4, ALogP 3.94, HBA 7, HBD 1, PSA 85.17, QED 0.57
Compound (SMILES): C[C@H](O)Cn1cc(-c2cn3ncc(C(N)=O)c(N[C@H]4CN(c5ncc(C#N)cn5)C[C@H]4C)c3n2)cn1
InChIKey: ZDBMCSJMCJKCOG-GLJUWKHASA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Kinase Assay: The assay reactions were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (100 mM HEPES pH 7.4, 10 mM MgCl2, 25 mM beta-glycerol phosphate 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of GST-JAK3 enzyme with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA to each sample. Each reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product Inhibition data were calculated by comparison with no enzyme control reactions for 100% inhibition, and vehicle-only treated reactions for 0% inhibition. The final concentration of reagents in the assay was: ATP, 8 μM; fluoresceinated peptide, 1.5 μM; GST-JAK3, 4.5 nM; and DMSO, 1.6%.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 487.53, ALogP 0.67, HBA 12, HBD 3, PSA 176.17, QED 0.34