lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): c1c2c(cc3c1OCO3)CN1CCc3cc4c(cc3[C@@H]1C2)OCO4
InChIKey: DEXCWVBNPJKCOQ-HNNXBMFYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Reception Binding Assay: Different concentrations (10^−5 M-10^−11 M) of the compound of the invention and corresponding isotope receptor ligand as well as receptor protein were loaded into the reaction tube and incubated in 30° C. water bath for 60 minutes. The reaction was terminated in a refrigerator. The reaction mixture was put in a Millipore filter (millipore) cell sample collector, filtered through suction filtration using GF/C glass fiber filter paper, and dried. The resulting sample was placed into 0.5 mL tube. 500 μL liquid scintillation fluid was added and intensity of radioactivity was determined by counting. For dopamine D1 receptor: isotope receptor ligands [3H] SCH23390 (85.0 Ci/mmol) (D1-selective, purchased from Amersham Corporation), D1 receptor protein expressed in HEK-293 cells; For D2 dopamine receptor: isotope receptor ligands [3H] Spiperone (77.0 Ci/mmol) (D2-selective, purchased from Amersham Corporation); D2 receptor protein expressed in HEK-293 cells; For 5-HT1A receptor: isotope receptor ligands [3H] 8-OH-DPAT; 5-HT1A receptor protein expressed in HEK-293 cells; For 5-HT2A receptor: isotope receptor ligands [3H]-Ketanserin; 5-HT2A receptor protein expressed in HEK-293 cells.
Measurement: IC50 = 1239.4 nM (pChEMBL 5.909999847412109)
Descriptors: MW 323.35, ALogP 2.8, HBA 5, HBD 0, PSA 40.16, QED 0.75
Compound (SMILES): CC(C)(C)OC(=O)N[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: FQRUKUIMJXRNOI-HAQNSBGRSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 > 1000 nM (pChEMBL 6)
Descriptors: MW 356.43, ALogP 3.16, HBA 6, HBD 2, PSA 97.2, QED 0.73
Compound (SMILES): CCn1nc(-c2cccc(-c3cc(OCC4CC4)cc(C(=O)NCCN4CCCCC4)c3)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: MTDITNPYKYPDNS-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibition of human recombinant PDE4B1 assessed as reduction in [3H]cAMP hydrolysis to [3H]AMP incubated for 60 mins by PDE-SPA assay
Measurement: IC50 = 0.15 nM (pChEMBL 9.819999694824219)
Descriptors: MW 704.66, ALogP 6.95, HBA 8, HBD 3, PSA 130.48, QED 0.15
Compound (SMILES): Cn1c(=O)n(-c2cccc(-c3c(Cl)cc(C(N)=O)c4[nH]c5cc(C(C)(C)O)ccc5c34)c2Cl)c(=O)c2cccc(F)c21
InChIKey: MHQPVCNAVZURHD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Jak2 Tyrosine Kinase Assay: The assays were performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (100 mM HEPES pH 7.4, 10 mM MgCl2, 25 mM beta-glycerolphosphate, 0.015% Brij 35 surfactant and 4 mM DTT). The reaction was initiated by the combination of Jak2 tyrosine kinase with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 minutes and terminated by adding 45 μL of 35 mM EDTA to each sample.
Measurement: IC50 = 3700 nM (pChEMBL 5.429999828338623)
Descriptors: MW 605.45, ALogP 5.76, HBA 6, HBD 3, PSA 123.11, QED 0.24
Compound (SMILES): O=C(CN1CCC(C(=O)c2ccc(OC3CCOCC3)cc2)CC1)NCc1nc2c(c(=O)[nH]1)COCC2
InChIKey: ONZUYVRREURPTH-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 2840 nM (pChEMBL 5.550000190734863)
Descriptors: MW 510.59, ALogP 1.61, HBA 8, HBD 2, PSA 122.85, QED 0.51
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(CN3CCOCC3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: MODQVRVNLINUQU-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 495.59, ALogP 3.21, HBA 9, HBD 1, PSA 104.09, QED 0.4
Compound (SMILES): CN1CC2(C1)C(=N)N[C@](C)(c1cc(NC(=O)c3ccc(Cl)cn3)ccc1F)CS2(=O)=O
InChIKey: JOSATGAYPZWFMY-IBGZPJMESA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Inhibition of human BACE2 preincubated for 30 mins followed by QSY7- EISEVNLDAEFC-Eu-amide substrate addition and measured after 1.5 hrs by HTRF assay
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 465.94, ALogP 2.02, HBA 6, HBD 3, PSA 115.25, QED 0.64
Compound (SMILES): Cc1cn(-c2ccc(-c3cn(CC(=O)Nc4cccc(Cl)c4Cl)nn3)cc2F)cn1
InChIKey: PRSMZNFUKBHAIS-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 for 3 to 5 mins by Fluo-4AM dye-based FLIPR assay
Measurement: EC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 445.29, ALogP 4.52, HBA 6, HBD 1, PSA 77.63, QED 0.49
Compound (SMILES): Cc1n[nH]c2ncc(C(=O)N3CCC[C@H]3c3ccc(Cl)cc3)cc12
InChIKey: ODRITQGYYWHQGM-INIZCTEOSA-N
Target: Cyclin-dependent kinase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: P49336
Assay: Inhibition of CDK8 in human LS174T cells expressing beta-catenin mutant assessed as suppression of WNT pathway by luciferase reporter gene assay
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 340.81, ALogP 3.9, HBA 3, HBD 1, PSA 61.88, QED 0.77
Compound (SMILES): O[C@H]1CCN(C[C@H]2CC[C@@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: FKHKOSJBNTWNIX-MJBXVCDLSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 > 1000 nM (pChEMBL 6)
Descriptors: MW 340.43, ALogP 1.95, HBA 6, HBD 2, PSA 82.34, QED 0.76
Compound (SMILES): Cc1ccn2nc([C@H](C)Nc3ncnc(N)c3-c3cnc(N)nc3)n(-c3ccccc3)c(=O)c12
InChIKey: VQWQZPAHKOHXCV-AWEZNQCLSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human N-terminal His6-tagged full length p110gamma using PIP2 as substrate preincubated for 30 mins followed by substrate addition by HTRF assay
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 454.5, ALogP 2.38, HBA 11, HBD 3, PSA 154.93, QED 0.36
Compound (SMILES): COCC(=O)N[C@H]1CC[C@H](CCN2CCN(c3nsc4ccccc34)CC2)CC1
InChIKey: DSNSWHGDQTZOTM-IYARVYRRSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Displacement of [3H]ketanserin from recombinant human 5-HT2A receptor expressed in HEK293 cell membranes after 60 mins by scintillation counting method
Measurement: Ki = 0.24 nM (pChEMBL 9.619999885559082)
Descriptors: MW 416.59, ALogP 3.13, HBA 6, HBD 1, PSA 57.7, QED 0.75
Compound (SMILES): C[C@@H]1C[C@@H]2C[C@H](C1)C[C@@H](N1[C@@H]3CCC[C@H]1C[C@@H](n1c(=O)c(N4CC(CC(=O)O)C4)nc4ccccc41)C3)C2
InChIKey: FPFHILQBEGTPLJ-NYALPDIWSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: In Vitro Delta-Opioid Receptor Binding Assay: Radioligand dose-displacement assays used 0.2 nM [3H]-Naltrindole (NEN; 33.0 Ci/mmole) with 10-20 μg membrane protein (recombinant delta opioid receptor expressed in CHO-K1 cells; Perkin Elmer) in a final volume of 500 μL binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 μM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 h at a temperature of about 25 °C. Binding reactions were determined by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by five filtration washes with 500 μL ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 1-2 hours. Fifty μL/well scintillation cocktail (MicroScint20, Packard) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki = 3990 nM (pChEMBL 5.400000095367432)
Descriptors: MW 518.7, ALogP 5.08, HBA 6, HBD 1, PSA 78.67, QED 0.59
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3ncccc3C(F)(F)F)CC2)C1
InChIKey: PTPWIXULKPNGSO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In vitro JAK Kinase Assay: 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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 550.55, ALogP 3.86, HBA 8, HBD 2, PSA 131.65, QED 0.39
Compound (SMILES): COc1cc(OC)nc(OCCn2cc(NC(=O)c3ncoc3-c3cccc(C)c3)cn2)n1
InChIKey: HXSNNYUKIYWXTE-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: 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 μg/mL G418, 100 U/mL penicillin, 100 μg/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 μM 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. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μl/well, incubated for 120 min and finally 10 μl/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 450.46, ALogP 2.99, HBA 10, HBD 1, PSA 126.42, QED 0.41
Compound (SMILES): O=S(=O)(CC[C@@H]1OCC[C@@]2(S(=O)(=O)c3ccc(Cl)cc3)c3c(F)ccc(F)c3OC[C@@H]12)CCN1CCNCC1
InChIKey: UVQHTQRPRAFTQL-CHZKFRDHSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of CYP3A4 in human liver microsomes preincubated for 30 mins after 30 secs by LC/MS/MS analysis
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 605.12, ALogP 2.8, HBA 8, HBD 1, PSA 102.01, QED 0.49
Compound (SMILES): CCn1nc(-c2cccc(-c3cncc(O)c3)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: SEGVDOJXNRJZCS-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibition of human recombinant PDE4B1 assessed as reduction in [3H]cAMP hydrolysis to [3H]AMP incubated for 60 mins by PDE-SPA assay
Measurement: IC50 = 0.085 nM (pChEMBL 10.069999694824219)
Descriptors: MW 497.34, ALogP 5.04, HBA 7, HBD 3, PSA 122.03, QED 0.36
Compound (SMILES): CCCCCC(O)c1ccc([C@H]2CCC(=O)[C@@H]2CCCc2ccc(C(=O)O)s2)cc1
InChIKey: BKEIZHJYCQKMOM-JAZPPYFYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Agonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 428.59, ALogP 6.15, HBA 4, HBD 2, PSA 74.6, QED 0.42
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3ccncc3C(F)(F)F)CC2)C1
InChIKey: HVPCXDYPRNQTOB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In vitro JAK Kinase Assay: 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 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 550.55, ALogP 3.86, HBA 8, HBD 2, PSA 131.65, QED 0.39
Compound (SMILES): Cc1cccc2cc([C@H](C)NC3CCOCC3)n(-c3ccccc3)c(=O)c12
InChIKey: JZWXUZDIBDPYCG-KRWDZBQOSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 362.47, ALogP 4.13, HBA 4, HBD 1, PSA 43.26, QED 0.76
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3ccccc3OC)ncc2C(F)(F)F)c1
InChIKey: ARSGAAANDMOADX-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13x ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1x kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10x stock, KB002A) and 0.2 mM DTT (DS001A). 5 uL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 25° C. with a 0.5 uL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 uL of the ATP/Tyr-Sox peptide substrate mix and monitored every 71 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 429.4, ALogP 5.12, HBA 6, HBD 3, PSA 88.17, QED 0.45
Compound (SMILES): COc1cnc2[nH]cc(C(=O)c3c(F)ccc(NS(=O)(=O)c4ccccc4F)c3F)c2c1
InChIKey: WRUFUIGQGIPKPM-UHFFFAOYSA-N
Target: Cytochrome P450 1A2 (SINGLE PROTEIN), Homo sapiens
UniProt: P05177
Assay: Inhibition of CYP1A2 (unknown origin) using CEC as substrate after 15 mins
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 461.42, ALogP 4.02, HBA 5, HBD 2, PSA 101.15, QED 0.42
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1NC1CC(F)(F)C1
InChIKey: KBQOSTHTFJGZBY-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 383.4, ALogP 3.43, HBA 4, HBD 3, PSA 82.7, QED 0.65
Compound (SMILES): CCOc1ccc(NC(=S)N(CCO)Cc2cc3cc(C)cc(C)c3[nH]c2=O)cc1
InChIKey: PSKUPWXOVUAUJD-UHFFFAOYSA-N
Target: Beta-glucuronidase (SINGLE PROTEIN), Homo sapiens
UniProt: P08236
Assay: Cell-Based Assay: PheG Assay: Two ß-glucuronidase activity assays were employed to examine the potency of inhibitors both in vitro and in cell-based studies. An absorbance assay based on the conversion of p-nitrophenyl glucuronide (PNPG) to p-nitrophenol (PNP) (Szasz (1967) Clin Chem 13:752-759), which absorbs at 410 nm, was employed as the primary in vitro assay. A secondary assay involving the conversion of phenolphthalein glucuronide (PheG) to phenolphthalein (Phe), which absorbs at 540 nm, was also employed. Id. Both assays were validated in vitro and in living cells, and the wavelengths monitored did not overlap with absorbance characteristics of putative inhibitors.
Measurement: EC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 425.55, ALogP 3.73, HBA 4, HBD 3, PSA 77.59, QED 0.5
Compound (SMILES): COC(=O)c1cnc(N2CCN(c3nnc(C(=O)c4ccccc4)c(C)c3C)C[C@H]2C)cn1
InChIKey: LWUAPVQWCGLGMN-OAHLLOKOSA-N
Target: Protein smoothened (SINGLE PROTEIN), Homo sapiens
UniProt: Q99835
Assay: Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 446.51, ALogP 2.62, HBA 9, HBD 0, PSA 101.41, QED 0.43
Compound (SMILES): Cc1noc(C)c1-c1ccc2c(c1)C(c1ccccc1)(N1CCC[C@H](O)C1)C(=O)N2
InChIKey: UXFXOEGGFKVCDW-XGLRFROISA-N
Target: Bromodomain testis-specific protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q58F21
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The assay was carried out in binding mixtures of the bromodomain protein, 0-10 μM compounds and tetra-acetylated histone peptide (SGRGAC.KGGAC.KGLGAC.KGGAAC.KRHGSGSK-biotin) in buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 0.1% BSA, 0.05% CHAPS, and detection reagents. The detection reagents, including streptavidin-labeled Tb cryptate and XL665-labeled anti-6×His antibody, were added after binding equilibrium was achieved for protein, compound and peptide. Upon further incubation for 1 hr, the TR-FRET signals were recorded on a BMG PHERAstar FS instrument.
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 403.48, ALogP 3.61, HBA 5, HBD 2, PSA 78.6, QED 0.7
Compound (SMILES): Nc1ccc(-c2ccc3ncc4c(=O)[nH]c(=O)n(-c5cccc(C(F)(F)F)c5)c4c3n2)cn1
InChIKey: OIOOKLQHEDGJOE-UHFFFAOYSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13131
Assay: Mobility Shift Assay: Agents: 1-fold kinase buffer without MnCl2: 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT. 1-fold kinase buffer with MnCl2: 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 10 mM MnCl2, 2 mM DTT. Termination solution: 100 mM HEPES, pH 7.5, 0.0015% Brij-35, 0.2% Coating Reagent#3, 50 mM EDTA. 2.5-fold kinase solution: The kinase was added to the 1-fold kinase buffer to form a 2.5-fold kinase solution. 2.5-fold substrate solution: FAM fluorescence-labelled polypeptide and ATP were added to the 1-fold kinase buffer to form a 2.5-fold substrate solution. Compounds: The final test concentration of the compound was 10 μM at maximum. Firstly, a 50-fold concentration (i.e. 500 μM) was prepared with 100% DMSO. The compound was diluted with 100% DMSO by 5 folds at 5 concentrations (successively 500 μM, 100 μM, 20 μM, 4 μM, and 0.8 μM). After diluting with 1-fold kinase buffer by 10 folds, 5 μl of the samples were transferred to a 384-well reaction plate. Two negative control wells and two positive control wells were set at each line respectively. Replication was made in a 96-well plate in five concentrations of 5-fold compound. 10% DMSO was added to the positive control well, and 5 μl of EDTA (250 mM) was added to the negative control well. Procedure: To the 384-well reaction plate was added 5-fold compound dissolved in 10% DMSO at 5 μl/well. To the 384-well reaction plate was added 2.5-fold kinase solution at 10 Î¼l/well. The plate was incubated at room temperature for 10 mins. To the 384-well reaction plate was added 2.5-fold substrate solution at 10 μl/well. The plate was incubated at 28° C. for a certain period. 25 μl termination solution was added to terminate the reaction.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 450.38, ALogP 3.29, HBA 7, HBD 2, PSA 119.55, QED 0.4
Compound (SMILES): C[C@H]1CN(c2ccc(C(C)(C)O)cn2)CCN1c1nnc(Cc2ccccc2Cl)c2c1CCC2
InChIKey: YVODGHXNTWUMSD-SFHVURJKSA-N
Target: Protein smoothened (SINGLE PROTEIN), Homo sapiens
UniProt: Q99835
Assay: Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 478.04, ALogP 4.55, HBA 6, HBD 1, PSA 65.38, QED 0.58
Compound (SMILES): CNS1(O)C[C@@](C)(c2cc(NC(=O)c3ccc(F)cn3)ccc2F)NC(=N)N1C
InChIKey: LIAVENAQWXAHHF-SFHVURJKSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: HTRF FRET Assay: BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30Â° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies).
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 424.48, ALogP 2.62, HBA 5, HBD 5, PSA 113.37, QED 0.52
Compound (SMILES): COc1cc(N2CCC(N(C)C)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: MIPKRMBRGKCCEI-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of recombinant human full length ErbB2 expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 674.76, ALogP 7.27, HBA 9, HBD 2, PSA 99.92, QED 0.17
Compound (SMILES): CC(=O)NCc1nc2c(-c3cnc[nH]3)cc(N3CCOCC3)nn2c1Cc1cccc(C(F)(F)F)c1C
InChIKey: GLORCTPRDHXJPK-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Binding affinity to human PI3Kgamma (144 to 1102 residues) expressed in mammalian expression system by KINOMEscan assay
Measurement: Kd > 30200 nM (pChEMBL 4.519999980926514)
Descriptors: MW 513.52, ALogP 3.51, HBA 7, HBD 2, PSA 100.44, QED 0.41
Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(C(F)(F)F)c2)C(=S)N1c1ccc(C(=O)NCCCCCCNS(N)(=O)=O)c(F)c1
InChIKey: HRMCCGXDCREFJC-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Inhibition of HDAC6 (unknown origin) assessed as inhibition of fluorogenic peptide deacetylation
Measurement: IC50 = 1120 nM (pChEMBL 5.949999809265137)
Descriptors: MW 628.67, ALogP 3.72, HBA 6, HBD 3, PSA 148.63, QED 0.2
Compound (SMILES): Cn1c(SCCCN2CC[C@]3(C[C@@H]3c3ccc(C(F)(F)F)cc3)C2)nnc1C1CC2CCC(C1)O2
InChIKey: IQKUCRAAVAKXAF-CEJRHITASA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Antagonist activity at human dopamine D2L receptor expressed in CHO cells coexpressing Galpha16 assessed as inhibition of dopamine-induced Ca2+ stimulation pre-incubated for 10 mins followed by dopamine challenge by Fluo-4AM based fluorometric analysis
Measurement: Ki = 537.03 nM (pChEMBL 6.269999980926514)
Descriptors: MW 506.64, ALogP 5.62, HBA 6, HBD 0, PSA 43.18, QED 0.36
Compound (SMILES): N#Cc1ccc(CN[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: VJNAQIVEIXLOPK-QAQDUYKDSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 371.45, ALogP 3.29, HBA 6, HBD 2, PSA 94.69, QED 0.57
Compound (SMILES): COc1cc(OC)cc(N(C[C@@H](O)CNC(C)C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: PUCMKYWRDKWWQJ-BOXHHOBZSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (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 476.58, ALogP 3.54, HBA 9, HBD 2, PSA 97.56, QED 0.36
Compound (SMILES): O=S(=O)(Nc1ccc(-c2nc3cc(NS(=O)(=O)c4ccccc4)ccc3o2)cc1)c1ccccc1
InChIKey: PESZCPYNSKOSJW-UHFFFAOYSA-N
Target: GroEL/GroES (PROTEIN COMPLEX), Escherichia coli
UniProt: Q7BGE6
Assay: Inhibition of Escherichia coli GroEL/ES-mediated refolding of the denatured rhodanese
Measurement: IC50 = 18000 nM (pChEMBL 4.75)
Descriptors: MW 505.58, ALogP 5.1, HBA 6, HBD 2, PSA 118.37, QED 0.32
Compound (SMILES): CN1CN([C@H]2CC[C@H](CNC3(C(F)(F)F)CC3)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: KNSNQUJTQSTIFH-HDJSIYSDSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 421.47, ALogP 3.66, HBA 4, HBD 2, PSA 64.26, QED 0.79
Compound (SMILES): COc1cc(O)cc(N(CCN)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: OGIVRWCGCBWJHX-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (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 390.45, ALogP 2.84, HBA 8, HBD 2, PSA 102.32, QED 0.52
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2C3CCC2COC3)nc(N2CCOCC2)n1
InChIKey: YJAFXQYRMGBMNV-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 0.376 nM (pChEMBL 9.430000305175781)
Descriptors: MW 553.59, ALogP 4.93, HBA 7, HBD 1, PSA 79.82, QED 0.48
Compound (SMILES): COc1cc(C2CCN(CCS(C)(=O)=O)CC2)c(C)cc1Nc1nccc(-c2c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: SDFYMETZQYDPPX-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of recombinant human full length ErbB2 expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 781.89, ALogP 7.28, HBA 11, HBD 2, PSA 140.05, QED 0.13
Compound (SMILES): CN(C)C/C=C/C(=O)N1CCCC1Cn1nc(-c2ccc(Oc3ccccc3)cc2)c2c(N)ncnc21
InChIKey: DINVXRZOGZOFRM-YRNVUSSQSA-N
Target: Tyrosine-protein kinase Lck (SINGLE PROTEIN), Homo sapiens
UniProt: P06239
Assay: HotSpot kinase assay: IC50s were determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 μM ATP.). For enzyme inhibition assays, compounds were tested in range of ten concentrations from 10 uM to 0.0005 uM using purified enzymes and the Hotspot kinase assay. Reaction conditions were 1 uM ATP, one hour incubation with inhibitor, and kinase activity detected using 33-ATP phosphorylation of an appropriately selected peptide substrate.
Measurement: IC50 = 2.43 nM (pChEMBL 8.609999656677246)
Descriptors: MW 497.6, ALogP 3.98, HBA 8, HBD 1, PSA 102.4, QED 0.37
Compound (SMILES): C[C@H]1[C@H]2Cc3ccc(C(N)=O)cc3[C@]1(C)CCN2CC1CCC1
InChIKey: PCGVHHYPMDIXBR-CYFNAFFESA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Opioid Receptor Binding Assay: The Ki (binding affinity) for opioid receptors was determined using a competitive displacement assay as previously described in Neumeyer (Journal of Med. Chem. 2012, p 3878), which is incorporated herein in its entirety. Briefly, membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed the cloned human opioid receptor were incubated with 12 different concentrations of the compound set forth herein in the presence of 0.25 nM [3H]DAMGO (see Tiberi et al., Can. J. Physiol. Pharmacol. 1988, Vol. 66, p 1368, which is incorporated by reference herein in its entirety) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO (see Gulati et al., Life Sci. 1990, Vol. 47, p 159, which is incorporated by reference herein in its entirety). Nonspecific binding was measured by inclusion of 10 M naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. IC50 values were calculated by least squares fit to a logarithm-probit analysis. Ki values of unlabelled compounds were calculated from the equation Ki=(IC50)/1+S where S=(concentration of radioligand)/(Kd of radioligand) (Cheng and Prusoff, 1973).
Measurement: Ki = 0.1 nM (pChEMBL 10)
Descriptors: MW 312.46, ALogP 3.11, HBA 2, HBD 1, PSA 46.33, QED 0.93
Compound (SMILES): Cc1cc(Nc2cc(C)c(C#N)c(NCCOc3ccccc3F)n2)n[nH]1
InChIKey: AWLBOIUGNGQJSU-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of CYP3A4 (unknown origin)
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 366.4, ALogP 3.67, HBA 6, HBD 3, PSA 98.65, QED 0.55
Compound (SMILES): CC(=O)N[C@H]1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: VAMYQOUPUFFEEZ-JYJNAYRXSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 > 1000 nM (pChEMBL 6)
Descriptors: MW 381.48, ALogP 2.09, HBA 6, HBD 2, PSA 91.21, QED 0.72
Compound (SMILES): Cc1ccccc1-n1c(-c2cc(C(=O)N(C)Cc3ccccc3)c(O)cc2O)n[nH]c1=O
InChIKey: XOWIZECDQINJIB-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Recombinant Hsp90 [His-Tev-huHsp90-alpha (9-236)] was immobilized on a Biacore CM5 chip at 25 degrees C and a flow rate of 30 uL/min using amine coupling at pH 4.50 according to Biacore standard protocol. Hsp90 was applied at a concentration of 20 ug/mL with 50uM 17-DMAG.Data sets were processed and analyzed using the software Biacore 4000 Evaluation. Solvent corrected and double-referenced association and dissociation phase data were fitted to a simple 1:1 interaction model with mass transport limitations
Measurement: Kd = 9.767 nM (pChEMBL 8.010000228881836)
Descriptors: MW 430.46, ALogP 3.22, HBA 6, HBD 3, PSA 111.45, QED 0.45
Compound (SMILES): CCOc1ccc(C(=O)C2CCN(CC(=O)NCc3nc4c(c(=O)[nH]3)COCC4)CC2)cc1
InChIKey: DBNHRWOXIIAGGS-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 3200 nM (pChEMBL 5.5)
Descriptors: MW 454.53, ALogP 1.45, HBA 7, HBD 2, PSA 113.62, QED 0.58
Compound (SMILES): COc1cc(OC)c(F)c(N(CCO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1F
InChIKey: JCQODDIJVHORMY-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (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 441.44, ALogP 3.46, HBA 8, HBD 1, PSA 85.53, QED 0.47
Compound (SMILES): O=C(Cn1c2c(c(-c3cccc(Cl)c3)cc1=O)C(=O)NC2)Nc1ccc(-c2nnn[nH]2)cc1
InChIKey: BUVUMAPNDQFBAF-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibition of human plasma Kallikrein using H-Pro-Phe-Arg-AMC as substrate measured for 30 mins by fluorometric assay
Measurement: IC50 = 43000 nM (pChEMBL 4.369999885559082)
Descriptors: MW 461.87, ALogP 2.23, HBA 7, HBD 3, PSA 134.66, QED 0.42
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3ccc(Cl)cc3F)CC2)C1
InChIKey: RNPPZDLOPXOTEW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In vitro JAK Kinase Assay: 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 < 10 nM (pChEMBL 8)
Descriptors: MW 534, ALogP 4.23, HBA 7, HBD 2, PSA 118.76, QED 0.4
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4ccc(OC(F)F)c(Cl)c4)cs3)cccc2n(C)c1=O
InChIKey: SHEZWPJUOWPXDN-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 506.92, ALogP 3.8, HBA 8, HBD 1, PSA 95.22, QED 0.43
Compound (SMILES): CNCCS(=O)(=O)CC[C@@H]1OCC[C@@]2(S(=O)(=O)c3ccc(Cl)cc3)c3c(F)ccc(F)c3OC[C@@H]12
InChIKey: RHWSQPZMDJIJDV-NYDSKATKSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of CYP3A4 in human liver microsomes preincubated for 30 mins after 30 secs by LC/MS/MS analysis
Measurement: IC50 = 8400 nM (pChEMBL 5.079999923706055)
Descriptors: MW 550.05, ALogP 3.11, HBA 7, HBD 1, PSA 98.77, QED 0.54
Compound (SMILES): N#Cc1ccc(CNC(=O)CCOCCN2CCN(c3ncccc3-c3ccccc3)CC2)cc1
InChIKey: HTCSLUIDBLHLBF-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Displacement of [3H]raclopride from human dopamine D2L receptor expressed in HEK293 cell membranes after 1 hr by microbeta counting analysis
Measurement: Ki = 537 nM (pChEMBL 6.269999980926514)
Descriptors: MW 469.59, ALogP 3.47, HBA 6, HBD 1, PSA 81.49, QED 0.46
Compound (SMILES): CC1=CC(=O)N(Nc2nc(-c3cccs3)nc3sc(C)c(C)c23)C1=O
InChIKey: QMZMEJFJABYIMJ-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Inhibition of FLT3-D835Y mutant (unknown origin)
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 370.46, ALogP 3.68, HBA 7, HBD 1, PSA 75.19, QED 0.71
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(Cc3cc4ccccc4nc3Cl)CC2)C1
InChIKey: VZYDRNUBBJZACH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In vitro JAK Kinase Assay: 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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 538.06, ALogP 4.61, HBA 8, HBD 1, PSA 102.55, QED 0.32