lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN1CCC(C)(C)c2ccc(Nc3nc(N4CCCCC4)nc4c3CCN(c3ncccc3C(F)(F)F)CC4)cc21
InChIKey: LMPHSOGPKNZCDE-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 565.69, ALogP 6.35, HBA 7, HBD 1, PSA 60.42, QED 0.4
Compound (SMILES): C[C@H](NC(=O)N(C)C)c1ccc(CN2Cc3ccc(OCC4CC4)cc3C(F)C2)cc1
InChIKey: AULYWDVQSAXTGR-KEJDIYNNSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 425.55, ALogP 4.83, HBA 3, HBD 1, PSA 44.81, QED 0.69
Compound (SMILES): CCCCC(O)c1ccc(N2C(=O)CC[C@@H]2COCc2ccc(C(=O)O)s2)cc1
InChIKey: LFGZMZXZOVNLKZ-DUSLRRAJSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Displacement of [3H]PGE2 from from human EP4 receptor expressed in HEK293 cells membranes incubated for 60 mins
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 417.53, ALogP 4.38, HBA 5, HBD 2, PSA 87.07, QED 0.6
Compound (SMILES): CC#C[C@@H](CC(=O)O)c1ccc(OCc2cccc(CN(Cc3ccsc3)C(C)C)c2)cc1
InChIKey: SMSIQLAGVMCTRR-SANMLTNESA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Agonist activity at human GPR40 expressed in HEK293 cells measured after 18 to 20 hrs by beta-gal based luciferase reporter gene assay
Measurement: EC50 = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 461.63, ALogP 6.32, HBA 4, HBD 1, PSA 49.77, QED 0.34
Compound (SMILES): Nc1ncc([C@H]2CC[C@@H](O)CC2)nc1-c1ccc(C(=O)NCc2cccc(Cl)n2)c(F)c1
InChIKey: CKWAOFOHKBFPRQ-AKAXFMLLSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 159 nM (pChEMBL 6.800000190734863)
Descriptors: MW 455.92, ALogP 3.86, HBA 6, HBD 3, PSA 114.02, QED 0.5
Compound (SMILES): N#Cc1c(N)nc(SCc2cccc(C(=O)NCCN)c2)c(C#N)c1-c1ccccc1
InChIKey: VPILLSACNTZHIJ-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Luciferase Assay: The stock cultures are grown, at 37° C. and under 5% CO2, in DMEM/F12 medium containing 10% FCS (fetal calf serum) and in each case split 1:10 after 2-3 days. Test cultures are seeded in 384-well plates with 2000 cells per well and grown at 37° C. for approx. 48 hours. The medium is then replaced with a physiological sodium chloride solution (130 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 20 mM HEPES, 1 mM magnesium chloride hexahydrate, 5 mM sodium bicarbonate, pH 7.4). The substances to be tested, which are dissolved in DMSO, are pipetted into the test cultures (maximum final concentration of DMSO in the test mixture: 0.5%) in a dilution series of from 5x10-11 M to 3x10-6 M (final concentration). 10 minutes later, forskolin is added to the A1 cells and all the cultures are subsequently incubated at 37° C. for four hours. After that, 35 ul of a solution which is composed of 50% lysis reagent (30 mM disodium hydrogenphosphate, 10% glycerol, 3% TritonX100, 25 mM TrisHCl, 2 mM dithiotreitol (DTT), pH 7.8) and 50% luciferase substrate solution (2.5 mM ATP, 0.5 mM luciferin, 0.1 mM coenzyme A, 10 mM tricine, 1.35 mM magnesium sulfate, 15 mM DTT, pH 7.8) are added to the test cultures, which are shaken for approx. 1 minute and the luciferase activity is measured using a camera system.
Measurement: EC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 428.52, ALogP 3.05, HBA 7, HBD 3, PSA 141.61, QED 0.49
Compound (SMILES): CCOc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)OCC=C(C)C)CC2
InChIKey: HIHMNVGNAPKSPZ-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 8.7 nM (pChEMBL 8.0600004196167)
Descriptors: MW 441.5, ALogP 4.98, HBA 4, HBD 1, PSA 76.07, QED 0.61
Compound (SMILES): COc1ccc(C(C)C)cc1C1=C(CN(Cc2cc(C(F)(F)F)cc(C(F)(F)F)c2)c2ncc(-c3cnn(C)c3)cn2)c2ccccc2CC1
InChIKey: KJTSLTMSJZXSKT-UHFFFAOYSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: Inhibition of recombinant CETP (unknown origin) assessed as inhibition of BODIPY-labeled cholesteryl transfer to fluorescent substrate by fluorimetric assay
Measurement: IC50 = 6630 nM (pChEMBL 5.179999828338623)
Descriptors: MW 691.72, ALogP 9.61, HBA 6, HBD 0, PSA 56.07, QED 0.14
Compound (SMILES): NC(=O)c1nn(-c2ccc3c(c2)OCO3)c2c1COc1ccc(NC(=O)c3ccncc3Cl)cc1-2
InChIKey: QCGNEEDJSOTNTJ-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus infected insect cells using biotinylated IkappaBalpha peptide as substrate in presence of [gamma-33P]-ATP after 30 mins by resin-based scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 489.88, ALogP 3.56, HBA 8, HBD 2, PSA 130.59, QED 0.45
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(N[C@H]5CCN(C6CC6)C5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: DGGBOFMYLNQDIL-UMSFTDKQSA-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 = 0.13 nM (pChEMBL 9.890000343322754)
Descriptors: MW 879.48, ALogP 8.47, HBA 11, HBD 3, PSA 166.04, QED 0.08
Compound (SMILES): COC(=O)[C@H](C)CN(c1sc2ccccc2c1C)S(=O)(=O)c1ccccc1
InChIKey: WREWEOWZYOZQJQ-CQSZACIVSA-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 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 403.53, ALogP 4.21, HBA 5, HBD 0, PSA 63.68, QED 0.58
Compound (SMILES): N#Cc1ccc(NC2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)c(F)c1
InChIKey: GTUQBUKJNCVDRI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS. From the plot of logarithm of a compound concentration and inhibitory activity, the IC50 was calculated.
Measurement: IC50 = 7.9 nM (pChEMBL 8.100000381469727)
Descriptors: MW 375.41, ALogP 3.75, HBA 6, HBD 2, PSA 94.69, QED 0.57
Compound (SMILES): COc1ccc(NC(=O)C2CCN(S(=O)(=O)c3cn(C(C)C)cn3)CC2)cc1OC
InChIKey: KLSQCSGIFSNAJS-UHFFFAOYSA-N
Target: Dihydrolipoyl dehydrogenase (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WHH9
Assay: Inhibition Assay: The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing Lpd (100 nM) and 100 mM sodium phosphate buffer (pH 7.0) was prepared. Another solution containing the substrate lipoamide (2 mM), 100 mM sodium phosphate buffer (pH 7.0), EDTA (4 mM), and NADH (200 uM) was also prepared. The detection reagent, DTNB was dissolved in DMSO and diluted to a concentration of 375 uM with a 100 mM sodium phosphate buffer (pH 7.0).4 uL/well of Lpd solution was dispensed into 1536-well clear bottom plates. Next, 50 nL of each test compounds (1 mM dissolved in DMSO) was added into each well. After one hour of incubation, 4 uL/well of lipoamide (LPA) solution was added, so that in each well, the concentrations of Lpd, LPA, and the test compound were 50 nM, 1 mM, and 6.25 uM, respectively.
Measurement: IC50 = 1830 nM (pChEMBL 5.739999771118164)
Descriptors: MW 436.53, ALogP 2.52, HBA 7, HBD 1, PSA 102.76, QED 0.72
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2c(C)ccc3ncccc23)cc(NC(=O)c2ccccn2)n1
InChIKey: CATNWFMLXOWVII-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: DELFIA Assay: R3 domains of human XIAP (covering amino acids 241 to 356; XIAP BIR3) and cIAPl (covering amino acids 256 to 363; cIAPl BIR3) were expressed and purified from Ecoli as GST-fusion proteins. Peptide AVPIAQKSE-Lys(Biotin), representing the N-terminus of mature human SMAC (SMAC peptide), was used as interaction partner in the protein- peptide interaction assay.BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonXlOO) for one hour at room temperature in the presence of inhibitiory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-Nl- antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluoresecence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 1418 nM (pChEMBL 5.849999904632568)
Descriptors: MW 440.51, ALogP 3.8, HBA 6, HBD 3, PSA 108.9, QED 0.42
Compound (SMILES): CC(C)Nc1cc(-n2ccc3cc(C#N)cnc32)ncc1C(=O)NCC(F)C1(O)COC1
InChIKey: ADGJSGULIJNUIP-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The 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 to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 438.46, ALogP 1.94, HBA 8, HBD 3, PSA 125.09, QED 0.51
Compound (SMILES): CN(CCC#N)c1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: VGGBFFPZFMLZAG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 414.52, ALogP 3.34, HBA 7, HBD 1, PSA 89.14, QED 0.52
Compound (SMILES): NCc1ccc(CNC(=O)[C@H](Cc2ccccc2)NC(=O)[C@@H](Cc2ccc(Cl)cc2)NC(=O)c2ccccc2)cc1
InChIKey: DFTDXVAGVUMEML-XZWHSSHBSA-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 = 521 nM (pChEMBL 6.28000020980835)
Descriptors: MW 569.11, ALogP 4.18, HBA 4, HBD 4, PSA 113.32, QED 0.21
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2CCN(c3ccc(NC(=O)c4ccccn4)c(OCC(=O)O)c3)CC2)cc1
InChIKey: ZMJMIKMXGHOZQY-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Antagonist activity at PGD2 receptor in human platelets assessed as inhibition of PGD2-mediated cAMP accumulation preincubated for 5 mins followed by PGD2 addition measured after 2 mins by HTRF assay
Measurement: IC50 = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 554.63, ALogP 3.1, HBA 8, HBD 2, PSA 138.37, QED 0.39
Compound (SMILES): Cc1cc(OCc2ccc(-c3ccnc4[nH]c5ccc(-c6ccc(N7CCN(C)CC7)cc6)cc5c34)cc2)ccc1Cl
InChIKey: QCDVYHMRAJKGHT-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 573.14, ALogP 8.34, HBA 4, HBD 1, PSA 44.39, QED 0.22
Compound (SMILES): Cc1ccc(NC(=O)c2cc(C)n(C(C)(C)C)n2)cc1-n1cc(-c2cncc(N3CCOCC3)c2)nn1
InChIKey: ANTPPDMHFTWHJV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: B-RAF (V600E) Kinase Assay: In a dilution series 10 uL/well of test substance solution are placed in a multiwell plate. The dilution series is selected so that generally a range of concentrations of 2 uM to 0.119 nM or 0.017 nM is covered. If necessary the initial concentration of 2 uM is changed to 50 uM, 10 uM or 0.4 uM or 0.2857 uM and further dilution is carried out accordingly. The final concentration of DMSO is 5%. 10 uL/well of the B-Raf (V600E)-kinase solution are pipetted in (containing 0.5 ng B-Raf (V600E)-kinase (e.g. from Upstate) in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.286 mM sodium orthovanadate, 10% glycerol, 1 mg/mL bovine serum albumin, 1 mM dithiothreitol) and the mixture is incubated for 1 h at RT under with shaking. The kinase reaction is started by the addition of 20 uL/well ATP solution [final concentration: 250 uM ATP, 30 mM Tris-HCl pH 7.5, 0.02% Brij, 0.2 mM sodium orthovanadate, 10 mM magnesium acetate, 0.1 mM EGTA, phosphatase cocktail (Sigma, #P2850, dilution recommended by the manufacturer)] and 10 uL/well MEK1 solution [containing 50 ng biotinylated MEK1 (prepared from purified MEK1 according to standard procedure, e.g. with EZ-Link Sulpho-NHS-LC-Biotin reagent, Pierce, #21335)] and carried out for 60 min at RT with constant shaking. The reaction is stopped by the addition of 12 uL/well of a 100 mM EDTA solution and incubation is continued for a further 5 min. 55 uL/well of the reaction solution are transferred into a streptavidin-coated plate (e.g. Streptawell HighBond, Roche, #11989685001) and shaken gently for 1 h at RT, in order to bind biotinylated MEK1 to the plate. After elimination of the liquid the plate is washed five times with 200 uL/well of 1Ã¿PBS and 100 uL/well solution of primary antibody plus europium-labelled secondary antibody [Anti Phospho-MEK (Ser217/221), Cell Signaling, #9121 and Eu-N1 labelled goat-anti-rabbit antibody, Perkin Elmer, #AD0105] is added, the primary antibody is diluted 1:2000 and the secondary antibody is diluted to 0.4-0.5 ug/mL in Delfia Assay Buffer (Perkin Elmer, #1244-111). After 1 h shaking at RT the solution is poured away and washed five times with 200 uL/well Delfia Wash Buffer (Perkin Elmer, #4010-0010/#1244-114). After the addition of 200 uL/well Enhancement Solution (Perkin Elmer, #4001-0010/#1244-105) the mixture is shaken for 10 min at RT and then measured in a Wallac Victor using the program "Delfia Time Resolved Fluorescence (Europium)". IC50 values are obtained from these dosage-activity curves using a software program (GraphPadPrizm).
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 500.61, ALogP 3.99, HBA 9, HBD 1, PSA 102.99, QED 0.44
Compound (SMILES): Cc1cc(-c2nc3ccc(C4(C(F)(F)F)CC4)cc3n2Cc2ccccn2)cn(C)c1=O
InChIKey: UICGVJNZCSIUIH-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (LysS, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
Measurement: IC50 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 438.45, ALogP 4.75, HBA 5, HBD 0, PSA 52.71, QED 0.46
Compound (SMILES): O=C(O)COC[C@H]1CC[C@@H](CNC(=O)N(c2ccccc2)c2ccccc2)CC1
InChIKey: FMSPYCIOLLLRCM-KDURUIRLSA-N
Target: Prostacyclin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43119
Assay: Agonist activity at human recombinant prostacyclin receptor expressed in CHO-K1 cells assessed as induction of cAMP accumulation incubated for 1 hr by HTRF assay
Measurement: EC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 396.49, ALogP 4.44, HBA 3, HBD 2, PSA 78.87, QED 0.69
Compound (SMILES): Cc1cccc2[nH]c(-c3ncsc3N3CCN(C(=O)Cn4ccc5cccnc54)[C@H](C)C3)nc12
InChIKey: SOSAHDDYEAPUCF-QGZVFWFLSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 471.59, ALogP 4.08, HBA 7, HBD 1, PSA 82.94, QED 0.43
Compound (SMILES): Cc1ncccc1-c1ccc(-c2nc(-c3ccc(Cl)c(Cl)c3)cs2)cc1C(=O)O
InChIKey: MCIFPCWLXPARFN-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E (SINGLE PROTEIN), Homo sapiens
UniProt: P06730
Assay: Binding Assay: Human eIF4E (aa 28-217) with a C-terminal His tag (HH-eIF4E) was expressed in E. coli in inclusion bodies. The protein was solubilized with 8 M urea and purified under denaturing conditions using nickel-charged HisTrap HP columns (GE Healthcare). The protein was refolded by diluting to approximately 0.25 mg/mL with 6 M urea, 20 mM Hepes pH 7.0, 500 mM NaCl, 1 mM DTT, 1 mM EDTA, and 0.5 M arginineÂ»HCl, and then dialyzing overnight into the same buffer without the urea. The protein was further dialyzed into 20 mM Hepes pH 6.5, 50 mM NaCl, 1 mM EDTA, and 1 mM DTT, filtered, and then concentrated using Hitrap SP sepharose FF columns (GE Healthcare). The protein was dialyzed into 20 mM Hepes pH 7.0, 500 mM NaCl, 5 mM DTT, and 10% glycerol and stored at -80 Â°C until use. Test compounds (1.6 mM stock in DMSO) were diluted 3 fold in series in DMSO. Compound solutions were diluted 4 fold in Assay Buffer (50 mM sodium phosphate, pH 6.5, 50 mM KC1, 1 mM DTT and 0.5 mg/mL gammaglobulin).
Measurement: IC50 = 4000 nM (pChEMBL 5.400000095367432)
Descriptors: MW 441.34, ALogP 6.85, HBA 4, HBD 1, PSA 63.08, QED 0.38
Compound (SMILES): CCc1cccc(NC(=O)Nc2ccc(-c3[nH]c4ccccc4c3C(N)=O)cc2)c1
InChIKey: VAKJINXSSWKNMD-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 2.7 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 μl per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 398.47, ALogP 5.14, HBA 2, HBD 4, PSA 100.01, QED 0.37
Compound (SMILES): CN(CCCCN1C(=O)CCc2ccccc21)Cc1ccccc1
InChIKey: FWSRLDTVTUGWKJ-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Cavia porcellus
UniProt: Q60492
Assay: Displacement of [3H]-(+)-pentazocine from sigma1 receptor in guinea pig brain cortex membranes after 120 mins by scintillation counting method
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 322.45, ALogP 3.88, HBA 2, HBD 0, PSA 23.55, QED 0.72
Compound (SMILES): CC(CC(=O)N1CCn2c(CCc3ccccc3)c(C(F)(F)F)c3cccc(c32)C1)C(F)(F)F
InChIKey: GCYPVXKJZFTPJS-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Endothelial Lipase Assay: To assay for cell surface lipase activity, cells expressing human endothelial lipase (EL) or LPL were plated in CellBIND® 384-well plates (Corning, Lowell, Mass.) in 25 μL serum free medium at a density of 2000 cells/well. After 18-24 hours incubation at 37° C., the medium was removed and replaced with 15 μL assay buffer [Hank's Buffered Saline Solution with 25 mM HEPES pH 7.2] and 15 μL PLA1 substrate for a final concentration of 10 μM using a Multidrop reagent dispenser. Fluorescence signal was monitored for 30 min at 37° C. on a Safire II plate reader in kinetic mode (60 cycles, kinetic interval: 30 seconds) with an excitation wavelength of 490 nm and an emission wavelength of 515 nm.
Measurement: IC50 = 101 nM (pChEMBL 7)
Descriptors: MW 482.47, ALogP 6.38, HBA 2, HBD 0, PSA 25.24, QED 0.39
Compound (SMILES): CC(C)OC(=O)/C=C\n1cnc(-c2cc(OCCN(C)C)cc(C(F)(F)F)c2)n1
InChIKey: FBDMJEQMMRUJJV-WAYWQWQTSA-N
Target: Exportin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14980
Assay: Inhibition of Crm1 in human U2OS cells expressing GFP-fused HIV1 Rev assessed as decrease in nuclear export of GFP-fused HIV1 Rev after 1 hr by Hoechst 33258 staining-based assay
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 412.41, ALogP 3.33, HBA 7, HBD 0, PSA 69.48, QED 0.49
Compound (SMILES): N#Cc1nc(-c2ccnc(Nc3ccc(NC(=O)CN4CCOCC4)nc3)n2)ccc1N1CC[C@H](F)C1
InChIKey: LKEHRNZWLGIXLL-KRWDZBQOSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 15 nM (pChEMBL 7.823999881744385)
Descriptors: MW 503.54, ALogP 2.37, HBA 10, HBD 2, PSA 132.19, QED 0.5
Compound (SMILES): Cc1cncc(C(=O)Nc2cccc(CN3CCC(C(=O)N4CC[C@H](F)C4)CC3)c2)c1
InChIKey: BLYLYYYSFJHHDJ-NRFANRHFSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 195 nM (pChEMBL 6.710000038146973)
Descriptors: MW 424.52, ALogP 3.42, HBA 4, HBD 1, PSA 65.54, QED 0.8
Compound (SMILES): CC(O)C(=O)N[C@@H](C)c1ccc(O[C@@H]2CCN(c3ccc(OCC4CC4)cc3)C2)cc1
InChIKey: BTCBAKVOIHWTHO-RDIXDNMASA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 200 μM f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 793 nM (pChEMBL 6.099999904632568)
Descriptors: MW 424.54, ALogP 3.69, HBA 5, HBD 2, PSA 71.03, QED 0.64
Compound (SMILES): CC1(C)Cc2nc(C3CCN(c4ncc(N5CCC(O)CC5)cn4)CC3)c([C@@H](F)c3ccc(C(F)(F)F)cc3)c(C3CCC(F)(F)CC3)c2[C@@H](O)C1
InChIKey: ANOPNZMZDISQDN-NHZFLZHXSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: CETP Inhibition Assay: A recombinant human CETP protein (manufactured by Roar Biomedical Inc.; 4.5 ng), the acceptor lipoprotein described in (2) above (32.5 ug) and 5,5'-dithio-bis-(2-nitrobenzoic acid) (7 mM, 15 ul) were taken in a 96 well plate and the total amount of the mixture was adjusted to 48.5 ul with the PBS solution. The test compound [DMSO solution (concentration: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 uM; 1.5 ul] was added to each well and the mixture was incubated in a thermostatic bath at 37° C. for 60 minutes. The reconstituted HDL (50 ul) described in (1) above was added to each well and the mixture was reacted in a thermostatic bath at 37° C. for 60 minutes. The 96 well plate was moved onto ice and a precipitation reagent [a mixed solution of magnesium chloride (60 mM) and 0.1% dextran sulfate [1/1 (v/v)]; 15 ul] was added to each well, and then the mixture was allowed to stand on ice for 15 minutes.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 731.83, ALogP 8.59, HBA 7, HBD 2, PSA 85.61, QED 0.25
Compound (SMILES): CNc1cc(-c2cn(C)nc2Oc2ccccn2)nn2c(C)nnc12
InChIKey: LUOSMBCXHFHGQV-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15nM GST-BRD4-BD1 protein (aa 44-168) or 150nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 336.36, ALogP 2.06, HBA 9, HBD 1, PSA 95.05, QED 0.61
Compound (SMILES): CC(C)(O)CCOc1cnc(N2CCC(c3nc4c(c(C5CCC(F)(F)CC5)c3[C@@H](F)c3ccc(C(F)(F)F)cc3)[C@@H](O)CC(C)(C)C4)CC2)nc1
InChIKey: XVUDQBUEUFZZQH-ZQAZVOLISA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: CETP Inhibition Assay: A recombinant human CETP protein (manufactured by Roar Biomedical Inc.; 4.5 ng), the acceptor lipoprotein described in (2) above (32.5 ug) and 5,5'-dithio-bis-(2-nitrobenzoic acid) (7 mM, 15 ul) were taken in a 96 well plate and the total amount of the mixture was adjusted to 48.5 ul with the PBS solution. The test compound [DMSO solution (concentration: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 uM; 1.5 ul] was added to each well and the mixture was incubated in a thermostatic bath at 37° C. for 60 minutes. The reconstituted HDL (50 ul) described in (1) above was added to each well and the mixture was reacted in a thermostatic bath at 37° C. for 60 minutes. The 96 well plate was moved onto ice and a precipitation reagent [a mixed solution of magnesium chloride (60 mM) and 0.1% dextran sulfate [1/1 (v/v)]; 15 ul] was added to each well, and then the mixture was allowed to stand on ice for 15 minutes.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 734.83, ALogP 9.17, HBA 7, HBD 2, PSA 91.6, QED 0.21
Compound (SMILES): COc1cc(N2CCC(O)CC2)ccc1Nc1nc2c(c(Nc3ccccc3S(=O)(=O)NC(C)C)n1)CCN2
InChIKey: OFLJPYMUAXJYGB-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 553.69, ALogP 3.59, HBA 10, HBD 5, PSA 140.74, QED 0.27
Compound (SMILES): CN[C@@H](C)C(=O)N[C@@H]1C(=O)N(Cc2c(OC)ccc3cc(Br)ccc23)c2ccccc2O[C@@]12CCOC2
InChIKey: HELQGTDTITZVDX-VVTZZXDESA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 568.47, ALogP 3.79, HBA 6, HBD 2, PSA 89.13, QED 0.47
Compound (SMILES): O=C(OC1C2CC3CC1CC(O)(C3)C2)N1CCC(c2cc[n+]([O-])cc2)C1
InChIKey: QYJRVXOBMULXMW-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: Scintillation Proximity Assay: Inhibition of microsomal preparations of 11.beta.-HSDl by compounds of the present invention, as described essentially previously (K. Solly, SS Mundt, HJ Zokian, GJ Ding, A. Hermanowski-Vosatka, B. Strulovici, and W. Zheng, High-Throughput Screening of 11-Beta-Hydroxyseroid Dehydrogenase Type 1 in Scintillation Proximity Assay Format. Assay Drug Dev Technol 3 (2005) 377-384) was measured. All reactions were carried out in a clear flexible PET Microbeta plates 96-well at room temperature (PerkinElmer). Assay, substrate solution (50mM HEPES, pH7.4,100mM KCl, 5mM NaCl, 2mM MgCl 2, 2mM NADPH and 160nM [3 H] cortisone (1Ci / mmol)) a 49ul and distribution, and starting from the 0.1mM on to start by combining the semi-log (half-log) multiple of the incremental test compound 1uL in DMSO previously diluted (8 points). After pre-incubation of 10 minutes, incubated human 11-HSD1 overexpress the enzyme containing CHO cells isolated from microsomes solution 50uL.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 358.44, ALogP 2.19, HBA 4, HBD 1, PSA 76.71, QED 0.65
Compound (SMILES): CC1(C)CC(=O)N(CCOc2ccc(F)c(C#N)c2)C1
InChIKey: GRUADGBFIDAGDY-UHFFFAOYSA-N
Target: Platelet-activating factor acetylhydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q13093
Assay: Inhibition of recombinant human Lp-PLA2 incubated for 20 mins by Thio-PAF assay
Measurement: IC50 = 15000 nM (pChEMBL 4.820000171661377)
Descriptors: MW 276.31, ALogP 2.33, HBA 3, HBD 0, PSA 53.33, QED 0.85