lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCC(N)CC3)n2)cn1
InChIKey: ZDKAFPXWZJQNPN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.87 nM (pChEMBL 9.0600004196167)
Descriptors: MW 388.48, ALogP 2.63, HBA 8, HBD 1, PSA 90.68, QED 0.58
Compound (SMILES): O=C(c1cc(Cl)ccc1-n1nccn1)N1CCOC[C@H]1Cc1cc(-n2nccn2)ccc1F
InChIKey: RWRHTRNNYAAULR-GOSISDBHSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 26000 nM (pChEMBL 4.579999923706055)
Descriptors: MW 467.89, ALogP 2.72, HBA 8, HBD 0, PSA 90.96, QED 0.45
Compound (SMILES): N#CCC1(n2ccc(-c3ncnc4[nH]ccc34)c2)CN(C2CCN(C(=O)c3ccc(F)c(F)c3)CC2)C1
InChIKey: JYTXMAGXSCRQTE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 501.54, ALogP 3.93, HBA 6, HBD 1, PSA 93.84, QED 0.45
Compound (SMILES): CCn1nc(-c2cccc(-c3cccc(C(=O)NCCCCCCCN(C)CCO)c3)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: OXMNWHWSPHHKPG-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 = 0.03 nM (pChEMBL 10.520000457763672)
Descriptors: MW 694.66, ALogP 6.55, HBA 8, HBD 4, PSA 141.48, QED 0.1
Compound (SMILES): COCCNc1cc2ncc(C(=O)Nc3cc(-c4cccs4)ccc3N)cc2cc1C1CC1
InChIKey: VTVFWPAHGJYNMJ-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC Enzyme Assay: Compounds for testing were diluted in DMSO to 50 fold the final concentration and a ten point three fold dilution series was made. The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM tris(2-carboxyethyl)phosphine) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer and pre-incubated with the compounds for 24 hours prior to addition of the substrate. The substrate tripeptide substrate 3 (synthesized in house) for each enzyme was equal to the Km as determined by a substrate titration curve. The enzyme and substrate concentrations used are given in Table 2. The substrates were diluted in assay buffer at 6x their final concentration with 0.3 uM sequencing grade trypsin (Sigma). The substrate/trypsin mix was added to the enzyme/compound mix, the plate was shaken for 60 seconds.
Measurement: IC50 = 1548 nM (pChEMBL 5.809999942779541)
Descriptors: MW 458.59, ALogP 5.73, HBA 6, HBD 3, PSA 89.27, QED 0.23
Compound (SMILES): O=C(c1c(F)ccc(NS(=O)(=O)c2cccnc2)c1F)c1c[nH]c2ncc(Cl)cc12
InChIKey: WTYQLNXQUJJGMD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf V600E mutant (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 448.84, ALogP 3.92, HBA 5, HBD 2, PSA 104.81, QED 0.45
Compound (SMILES): O=S(=O)(C(F)F)N1CCN(c2ccc(OC[C@@H]3CO[C@@](Cn4ccnc4)(c4ccc(Cl)cc4Cl)O3)cc2)CC1
InChIKey: NRJZYAWVYYNCON-PXDATVDWSA-N
Target: Steroid 21-hydroxylase (SINGLE PROTEIN), Homo sapiens
UniProt: P08686
Assay: Inhibition of recombinant CYP21 (unknown origin) overexpressed in human AD293 cells assessed as reduction in 11-deoxycortisol formation preincubated for 60 mins followed by addition of 17-alpha-hydroxyprogesterone as substrate measured after 45 mins by LC-MS/MS analysis
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 603.48, ALogP 4.21, HBA 8, HBD 0, PSA 86.13, QED 0.36
Compound (SMILES): Cc1cnn(C)c1S(=O)(=O)N1CCC(Oc2ccc(Cl)cc2Cl)CC1
InChIKey: QCMDYTSNEDLAHY-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Biological Assay: The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro.Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following to excitation at 485 nM.
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 404.32, ALogP 3.27, HBA 5, HBD 0, PSA 64.43, QED 0.78
Compound (SMILES): C=CC(=O)Nc1cc(-c2cnc3[nH]nc(C)c3c2)ccc1Cl
InChIKey: IIVMYNDXTSFVTC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 312.76, ALogP 3.71, HBA 3, HBD 2, PSA 70.67, QED 0.72
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3Cl)ncnc2cc1OC[C@H]1CN2CCCC[C@H]2CO1
InChIKey: OXOZNJXEGUXWOO-LSDHHAIUSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 568.3, ALogP 6.08, HBA 7, HBD 1, PSA 68.74, QED 0.36
Compound (SMILES): COc1cc(N2CCN3CCOC[C@@H]3C2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: QUDNSEPGTSESQY-MHZLTWQESA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR 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 688.74, ALogP 6.26, HBA 10, HBD 2, PSA 109.15, QED 0.19
Compound (SMILES): CCn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCN[C@@H](C)C3)n2)cn1
InChIKey: JVWOWDAKPHFJQW-HNNXBMFYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.349 nM (pChEMBL 9.460000038146973)
Descriptors: MW 388.48, ALogP 2.5, HBA 8, HBD 1, PSA 76.69, QED 0.58
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4ccc(C5CCCCC5)cc4)cs3)cccc2n(C)c1=O
InChIKey: AVGRXCGZLZSSPC-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NET8
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 488.61, ALogP 4.59, HBA 7, HBD 1, PSA 85.99, QED 0.45
Compound (SMILES): CC(C)COc1ccc([C@H](C)Nc2nccc(N3C(=O)OC[C@@H]3[C@@H](C)O)n2)c(F)c1
InChIKey: LJIFTDXMEPVARJ-PMUMKWKESA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R(−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM. Curve fitting for dose response IC50 determinations was done using a 4-parameter logistic model: y=min+((max−min)/1+(x/IC50)slope).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 418.47, ALogP 3.53, HBA 7, HBD 2, PSA 96.81, QED 0.68
Compound (SMILES): C=CC(=O)N1CCc2[nH]c3c(C(N)=O)c(-c4ccc(Oc5ccccc5)cc4)nn3c2C1
InChIKey: SEEJRWBTNZCBHF-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of EGFR (unknown origin) preincubated for 1 hr followed by Biotin-AVLESEEELYSSARQ-NH2 substrate addition in presence of ATP and measured after 1 hr by TR-FRET assay
Measurement: IC50 < 0.25 nM (pChEMBL 9.60200023651123)
Descriptors: MW 427.46, ALogP 3.29, HBA 5, HBD 2, PSA 105.72, QED 0.48
Compound (SMILES): CCCOc1cc2c(cc1OC)CCN1Cc3[nH]c4ccc(OC)cc4c3CC21
InChIKey: GVCJJVSREOMICJ-UHFFFAOYSA-N
Target: Alpha-1D adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25100
Assay: Antagonist activity at human full-length N-terminal SNAP-tagged alpha1D adrenoceptor expressed in HEK293 cells assessed as inhibition of agonist-induced calcium mobilization preincubated for 10 mins followed by agonist addition by fluo-4 AM dye-based assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 392.5, ALogP 4.63, HBA 4, HBD 1, PSA 46.72, QED 0.69
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Cl)cc3F)ncnc2cc1OCC1CN(C)CCO1
InChIKey: UTCBITIGWNRGAW-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 467.33, ALogP 4.54, HBA 7, HBD 1, PSA 68.74, QED 0.53
Compound (SMILES): Cc1ncc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)c(=O)n(C)n1
InChIKey: KPZHLNWOKWZIHG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
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 = 6.933 nM (pChEMBL 8.15999984741211)
Descriptors: MW 473.46, ALogP 3.26, HBA 7, HBD 1, PSA 89.35, QED 0.63
Compound (SMILES): CCC(=O)NC1CCCc2c(-c3ccc(C(F)(F)F)c(F)c3)cncc21
InChIKey: MXZPGNOPALOWQW-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Cellular Enzyme Assay: G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 ug/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 1813 nM (pChEMBL 5.739999771118164)
Descriptors: MW 366.36, ALogP 4.81, HBA 2, HBD 1, PSA 41.99, QED 0.79
Compound (SMILES): CC1CCC(CN(CCN2[C@@H]3CC[C@H]2C[C@@H](c2cccc(C(N)=O)c2)C3)C(=O)CS(C)(=O)=O)CC1.O=C(O)C(F)(F)F
InChIKey: GRRYPKBKVIGDER-BGRHAWAUSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 503.71, ALogP 3.2, HBA 5, HBD 1, PSA 100.78, QED 0.56
Compound (SMILES): COc1cc(N2CCC(C3CCN(CCF)CC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: RDGFRNBJLDAZEJ-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR 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 760.86, ALogP 8.64, HBA 9, HBD 2, PSA 99.92, QED 0.14
Compound (SMILES): CC1(CC(=O)N2CCC3(CC2)CC3CNC(=O)N2Cc3ccncc3C2)CCOCC1
InChIKey: YQPSPAFFTSSKRG-UHFFFAOYSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Reversible inhibition of CYP2C9 in human liver microsomes using (S)-warfarin as substrate in presence of NADPH by LC-MS/MS analysis
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 426.56, ALogP 2.94, HBA 4, HBD 1, PSA 74.77, QED 0.8
Compound (SMILES): CCc1ccc(-c2csc(NC(=O)Cc3cccc4c3c(=O)n(C)c(=O)n4C)n2)cc1
InChIKey: KFPIBSJOVPWQQT-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NET8
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 434.52, ALogP 3.1, HBA 7, HBD 1, PSA 85.99, QED 0.52
Compound (SMILES): Cc1ncccc1-c1nnc(SCCCN2CC[C@]3(C[C@@H]3c3ccc(C(F)(F)F)cc3)C2)n1C
InChIKey: MJUZYNLBBPDZMI-QPPBQGQZSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG transfected in HEK293 cells assessed as reduction in tail current by patch clamp assay
Measurement: IC50 = 6309.57 nM (pChEMBL 5.199999809265137)
Descriptors: MW 487.6, ALogP 5.57, HBA 6, HBD 0, PSA 46.84, QED 0.32
Compound (SMILES): COc1ccc(CN2CCC(c3ccc4[nH]c(C(=O)N5CCN(Cc6ccc(C(F)(F)F)cc6)CC5)nc4c3)CC2)cn1
InChIKey: KKRRTZFDLYCBDK-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Cell Proliferation Inhibition Assay: In order to make human PIK3 CA mutation-positive breast cancer cell line MDA-MB-453 cells become 500 cells per well in a 384 well non-adhesive plate (Lipidure-Coat plate AT-384; manufactured by NOF Corporation), 36 μl/well was seeded in Leibovitz's L-15 medium including 10% fetal bovine serum (manufactured by Life technologies Corp.), followed by culturing at 37° C. overnight in the presence of CO2. The following day, the test compound (the test compound had 11 steps in a final concentration from 10000 nM to 0.1 nM), and DMSO which is a solvent for the test compound as a negative control were diluted with the medium, and 4 μl was added to cells. Thereafter, the cells were cultured for 4 days at 37° C. in the absence of CO2, and a cytometric reagent (CellTiter-Glo Luminescent Cell Viability assay manufactured by Promega Corporation) was added. The resultant product was stirred for 30 minutes, and then measurement was carried out using a luminescence measurement apparatus
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 592.67, ALogP 5.32, HBA 6, HBD 1, PSA 77.59, QED 0.31
Compound (SMILES): Cc1cnn(C)c1-c1cc(C(=O)N[C@H](CN)Cc2cccc(F)c2)oc1Cl
InChIKey: UNQJIIVQNBKFRB-AWEZNQCLSA-N
Target: RAC-gamma serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y243
Assay: Inhibition of full length human AKT3 expressed in Sf9 cells assessed as reduction in substrate phosphorylation using biotin-ahx-ARKRERAYSFGHHA-amide substrate and [gamma-33P]ATP incubated for 40 mins by top count microplate scintillation counting method
Measurement: IC50 <= 25.12 nM (pChEMBL 7.599999904632568)
Descriptors: MW 390.85, ALogP 3.08, HBA 5, HBD 2, PSA 86.08, QED 0.68
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1cscn1
InChIKey: SHQICENFGOEXMY-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 433.32, ALogP 5.72, HBA 7, HBD 1, PSA 69.16, QED 0.42
Compound (SMILES): N#CCNC[C@H]1CC[C@H](N2CN(CC(F)(F)F)C(=O)c3cnc4[nH]ccc4c32)CC1
InChIKey: WUFVCJVJPCLFKL-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 420.44, ALogP 3.02, HBA 5, HBD 2, PSA 88.05, QED 0.57
Compound (SMILES): COCCNc1cc(NC(=O)N2CCCc3cc(CN4CCOCC4=O)c(C=O)nc32)ncc1C#N
InChIKey: ZTYSXMGILYNNJX-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Inhibition of FGFR4 (unknown origin) incubated for 60 mins by caliper microfluidic mobility shift technology based assay
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 493.52, ALogP 1.56, HBA 9, HBD 2, PSA 149.78, QED 0.41
Compound (SMILES): CC(C)c1cccc(NC(=O)Nc2ccc(-c3cc4ccccc4n3C(N)=O)cc2)c1
InChIKey: QEUJPSURIIRPKP-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: Kinase Assay: Biochemical PDGFRβ kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 36 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b protein (Millipore). Following a 60 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 412.49, ALogP 6, HBA 3, HBD 3, PSA 89.15, QED 0.38
Compound (SMILES): CC(C)(C)OC(=O)NCc1nc(-c2nc(C(=O)NCCCCCC(=O)NO)cs2)cs1
InChIKey: CQLJYHNTHMSMLR-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzyme Assay: Enzyme activity of HDAC1,3 is determined using the substrate Ac-Lys-Tyr-Lys(Ac)-AMC while the enzyme activity of HDAC6 is assayed using the substrate Boc-lys(Ac)-AMC. The reaction is carried out in flat-bottom, 96-well, or 384-well microplates. After the substrate was deacetylated by HDACs, the product AMC which was obtained from hydrolysis by trypsin can generate fluorescence signal. Measurement is taken in using an multilabe plate reader a 355 nm excitation filter and a 460 nm emission filter. The initial rate of fluorescence should accurately reflect the rate of product formation and enzyme activity. Sample processing: The sample was dissolved in DMSO and kept at a low temperature. DMSO in the final system is limited under a low concentration which won't affect the enzyme activity.
Measurement: IC50 = 566 nM (pChEMBL 6.25)
Descriptors: MW 469.59, ALogP 3.09, HBA 9, HBD 4, PSA 142.54, QED 0.24
Compound (SMILES): COc1cc(OC)cc(N(CC2(O)CCNCC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: JNTQLKRLUHCITH-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 absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 474.57, ALogP 3.3, HBA 9, HBD 2, PSA 97.56, QED 0.42
Compound (SMILES): C[C@@H]1CN(Cc2ccc(F)c(F)c2)c2ccccc2[C@H](C)N1C(=O)Nc1cccnc1
InChIKey: FESPTXQLNNHGLK-SJORKVTESA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1I (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P0X4
Assay: Channel blocking activity at recombinant human CaV 3.3 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 422.48, ALogP 5.36, HBA 3, HBD 1, PSA 48.47, QED 0.62
Compound (SMILES): CO[C@H]1CC[C@]2(C=C(c3cc(-c4ccc(Cl)cc4)ccc3C)C(=O)N2)CC1
InChIKey: PKHPLDHMCZUPTF-OKDJAKQTSA-N
Target: Acetyl-CoA carboxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13085
Assay: ACC1 Enzyme Assay B1: For the assay, 50 nl of a 100-times concentrated solution of the test substance in DMSO were pipetted into a white low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 ul of a solution of hACC1 in assay buffer [50 mM HEPES/NaOH pH 7.5, 2 mM MgCl2, 2 mM potassium citrate, 12 mM NaHCO3, 2 mM dithiothreitol (DTT), 0.005% (w/v) bovine serum albumin (BSA)] were added and the mixture was incubated for 15 min to allow prebinding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 2.5 ul of a solution of adenosine triphosphate (ATP, 100 uM=>final concentration in 5 ul of assay volume: 50 uM) and acetyl-CoA (20 uM=>final concentration in 5 ul assay volume: 10 uM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of 45 min. The concentration of the hACC1 was adapted to the respective activity of the enzyme and adjusted such that the assay operated in the linear range. Typical concentrations were in the range of 1.75 ng/ul. The reaction was stopped by addition of 2.5 ul of the "ADP-GLO reagent" (1:1.5-times diluted), and the resulting mixture was incubated at 22° C. for 1 h to convert the unreacted ATP completely into cAMP. 2.5 ul of the "kinase detection reagent" were then added (1.2-times more concentrated than rec mmended by the manufacturer), the resulting mixture was incubated at 22° C for 1 h.
Measurement: IC50 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 381.9, ALogP 5.16, HBA 2, HBD 1, PSA 38.33, QED 0.8
Compound (SMILES): CC(C)Cc1sc(-c2nccs2)nc1C(=O)NCCCNC(=O)CS
InChIKey: MVRMBYQFBFDOBQ-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzyme Assay: Enzyme activity of HDAC1,3 is determined using the substrate Ac-Lys-Tyr-Lys(Ac)-AMC while the enzyme activity of HDAC6 is assayed using the substrate Boc-lys(Ac)-AMC. The reaction is carried out in flat-bottom, 96-well, or 384-well microplates. After the substrate was deacetylated by HDACs, the product AMC which was obtained from hydrolysis by trypsin can generate fluorescence signal. Measurement is taken in using an multilabe plate reader a 355 nm excitation filter and a 460 nm emission filter. The initial rate of fluorescence should accurately reflect the rate of product formation and enzyme activity. Sample processing: The sample was dissolved in DMSO and kept at a low temperature. DMSO in the final system is limited under a low concentration which won't affect the enzyme activity.
Measurement: IC50 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 398.58, ALogP 2.63, HBA 7, HBD 3, PSA 83.98, QED 0.45
Compound (SMILES): CN(C)C/C=C/C(=O)Nc1cc2c(Nc3ccc(F)c(Cl)c3)ncnc2cn1
InChIKey: GCUYIMVZVRRUPD-ONEGZZNKSA-N
Target: Receptor tyrosine-protein kinase erbB-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15303
Assay: Irreversible inhibition of GST-tagged ERBB4 (unknown origin) (Gly-259 to Gly-690 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 400.85, ALogP 3.62, HBA 6, HBD 2, PSA 83.04, QED 0.61
Compound (SMILES): Cn1c(=O)n(C2CC2)c2c3cc(-c4cnc(N)nc4)ccc3ncc21
InChIKey: HZYTVQKACFDSMV-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Cellular Assay: Assay c) BT474 cells (human breast ductal carcinoma, ATCC HTB-20) were seeded into black 384 well plates (Costar, #3712) at a density of 5600 cells/well in DMEM containing 10% FBS and 1% glutamine and allowed to adhere overnight. The following morning compounds in 100% DMSO were added to assay plates by acoustic dispensing. After a 2 h incubation at 37° C. and 5% CO2, the medium was aspirated and the cells were lysed with a buffer containing 25 mM Tris, 3 mM EDTA, 3 mM EGTA, 50 mM sodium fluoride, 2 mM Sodium orthovanadate, 0.27 M sucrose, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 0.5% Triton X-100 and complete protease inhibitor cocktail tablets (Roche #04 693 116 001, used 1 tab per 50 ml lysis buffer). After 20 minutes, the cell lysates were transferred into ELISA plates (Greiner #781077) which had been pre-coated with an anti total-AKT antibody in PBS buffer and non-specific binding was blocked with 1% BSA in PBS containing 0.05% Tween 20. Plates were incubated over night at 4° C. The next day the plates were washed with PBS buffer containing 0.05% Tween 20 and further incubated with a mouse monoclonal anti-phospho AKT T308 for 2 h. Plates were washed again as above before addition of a horse anti-mouse-HRP conjugated secondary antibody. Following a 2 h incubation at r.t., plates were washed and QuantaBlu substrate working solution (Thermo Scientific #15169, prepared according to provider's instructions) was added to each well. The developed fluorescent product was stopped after 60 minutes by addition of Stop solution to the wells. Plates were read using a Tecan Satire plate reader using 325 nm excitation and 420 nm emission wavelengths respectively. Except where specified, reagents contained in the Path Scan Phospho AKT (Thr308) sandwich ELISA kit from Cell Signalling (#7144) were used in this ELISA assay.
Measurement: IC50 = 247 nM (pChEMBL 6.610000133514404)
Descriptors: MW 332.37, ALogP 2.26, HBA 7, HBD 1, PSA 91.62, QED 0.61
Compound (SMILES): N#Cc1ccc(CN[C@H]2CC[C@@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: VJNAQIVEIXLOPK-CALCHBBNSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 371.45, ALogP 3.29, HBA 6, HBD 2, PSA 94.69, QED 0.57
Compound (SMILES): O=C(O)c1ccc(NC(=O)C(Cc2ccncc2)n2ccc(-c3cc(Cl)ccc3C(F)(F)F)cc2=O)cc1
InChIKey: VOEMZDCOWSMTBH-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 541.91, ALogP 5.7, HBA 5, HBD 2, PSA 101.29, QED 0.31
Compound (SMILES): OC(c1ccc(Cl)cc1)(c1ccc(Cl)cc1)c1ccc2nccc(/C=C/c3ccccc3)c2c1
InChIKey: XKNGJBBRHVEYIR-VOTSOKGWSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Binding Assay: Experimental Procedure CB-1 Membrane Binding: Into Greiner V bottom polypropylene plates, hCB1-CHO-K1 membranes (2 ug/well final concentration) in assay buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2 and 0.5 mg/ml (0.05%) Ultra fatty acid free BSA) were dispensed. Membranes were purchased from Perkin Elmer. Test compounds were then added to each well and then [3H] CP 55, 940 (0.4 nM final well concentration) in assay buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2 and 0.5 mg/ml (0.05%) Ultra fatty acid free BSA) was added. Samples were mixed and incubated for 90 min at 30° C. in the Greiner V Bottom Polypropylene plate. After incubation, assay reagents were transferred to a blocked 384 well polypropylene filter plates. The binding reaction was stopped by filtration and washed seven times with ice cold rinse buffer. Filter plates were then dried overnight at room temperature. The next day, plate bottoms were sealed with plate tape and 15 ul MicroScint 20 was added to each well. Plates were incubated for 2 h and radioactivity was measured by Topcount.
Measurement: EC50 = 52.1 nM (pChEMBL 7.28000020980835)
Descriptors: MW 482.41, ALogP 8, HBA 2, HBD 1, PSA 33.12, QED 0.26
Compound (SMILES): CC#CC(=O)Nc1cccc(-c2cnc3[nH]ccc3c2)n1
InChIKey: BTUKQVNRDONJAO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 276.3, ALogP 2.59, HBA 3, HBD 2, PSA 70.67, QED 0.71
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4cn[nH]c4)cc23)c1F
InChIKey: MPBADXYYEQZJGR-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of C-Raf1 (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 445.45, ALogP 3.61, HBA 5, HBD 3, PSA 120.6, QED 0.38
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2sccc2N)C(=O)N[C@@H](Cc2csc3ccccc23)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: CYVSDXJVCZURLD-IHLOFXLRSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: In Vitro Inhibition Assay: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 7100 nM (pChEMBL 5.150000095367432)
Descriptors: MW 655.85, ALogP 4.79, HBA 8, HBD 5, PSA 148.57, QED 0.12
Compound (SMILES): Cc1cc(C(=O)N2CCOC[C@H]2Cc2cc(-c3ncon3)ccc2F)c(-n2nccn2)cc1C
InChIKey: PUBPGWFEGLTPSV-LJQANCHMSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 29000 nM (pChEMBL 4.539999961853027)
Descriptors: MW 462.49, ALogP 3.16, HBA 8, HBD 0, PSA 99.17, QED 0.45
Compound (SMILES): N#Cc1cnc2[nH]cc(C(=O)c3c(F)ccc(NS(=O)(=O)c4c(F)cccc4F)c3F)c2c1
InChIKey: KULMRXQPCFRJJZ-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of C-Raf1 (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 474.39, ALogP 4.02, HBA 5, HBD 2, PSA 115.71, QED 0.33
Compound (SMILES): CNCCCN(c1cc(OC)cc(OC)c1)c1ccc2ncc(-c3cnn(C)c3)nc2c1
InChIKey: WEASQIMFKRBYLI-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 absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 575.44 nM (pChEMBL 6.239999771118164)
Descriptors: MW 432.53, ALogP 3.8, HBA 8, HBD 1, PSA 77.33, QED 0.4
Compound (SMILES): O=C(Cc1ccc(-c2n[nH]c(=O)c3ccccc23)cc1)Nc1nc2ccccc2o1
InChIKey: JPHMTCGRECDTPW-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 396.41, ALogP 3.91, HBA 5, HBD 2, PSA 100.88, QED 0.48
Compound (SMILES): COc1cc2c(Nc3cccc(Cl)c3Cl)ncnc2cc1OCC1CN(C(C)=N)CCCO1
InChIKey: BSSZGBUEIGRDHQ-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 490.39, ALogP 5.16, HBA 7, HBD 2, PSA 92.59, QED 0.36
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Br)cc3F)ncnc2cc1OC[C@@H]1CN2CCC[C@H]2CO1
InChIKey: NGRFKPHUUUCTAT-KBPBESRZSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 537.82, ALogP 5.18, HBA 7, HBD 1, PSA 68.74, QED 0.43
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3ccc(-c4cnn(C)c4)cc3F)nn2[C@@H]2CCOC2)C1
InChIKey: NKOJNOBJGYTLLZ-QGZVFWFLSA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Displacement of biotinylated ligand from recombinant His-tagged EP300 (unknown origin) measured after 10 mins by TR-FRET assay
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 424.48, ALogP 3.03, HBA 7, HBD 1, PSA 77.21, QED 0.7
Compound (SMILES): O=C(O)CCc1cc2cc(-c3cccc(OC4CCC4)c3)ccc2o1
InChIKey: AIGFQVBWSVEVLF-UHFFFAOYSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Agonist activity at human GPR40 measured after 60 mins by IP1-HTRF assay
Measurement: EC50 = 2504 nM (pChEMBL 5.599999904632568)
Descriptors: MW 336.39, ALogP 5.05, HBA 3, HBD 1, PSA 59.67, QED 0.68