lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Oc1ccc(Cl)cc1/C=N/c1nc(-c2ccccc2)cs1
InChIKey: JBEDXEOJJBSVPS-GIJQJNRQSA-N
Target: Urease (SINGLE PROTEIN), Canavalia ensiformis
UniProt: P07374
Assay: Inhibition Assay: Reaction mixtures consisting of 25 μL (1 unit/well) of Jack bean (Canavalia ensiformis) urease, 55 μL of buffer at pH 6.8, 100 mM of urea, and 5 μL of various concentrations of test compounds were incubated at 30° C. for 15 min in 96-well plates. Subsequently 45 μL phenol reagents (1% w/v phenol and 0.005% w/v sodium nitroprussside), and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% w/v NaOCl) were added to each well. Urease activity through indophenols method was confirmed by the production of ammonia, as described by Weatherburn [12]. After 50 min, the increasing absorbance at 630 nm was measured in a microplate reader SpectraMax M2 (Molecular Devices, CA, USA). All reactions were performed in triplicate in a final volume of 200 μL.
Measurement: IC50 = 8130 nM (pChEMBL 5.090000152587891)
Descriptors: MW 314.8, ALogP 4.92, HBA 4, HBD 1, PSA 45.48, QED 0.7
Compound (SMILES): CC1Cc2c(Cl)cccc2N1C(=O)Cc1nc(N2CCOCC2)cc(=O)n1C
InChIKey: XMQJMBYTKNDURO-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Sandwich Immunoassay: This test is based on measurement of the expression of AKT protein phosphorylated on serine 473 (P-AKT-S473) in the PC3 human prostate carcinoma line, by the technique based on a sandwich immunoassay using the Kit MSD Multi-spot Biomarker Detection of Meso Scale Discovery: phospho-Akt kits (Ser473) whole cell lysate (# K151 CAD) or phospho-Akt (Ser473) / Akt whole cell lysate Total (# K15100D). The primary antibody specific for P-AKT-S473 (Kit # K151 CAD) is coated onto an electrode in each well of 96-well plates of the MSD kit: after adding a protein lysate in each well, the revelation of the signal done by the addition of a secondary antibody labeled with an electrochemiluminescent detection compound. The procedure is described in the kit.On day 1, the PC3 cells are inoculated in 96-well plates (TPP # 92096) at a concentration of 35000 cells / well in 200 uL DMEM medium (DMEM Gibco # 1 1960-044) containing 10% fetal calf serum (FBS Gibco, # 10500-056) and 1% glutamine.
Measurement: IC50 = 242 nM (pChEMBL 6.619999885559082)
Descriptors: MW 402.88, ALogP 1.79, HBA 6, HBD 0, PSA 67.67, QED 0.78
Compound (SMILES): CC(C)(C)c1ccc(N2C(=O)CC[C@@H]2COCc2ccc(C(=O)O)s2)cc1
InChIKey: BKVIUNNZVOCFGC-MRXNPFEDSA-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 387.5, ALogP 4.46, HBA 4, HBD 1, PSA 66.84, QED 0.79
Compound (SMILES): O=C(O)C(=O)Nc1ccc2nc(NC(=O)c3cc(Br)c(Br)[nH]3)sc2c1
InChIKey: ZDVZBRWEKLWNNU-UHFFFAOYSA-N
Target: DNA gyrase subunit B (SINGLE PROTEIN), Escherichia coli
UniProt: P0AES6
Assay: Inhibition of Escherichia coli DNA gyrase B ATP binding site assessed as reduction in supercoiling of relaxed pNO1 DNA after 30 mins in presence of biotinylated oligonucleotide by sybrGOLD staining based fluorescence assay
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 488.12, ALogP 3.42, HBA 5, HBD 4, PSA 124.18, QED 0.42
Compound (SMILES): Cc1cc(NC(=O)N2CCC(N3CC(CC#N)(n4cc(-c5ncnc6[nH]ccc56)cn4)C3)CC2)n(C)n1
InChIKey: KVMNGYIMVRBSMH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 499.58, ALogP 2.48, HBA 9, HBD 2, PSA 136.58, QED 0.43
Compound (SMILES): COc1c(Nc2ncc(Cl)c(N[C@H]3[C@@H](C(N)=O)[C@@H]4C=C[C@H]3C4)n2)ccc2c1CCC[C@@H](N1CCOCC1)C2
InChIKey: LIBCMUMCEYECFM-QZYSWDHYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of recombinant human INSR expressed in baculovirus using ATP as substrate after 15 mins by TRF assay
Measurement: IC50 = 103 nM (pChEMBL 6.989999771118164)
Descriptors: MW 539.08, ALogP 3.55, HBA 8, HBD 3, PSA 114.63, QED 0.36
Compound (SMILES): Cc1cc2ccc1[C@@H](C)COC(=O)Nc1cc(F)c(S(=O)(=O)C(C)C)c(c1)CN(C)C(=O)[C@@H]2Nc1cc(F)c2c(N)nccc2c1
InChIKey: HLPISXDQETYZEV-ADXZGYQBSA-N
Target: Coagulation factor VII (SINGLE PROTEIN), Homo sapiens
UniProt: P08709
Assay: In Vitro Assay: Tissue kallikrein-1 (37° C) activity was determined in reactions containing 0.05 nM enzyme and 90 μM substrate (H-D-Val-Leu-Arg-AFC) in buffer (0.1M sodium phosphate pH 7.4, 0.2 M NaCl, 0.5% PEG 8000, and 1% DMSO). Assays were performed using 96-well microtiter plates (COSTAR 3600, CORNING, NY, USA) and a thermostatic temperature controlled plate reader (SPECTRAMAX® Gemini, Molecular Devices, Sunnyvale, Calif., USA). Fluorescence was monitored using 400 nm excitation and 505 nm emission wavelengths.
Measurement: Ki = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 651.74, ALogP 6.06, HBA 8, HBD 3, PSA 143.72, QED 0.24
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1ccc(N2C[C@H](O)[C@@H](O)C2)c(-c2cccnc2)c1
InChIKey: TXRYJDJETFXTIP-SFTDATJTSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper ABL : The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 uL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 uM ATP, 4 uM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 uL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 uL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 459.42, ALogP 3.44, HBA 6, HBD 3, PSA 94.92, QED 0.54
Compound (SMILES): C[C@H](Nc1ncc(F)c(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1ccc(Cl)cc1Cl
InChIKey: CEORPLNBANSUEU-ATEUNZGCSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 145 nM (pChEMBL 6.840000152587891)
Descriptors: MW 415.25, ALogP 3.8, HBA 6, HBD 2, PSA 87.58, QED 0.77
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1ccc(OCCN2CCCC2)c(-c2cncnc2)c1
InChIKey: WPSWNDSKLXFMIS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Inhibition of ABL1 (64 to 515 residues)(unknown origin) expressed in Escherichia coli using FITC-Ahx-EAIYAAPFAKKK-NH2 peptide as substrate after 60 mins by caliper assay
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 472.47, ALogP 4.77, HBA 6, HBD 1, PSA 76.58, QED 0.51
Compound (SMILES): c1ccc(Oc2ccc(Cn3cnnc3-c3ccc4[nH]cnc4c3)cc2)cc1
InChIKey: CVGBPSGVCJCEFD-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase-like protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NXS2
Assay: Spectrophotometric Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R. C. J. 1989 J Neurosci Methods 30, 23-28) utilizing glutamate dehydrogenase as auxiliary enzyme. Samples consisted of the respective QC substrate, 0.3 mM NADH, 14 mM α-Ketoglutaric acid and 30 U/ml glutamate dehydrogenase in a final volume of 250 ul. Reactions were started by addition of QC and perused by monitoring of the decrease in absorbance at 340 nm for 8-15 min.The initial velocities were evaluated and the enzymatic activity was determined from a standard curve of ammonia under assay conditions. All samples were measured at 30° C., using either the SPECTRAFluor Plus or the Sunrise (both from TECAN) reader for microplates. Kinetic data was evaluated using GraFit software.
Measurement: IC50 = 480 nM (pChEMBL 6.320000171661377)
Descriptors: MW 367.41, ALogP 4.66, HBA 5, HBD 1, PSA 68.62, QED 0.49
Compound (SMILES): CNC(=O)c1cc(OC)cc(N(CCO)c2ccc3ncc(-c4cnn(CCOC)c4)nc3c2)c1
InChIKey: AIVBMTJXLHCJTQ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 476.54, ALogP 2.64, HBA 9, HBD 2, PSA 114.63, QED 0.36
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1cnc(N2CC[C@H](CO)C2)c(-c2ccc(CF)nc2)c1
InChIKey: CCOWODSAVFJFEA-HNNXBMFYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper ABL : The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 uL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 uM ATP, 4 uM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 uL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 uL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 490.46, ALogP 4.58, HBA 6, HBD 2, PSA 87.58, QED 0.47
Compound (SMILES): COc1cc(N2CCCCC2=O)ccc1Nc1nc2c(c(Nc3ccccc3S(=O)(=O)NC(C)C)n1)CCN2
InChIKey: ILDBEOWNTWUXNR-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
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 551.67, ALogP 4.14, HBA 9, HBD 4, PSA 137.58, QED 0.31
Compound (SMILES): O=C(Nc1ccc(C2CCNC2)cc1)c1cccc(Cl)c1
InChIKey: OMWKYYJZUDVQJA-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat 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 = 7.7 nM (pChEMBL 8.109999656677246)
Descriptors: MW 300.79, ALogP 3.67, HBA 2, HBD 2, PSA 41.13, QED 0.91
Compound (SMILES): COc1cc(N2CCOCC2)ccc1Nc1nc2c(c(Nc3ccccc3S(=O)(=O)NC3CCC3)n1)C1(CC1)CN2
InChIKey: SVLPSYRFJDMNJP-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
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 577.71, ALogP 4.1, HBA 10, HBD 4, PSA 129.74, QED 0.3
Compound (SMILES): CN1CCC[C@H]1C(=O)N1CCC(CCOc2ncc(-c3nc(C#N)nc4c3ncn4C)cc2C(F)(F)F)CC1
InChIKey: VZIIZGWZMUIFAB-IBGZPJMESA-N
Target: Cathepsin S (SINGLE PROTEIN), Mus musculus
UniProt: O70370
Assay: Inhibition Assay: First, spleen cells collected from C57BL/6 mice were seeded in a 96-well plate so as to be a 1× 105 cells/well, then, 5-fold dilution of 10 mM DMSO solution of the test compound was serially performed with RPMI 1640 medium (including 10% fetal bovine serum (FCS), 5×10^−5 M 2-mercaptoethanol, 50 IU/mL penicillin and 50 μg/mL streptomycin) nine times such that the final concentration becomes 0.026 nM to 10 μM, and the resultant products were added thereto (the final DMSO concentration was 0.1%). At the same time, LPS (Sigma L4005) was added to the wells such that the final concentration becomes 2 μg/mL, and culture was performed at 37° C. for 48 hours in 5% CO2. After culturing, the cells were stained at 4° C. for 20 minutes with PE-labeled streptavidin (BD BIOSCIENCE 554061) and biotin-labeled YAe antibody (EBIOSCIENCE 13-5741-85) together with FITC-labeled anti-mouse B220 antibody (BD BIOSCIENCE 553088), and an expression level (fluorescence intensity of YAe-bioti
Measurement: IC50 = 0.85 nM (pChEMBL 9.069999694824219)
Descriptors: MW 542.57, ALogP 3.42, HBA 9, HBD 0, PSA 113.06, QED 0.47
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCN2CC[C@]3(C[C@H]3c3ccc(F)cc3F)C2)n1C
InChIKey: XIHOJUYAGODVFH-JTSKRJEESA-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 = 25.12 nM (pChEMBL 7.599999904632568)
Descriptors: MW 445.54, ALogP 4.42, HBA 7, HBD 0, PSA 59.98, QED 0.4
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccnc(C(F)(F)F)c4)cn3CCN3CCC3)CC2)c1-c1cnoc1
InChIKey: MNAVNDWRKFKOGX-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 539.57, ALogP 4.08, HBA 10, HBD 1, PSA 115.02, QED 0.37
Compound (SMILES): Cc1nn(Cc2ccc(Oc3ccccc3)cc2)c2nc(-c3cccs3)cc(C(=O)O)c12
InChIKey: AOKJSDQDESVTNU-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 homolog (SINGLE PROTEIN), Mus musculus
UniProt: P97287
Assay: Fluorescence Polarization-Based Binding Assay: Sensitive and quantitative FP-based binding assays were developed and optimized to determine the binding affinities of small-molecule inhibitors to the recombinant Mcl-1, A1/Bfl-1, Bcl-w, Bcl-2, and Bcl-xL proteins. The concentrations of the proteins used in the competitive binding experiments were 10 nM for Mcl-1, 80 nM for Bcl-xL, and 60 nM for Bcl-2. The fluorescent probes, Flu-BID or FAM-BID were fixed at 2 nM for all assays, which binds with Kd values of 2.3 nM, 14.2 nM and 24.1 nM against Mcl-1, Bcl-2 and Bcl-xL respectively. 5 μL of the tested compound in DMSO and 120 μL of protein/probe complex in the assay buffer (20 mM phosphate pH 7.4, 50 mM NaCl, 1 mM EDTA. 0.05% Pluronic F68)s) were added to 96 well black assay plates, incubated at room temperature for 3 h and the polarization values (mP) were measured at an excitation wavelength at 485 nm and an emission wavelength at 530 nm using the plate reader Synergy H1 Hybrid, BioTek.
Measurement: IC50 = 6760 nM (pChEMBL 5.170000076293945)
Descriptors: MW 441.51, ALogP 6.01, HBA 6, HBD 1, PSA 77.24, QED 0.35
Compound (SMILES): CN1CCCN(c2cncc(-c3cc(CCO)no3)c2)CC1
InChIKey: ZDANANXJEHBAHQ-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor; alpha2/beta4 (PROTEIN COMPLEX), Rattus norvegicus
UniProt: P12392
Assay: Displacement of [3H]epibatidine from rat alpha2beta4 nAChR by liquid scintillation counting
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 302.38, ALogP 1.41, HBA 6, HBD 1, PSA 65.63, QED 0.92
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NC5CCN(CC(F)(F)F)CC5)c([N+](=O)[O-])c4)c(Oc4cccc5[nH]ccc45)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: DKXBXPNDIWPVSD-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 59.64 nM (pChEMBL 7.21999979019165)
Descriptors: MW 934.48, ALogP 9.86, HBA 10, HBD 3, PSA 153.15, QED 0.08
Compound (SMILES): Cn1cc(-c2ccc(OCC3CC3)c(-c3cn(C)c(=O)c4[nH]ccc34)c2)cn1
InChIKey: MMMAOGOGNTXXRN-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 625 nM (pChEMBL 6.199999809265137)
Descriptors: MW 374.44, ALogP 3.72, HBA 5, HBD 1, PSA 64.84, QED 0.58
Compound (SMILES): NC(=O)CCC(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)CC1CCCCC1
InChIKey: RTMJBVBJJCDQGF-NUNAXRQHSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 441.62, ALogP 3.78, HBA 4, HBD 2, PSA 86.87, QED 0.61
Compound (SMILES): CN1CCCc2ccc(NC(=O)N3CCC(F)(c4ncccc4F)CC3)cc21
InChIKey: UUQIOFAMJIAZCY-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 ("wash buffer"), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 ("assay buffer"). Plates are then transferred to a FLIPR for assay. The test compound is diluted in assay buffer, and 50 μL of the resultant solution is added to the cell plates and the solution is monitored for two minutes. The final concentration of the test compound is adjusted to range from about 50 picoM to about 3 μM. Agonist buffer (wash buffer titrated with 1N HCl to provide a solution having a pH of 5.5 when mixed 1:1 with assay buffer) (0.1 mL) is then added to each well, and the plates are incubated for 1 additional minute.
Measurement: IC50 = 1175.1 nM (pChEMBL 5.929999828338623)
Descriptors: MW 386.45, ALogP 4.1, HBA 3, HBD 1, PSA 48.47, QED 0.85
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1ccccc1C(F)(F)F
InChIKey: AUFAMKXCMIVFGS-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 808 nM (pChEMBL 6.090000152587891)
Descriptors: MW 393.41, ALogP 4.22, HBA 3, HBD 3, PSA 84.22, QED 0.46
Compound (SMILES): Cn1c(Nc2c(F)cccc2Cl)nc2cc(C(=O)Nc3cccc(C(F)(F)F)c3)c3ncccc3c21
InChIKey: QHNDTJXPCKDOAM-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition of mPGES-1 in human A549 cells assessed as reduction in interleukin-1 beta-induced PGE2 production incubated for 30 mins followed by IL-1beta stimulation for 16 to 20 hrs in presence of 2% fetal bovine serum by HTRF assay
Measurement: IC50 = 153.2 nM (pChEMBL 6.820000171661377)
Descriptors: MW 513.88, ALogP 6.93, HBA 5, HBD 2, PSA 71.84, QED 0.26
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(C(F)(F)F)c(Nc2ccc(N3CCN(C(=O)CO)CC3)cc2OC)n1
InChIKey: SFKOLTOIRPVPKB-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 658.68, ALogP 3.31, HBA 11, HBD 3, PSA 135.63, QED 0.31
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4ccn5c6ccc(O)nc6cc5c4n3)cc12
InChIKey: WVPBVKDXGKLKCU-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 33.86 nM (pChEMBL 7.46999979019165)
Descriptors: MW 609.64, ALogP 5.72, HBA 8, HBD 2, PSA 130.04, QED 0.26
Compound (SMILES): CNc1nc(NCc2ccc(NC(=O)c3ccc(F)cc3Cl)cc2)c2cc(C)ccc2n1
InChIKey: RRZIAHBPWFIHEA-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf22C11 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 > 1389 nM (pChEMBL 5.85699987411499)
Descriptors: MW 449.92, ALogP 5.64, HBA 5, HBD 3, PSA 78.94, QED 0.35
Compound (SMILES): CC(C)CNc1cc(NC2CC2)n2ncc(/C=C3\SC(=O)NC3=O)c2n1
InChIKey: CERNOHHTNQVWNF-XGICHPGQSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Inhibition Assay: 30 uM ATP: In a final reaction volume of 50 ul, recombinant PIM-1 (1 ng) was incubated with 12 mM MOPS pH 7.0, 0.4 mM EDTA, glycerol 1%, brij 35 0.002%, 2-mercaptoethanol 0.02%, BSA 0.2 mg/ml, 100 uM KKRNRTLTK (SEQ ID NO.: 5), 10 mM MgAcetate, 30 uM ATP, [γ-33P-ATP] (specific activity approx. 500 cpm/pmol), DMSO 4% and test inhibitor compound at the required concentration. The reaction was initiated by the addition of the Magnesium ATP mixture. After 40 min incubation at 23° C., the reactions were quenched by the addition of 100 ul 0.75% Phosphoric acid, and the labeled peptide collected by filtration through a phosphocellulose filter plate. The plate was washed 4 times with 0.075% phosphoric acid (100 ul per well) and then, after the addition of scintillation fluid (20 ul per well), the counts were measured by a scintillation counter.
Measurement: IC50 = 2063.8 nM (pChEMBL 5.679999828338623)
Descriptors: MW 372.45, ALogP 2.7, HBA 8, HBD 3, PSA 100.42, QED 0.67
Compound (SMILES): NC(=O)c1cc(-c2cn3cc(Br)ccc3n2)ccc1O
InChIKey: JLJIFIKOVCMCTP-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Inhibition of DYRK1A (unknown origin)
Measurement: IC50 = 2600 nM (pChEMBL 5.579999923706055)
Descriptors: MW 332.16, ALogP 2.57, HBA 4, HBD 2, PSA 80.62, QED 0.76
Compound (SMILES): COc1cc(Nc2nn(C3(CC#N)CCN(C(=O)OC(C)(C)C)CC3)cc2C(N)=O)ccn1
InChIKey: VFPOCCFTSWBEBY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 146 nM (pChEMBL 6.840000152587891)
Descriptors: MW 455.52, ALogP 2.77, HBA 9, HBD 2, PSA 148.39, QED 0.68
Compound (SMILES): Cc1nn(C)c(C)c1N(C)S(=O)(=O)c1c(Cl)cc(-c2cccc(CNC3CCCCC3)c2)cc1Cl
InChIKey: BOIVPYGWJBISQP-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30419
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 4480 nM (pChEMBL 5.349999904632568)
Descriptors: MW 535.54, ALogP 6.26, HBA 5, HBD 1, PSA 67.23, QED 0.39
Compound (SMILES): COc1cccc(C2(C(F)(F)F)CC(c3ccc(C)cc3)=C(C#N)C(=O)N2)c1
InChIKey: VAIQMICSZYVISL-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibition of human recombinant MGAT2 expressed in Sf9 cell membrane using 2-monooleglycerol and [H3]-oleoyl-CoA as substrates incubated for 20 mins by scintillation proximity assay
Measurement: IC50 = 1905 nM (pChEMBL 5.71999979019165)
Descriptors: MW 386.37, ALogP 4.26, HBA 3, HBD 1, PSA 62.12, QED 0.86
Compound (SMILES): CC(=O)NC1=NN(C(C)=O)C(c2cn(-c3ccccc3)nc2-c2ccc([N+](=O)[O-])cc2)S1
InChIKey: RYXOFWLXQLIRLX-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35354
Assay: Inhibition of recombinant human COX2 assessed as reduction in PGF2alpha production from PGH2 by enzyme immunoassay
Measurement: IC50 = 20000 nM (pChEMBL 4.699999809265137)
Descriptors: MW 450.48, ALogP 3.45, HBA 8, HBD 1, PSA 122.73, QED 0.48
Compound (SMILES): COc1cc(-c2cn(CC(=O)Nc3cccc(Cl)c3Cl)nn2)ccc1-n1cnc(C)c1
InChIKey: XWKVYBKJUXFKMD-UHFFFAOYSA-N
Target: Probable G-protein coupled receptor 142 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z601
Assay: Agonist activity at human GPR142 expressed in CHO cells measured after 30 to 60 mins by IP-One assay
Measurement: EC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 457.32, ALogP 4.39, HBA 7, HBD 1, PSA 86.86, QED 0.46
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#CC1CCCCC1)c1nc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: XIJMBFHREMJADV-SFHVURJKSA-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: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., 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).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 499.02, ALogP 5.52, HBA 7, HBD 2, PSA 98.72, QED 0.36
Compound (SMILES): N#Cc1c(N)nc(N)nc1N[C@H](c1nc2c(F)ccc(Cl)c2c(=O)n1-c1ccc(N)nc1)C1CC1
InChIKey: VOKQIKXWIGKAPD-INIZCTEOSA-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: 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 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., 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; 10lps delay and 500 μs read window).
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 492.91, ALogP 2.54, HBA 11, HBD 4, PSA 187.44, QED 0.32
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cc4ccccc4s3)CC2)C1
InChIKey: CMBZNHSLRJYIHI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of human N-terminal His-tagged JAK2 catalytic domain (828 to 1132 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 10 nM (pChEMBL 8)
Descriptors: MW 522.64, ALogP 4.27, HBA 8, HBD 1, PSA 106.73, QED 0.37
Compound (SMILES): C[C@]1(N2CCc3c(-c4cnc(N)nc4)nc(N4CCOCC4)nc32)CCN(S(C)(=O)=O)C1
InChIKey: ROYQBLPXACEGTQ-FQEVSTJZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition of recombinant human N-terminal Myr signal and C-terminal Flag-tagged PI3K p110beta expressed in rat Rat1 cells
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 460.56, ALogP 0.14, HBA 10, HBD 1, PSA 130.67, QED 0.67
Compound (SMILES): COc1cccc(OC)c1C1CCCC(=O)N1Cc1ccc(-c2ccccc2)s1
InChIKey: GSZNHHZGYCSYMK-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 407.54, ALogP 5.69, HBA 4, HBD 0, PSA 38.77, QED 0.53
Compound (SMILES): CS(=O)(=O)c1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: ZSQDUTTYOZJDEM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 410.5, ALogP 2.39, HBA 7, HBD 1, PSA 96.25, QED 0.55
Compound (SMILES): O=C(OC(c1ccc(Cl)c(Cl)c1)C1CNC1)c1ccccc1
InChIKey: XXXHDRXZALCTPU-UHFFFAOYSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Binding Assay: NE transporter binding assay for evaluating binding of the compound to the norepinephrine transporter was carried out using human recombinant norepinephrine transporter membrane (PerkinElmer Life and Analytical Sciences, USA) expressed in MDCK cells and radioisotope [3H]Nisoxetine (PerkinElmer).That is, the test drug, 6 nM [3H]Nisoxetine, norepinephrine transporter membrane (11 ug/well), 120 mM NaCl and 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM KCl were added to obtain a reaction mixture with a final volume of 0.25 ml. After incubation for 60 minutes at 4° C., the mixture was quickly passed through a Filtermat A glass fiber filter pre-soaked with 0.5% (w/v) PEI (polyethyleneimine) using Inotech Harvester (Inotech) to terminate the reaction. After washing with cold washing buffer (50 mM Tris-HCl, pH 7.4, 0.9% NaCl) solution, the filter was covered with MeltiLex and sealed in a sample bag. After drying in an oven, radioactivity was counted using MicroBeta Plus (Wallac).
Measurement: IC50 = 214 nM (pChEMBL 6.670000076293945)
Descriptors: MW 336.22, ALogP 4.11, HBA 3, HBD 1, PSA 38.33, QED 0.85