lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Fc1cccc(Nc2nc3cc(-c4nnn[nH]4)ccc3c3sccc23)c1
InChIKey: XVEQFFZCOYXBJG-UHFFFAOYSA-N
Target: Casein kinase II subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P68400
Assay: Modulation of Endogenous Assay: The human leukemia Jurkat T-cell line was maintained in RPMI 1640 (Cambrex) supplemented with 10% fetal calf serum and 50 ng/ml Geutamycin. Before treatment cells were washed, resuspended at a density of about 106 cells/milliliter in medium containing 1% fetal calf serum and incubated in the presence of indicated mounts of drug for two hours. Cells were recovered by centrifugation, lysed using a hypotonic buffer (20 mM Tris/HCl pH 7.4; 2 mM EDTA; 5 mM EGTA; 10 mM mercaptoethanol; 10 mM NaF; 1 uM Okadaic acid; 10% v/v glycerol; 0.05% NP-40; 1% Protease Inhibitor Cocktail) and protein from the cleared lysate was diluted to 1 microgram per microliter in Assay Dilution Buffer (ADB; 20 mM MOPS, pH 7.2, 25 mM β-glycerolphosphate, 5 mM EGTA, 1 mM sodium orthovanadate and 1 mM dithiothreitol). To 20 microliters of diluted protein was added 10 microliters of substrate peptide (RRRDDDSDDD, dissolved in ADB at a concentration of 1 mM) and 10 microliters of PKA Inhibitor cocktail (Upstate).
Measurement: IC50 = 4426 nM (pChEMBL 5.349999904632568)
Descriptors: MW 362.39, ALogP 4.51, HBA 6, HBD 2, PSA 79.38, QED 0.49
Compound (SMILES): Nc1ncc(-c2ccc(-c3ccccc3S(=O)(=O)c3ccnc(N)n3)cc2F)cn1
InChIKey: KTFBCUYDEWVMDA-UHFFFAOYSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: Homogeneous Time Resolved Fluorescence Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBacl (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator [5-[({[2-(2-{3-[3-(tert-Butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid]. The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 ug/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid].
Measurement: Ki = 1296 nM (pChEMBL 5.889999866485596)
Descriptors: MW 422.45, ALogP 2.74, HBA 8, HBD 2, PSA 137.74, QED 0.48
Compound (SMILES): Nc1ncc(-c2ccc(-c3cccnc3C(=O)N3CCOCC3)cc2F)cn1
InChIKey: FJGDZBYDXBZXPL-UHFFFAOYSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: Homogeneous Time Resolved Fluorescence Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBacl (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator [5-[({[2-(2-{3-[3-(tert-Butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid]. The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 ug/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid].
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 379.4, ALogP 2.4, HBA 6, HBD 1, PSA 94.23, QED 0.75
Compound (SMILES): Nc1cnc(-c2ccc(-c3cccnc3S(=O)(=O)C3CCNC3)cc2F)cn1
InChIKey: KYQLDZSZMJMBND-UHFFFAOYSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: Homogeneous Time Resolved Fluorescence Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBacl (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator [5-[({[2-(2-{3-[3-(tert-Butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid]. The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 ug/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid].
Measurement: Ki = 106.5 nM (pChEMBL 6.96999979019165)
Descriptors: MW 399.45, ALogP 2.06, HBA 7, HBD 2, PSA 110.86, QED 0.69
Compound (SMILES): Nc1cnc(-c2ccc(-c3cccnc3S(=O)(=O)C3CCCC3)cc2F)cn1
InChIKey: UEQZKVCYCZQCHV-UHFFFAOYSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: Homogeneous Time Resolved Fluorescence Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBacl (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator [5-[({[2-(2-{3-[3-(tert-Butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid]. The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 ug/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid].
Measurement: Ki = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 398.46, ALogP 3.64, HBA 6, HBD 1, PSA 98.83, QED 0.72
Compound (SMILES): COc1ccc(C(=O)N[C@H](C)c2cccc(C(=O)Nc3nc4c(s3)CN(C)CC4)c2)cc1C
InChIKey: NOGILCBALCKFRT-MRXNPFEDSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Kinase Inhibition Assay: The following assay protocol is for measuring the phosphorylation of a peptide substrate (FAM-KKLRRTLSVA-OH wherein FAM is carboxyfluorescein). The peptide is >98% purity by Capillary Electrophoresis. The peptide is phosphorylated by the protein kinase ROCK1 or ROCK2. The ROCK1 or ROCK2 enzyme, substrate, and cofactors (ATP and Mg2+) are combined in a well of a microtiter plate and incubated for 3 hours at 25° C. in the presence or absence of an inhibitor compound. At the end of the incubation, the reaction is quenched by the addition of an EDTA-containing buffer. The substrate and product are separated and quantified electrophoretically using the microfluidic-based LABCHIP® 3000 Drug Discovery System from Caliper Life Sciences (Hopkinton, Mass.).The components of the assay mixture are:100 mM HEPES, pH 7.50.1% BSA0.01% Triton X-1001 mM DTT10 mM MgCl210 μM Sodium Orthovanadate10 LM Beta-Glycerophosphate5 μM ATP (for ROCK1) or 7 μM ATP (for ROCK2)1% DMSO (from compound)1.25 μM FAM-KKLRRTLSVA-OH3 nM ROCK1 or 2.5 nM ROCK2 enzymeSubstrate and product peptides present in each sample are separated electrophoretically using the LABCHIP® 3000 capillary electrophoresis instrument. As substrate and product peptides are separated two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured reflecting enzyme activity. Capillary electrophoregramms (RDA acquisition files) are analyzed using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). The kinase activity in each sample is determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product peptide and S is the peak height of the substrate peptide. For each compound, enzyme activity is measured at various concentrations (12 concentrations of compound spaced by 3× dilution intervals).
Measurement: IC50 = 22.5 nM (pChEMBL 7.650000095367432)
Descriptors: MW 464.59, ALogP 4.19, HBA 6, HBD 2, PSA 83.56, QED 0.57
Compound (SMILES): CCN(c1cc(-c2cnn(C)c2)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)[C@H]1CC[C@H](N(C)C)CC1
InChIKey: VUEDRHYYGUZNMA-SOAUALDESA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 518.71, ALogP 4.33, HBA 6, HBD 2, PSA 86.26, QED 0.46
Compound (SMILES): CCN(c1cc(-c2ccc(N3CCNCC3)nc2)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)C1CCOCC1
InChIKey: FTUGIYDOSRLWHX-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 11.2 nM (pChEMBL 7.949999809265137)
Descriptors: MW 558.73, ALogP 3.71, HBA 7, HBD 3, PSA 102.59, QED 0.39
Compound (SMILES): CCN(c1cc(-c2cnccn2)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)[C@H]1CC[C@H](N(C)C)CC1
InChIKey: FIXJORJSCJZMFR-RQNOJGIXSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 516.69, ALogP 4.39, HBA 6, HBD 2, PSA 94.22, QED 0.46
Compound (SMILES): CCN(c1cc(-c2cnc(C)cn2)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)[C@H]1CC[C@H](N(C)C)CC1
InChIKey: DBJMXRJCUHUFBX-SOAUALDESA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 530.72, ALogP 4.69, HBA 6, HBD 2, PSA 94.22, QED 0.44
Compound (SMILES): CCCc1cc(C)[nH]c(=O)c1CNC(=O)c1cc(-c2ccc(CN3CCOCC3)cc2)cc(N(CC)C2CCOCC2)c1C
InChIKey: GAYPXEWQCCLAHL-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 600.8, ALogP 5.38, HBA 6, HBD 2, PSA 86.9, QED 0.31
Compound (SMILES): CCN(c1cc(-c2ccc(CN3CCOCC3)cc2)cc(C(=O)NCc2c(C)c(Br)c(C)[nH]c2=O)c1C)C1CCOCC1
InChIKey: OCCONAFXMOLOEB-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 973.5 nM (pChEMBL 6.010000228881836)
Descriptors: MW 651.65, ALogP 5.5, HBA 6, HBD 2, PSA 86.9, QED 0.32
Compound (SMILES): CCN(c1cc(-c2ccc(CN3CCOCC3)cc2)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1Cl)C1CCOCC1
InChIKey: DGCNEYPMLOTXMG-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 593.17, ALogP 5.08, HBA 6, HBD 2, PSA 86.9, QED 0.36
Compound (SMILES): CCN(c1cc(-c2ccc(CN3CCOCC3)cc2)cc(C(=O)NCc2c(C)c(F)c(C)[nH]c2=O)c1C)C1CCOCC1
InChIKey: MVMFNMZLRJXPHI-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 92.2 nM (pChEMBL 7.039999961853027)
Descriptors: MW 590.74, ALogP 4.87, HBA 6, HBD 2, PSA 86.9, QED 0.37
Compound (SMILES): CCN(c1cc(-c2ccc(C(=O)NCCCO)cc2)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)[C@H]1CC[C@H](N(C)C)CC1
InChIKey: LPHBHYGFGVLDSX-WXUXXXNLSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 615.82, ALogP 4.71, HBA 6, HBD 4, PSA 117.77, QED 0.22
Compound (SMILES): CCCc1cc(C)[nH]c(=O)c1CNC(=O)c1cc(-c2ccc(N3CCN(C)CC3)nc2)cc(N(CC)C2CCOCC2)c1C
InChIKey: KAQCDEYIHYEDMG-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 600.81, ALogP 4.69, HBA 7, HBD 2, PSA 93.8, QED 0.35
Compound (SMILES): CCCc1cc(C)[nH]c(=O)c1CNC(=O)c1cc(-c2ccc(N3CCNCC3)nc2)cc(N(CC)C2CCOCC2)c1C
InChIKey: SMGSYCJYJLHFGP-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 586.78, ALogP 4.35, HBA 7, HBD 3, PSA 102.59, QED 0.32
Compound (SMILES): Cc1nccc(-c2ccc(C(=O)N3CCOCC3)c(Cl)c2)c1C#Cc1ccc(N)nc1
InChIKey: UDIUKSLSDVCBFN-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: HTS Assay: The GSK3beta primary screen was conducted in assay ready 1536 plates (Aurora 29847) that contain 2.5 mL/well of 10 mM compound. Human GSK3beta as a GST fusion expressed in baculoviral system was purchased from BPS Bioscience (San Diego, Calif.). The GSK3beta peptide substrate was from American Peptide (Sunnyvale, Calif.; Cat 311153). 1 L/well of CABPE (22.5 nM GSK3beta , 8 uM peptide in AB buffer (12.5 mM DTT, 0.25 mg/mL BSA, 0.5 unit/mL Heparin)), 0.5 L/well of 125 uM of ATP, and 1 L/well of positive control 50 uM of GW8510 (positive control) or AB (DMSO only neutral control) in respective wells according to plate design using BioRAPTR (Beckman, Brea, Calif.). Reactions were incubated at room temperature for 60 minutes. 2.5 uL/well of ADP-Glo (Promega, V9103) was added with BioRAPTR, and incubated at room temperature for 40 minutes followed by addition of 5 uL/well of ADP-Glo detection reagent (Promega, V9103) with Combi nL (Thermo, Waltham, Mass.).
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 432.91, ALogP 3.56, HBA 5, HBD 1, PSA 81.34, QED 0.63
Compound (SMILES): Cc1nccc(-c2ccc(C(=O)N(C)C)c(Cl)c2)c1C#Cc1ccc(N)nc1
InChIKey: MNHCYTKOZZNQIL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: TR-FRET Assay: Kinase reaction conditions:400 nM GFP-4E-BP1, 8 uM ATP, -150 ng/mL mTOR, 50 mM HEPES pH 7.5, 0.01% Polysorbate 20, 1 mM EGTA, 10 mM MnC12, and variable amounts of test compounds.Preparation of Reagents:Note: Thaw and keep mTOR, the substrate, ATP, and the antibody on ice prior to making working dilutions. Working dilutions of these components can be kept at room temperature for short periods of time the day of use.1. Add 2 ml of 5X Assay Buffer to 8 ml water to prepare 10 ml of IX Assay Buffer. Note: The concentration of IX Assay Buffer is 50 mM HEPES pH 7.5, 0.01% Polysorbate 20, 1 mM EGTA, and 10 mM MnC12.2. Prepare Antibody/EDTA Solution by first adding 2.75 ul of Tb-anti p4E-BPl Antibody to 2397 ul of LanthaScreenTM TR-FRET Dilution Buffer. Then, add 100 ul of 0.5 M EDTA. 3. Prepare 4X Substrate/Enzyme Solution by first adding 72 ul of GFP-4E-BP1 (22 uM) to 926 ul of IX Assay Buffer. Then, add 1.6 ul of mTOR (0.45 mg/mL). 4.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 390.87, ALogP 3.79, HBA 4, HBD 1, PSA 72.11, QED 0.68
Compound (SMILES): COC(=O)[C@H]1CCCN1C(=O)c1cccc(Nc2c(N[C@@H](c3ccc(C)o3)C3(C)COC3)c(=O)c2=O)c1O
InChIKey: SIUQYAWDXLUCLD-OSPHWJPCSA-N
Target: C-X-C chemokine receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25024
Assay: In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Measurement: IC50 = 604 nM (pChEMBL 6.21999979019165)
Descriptors: MW 523.54, ALogP 2.6, HBA 10, HBD 3, PSA 147.41, QED 0.23
Compound (SMILES): Cc1ccc(C(Nc2c(Nc3ccc(Cl)c(S(=O)(=O)N(C)C)c3O)c(=O)c2=O)C2(C)COC2)o1
InChIKey: YWEPAFKNLSBBBU-UHFFFAOYSA-N
Target: C-C chemokine receptor type 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P51684
Assay: FLIPR Assay: The experiments were carried out on the FLIPR TETRA.RTM. platform from Molecular Devices. After the basal level had been read, the compounds were added to the cells expressing the chemokine receptor of interest and the agonist activity was read at 10 seconds. After a further incubation for 10 minutes, the cells were activated, with a concentration equivalent to the AC80, using a reference agonist in order to detect whether this compound exhibits antagonist activity.Each cell line expressing a chemokine receptor was established on the basis of the Chem-1 cell stably expressing the recombinant form of the chemokine receptor and also an associated G protein, with the aim of coupling the receptor to the calcium signalling pathway. 21 receptors belonging to the chemokine receptor family (CCRs and CXCRs) were analyzed. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC.sub.50).
Measurement: IC50 = 3.4 nM (pChEMBL 8.470000267028809)
Descriptors: MW 509.97, ALogP 2.73, HBA 9, HBD 3, PSA 138.18, QED 0.31
Compound (SMILES): CCC(=N\O)/C(C)=C/c1ccc(F)c(C)c1
InChIKey: WRHHATMLNUQQTB-MZRBSKCNSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99720
Assay: GPCRScan assay: inhibition of Sigma 1
Measurement: Ki = 501.19 nM (pChEMBL 6.300000190734863)
Descriptors: MW 221.27, ALogP 3.78, HBA 2, HBD 1, PSA 32.59, QED 0.47
Compound (SMILES): Cc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4ccccc4)n3)c3ccccc23)cc1
InChIKey: DBXJNQABFNNKPN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase HCK (SINGLE PROTEIN), Homo sapiens
UniProt: P08631
Assay: Enzyme Inhibition Assay: The kinase enzyme binding activities of compounds disclosed herein were determined using a proprietary assay which measures active site-directed competition binding to an immobilized ligand (Fabian, M. A. et al., Nature Biotechnol., 2005, 23:329-336). These assays were conducted by DiscoverX (formerly Ambit; San Diego, Calif.). The Kd value (Dissociation constant value) was calculated as the index of affinity of the compounds to each kinase. The enzyme inhibitory activities of compounds disclosed herein were determined by FRET using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen Ltd., Paisley, UK). In all cases, the site-specific protease cleaves non-phosphorylated peptide only and eliminates the FRET signal. Phosphorylation levels of each reaction were calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor), for which low ratios indicate high phosphorylation and high ratios indicate low phosphorylation levels.
Measurement: Kd = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 583.7, ALogP 8.6, HBA 7, HBD 3, PSA 105.99, QED 0.17
Compound (SMILES): Cc1nc(C(=O)Nc2ccc(-c3ccc(C4CCC(CC(=O)O)OC4)cc3)cc2)c(C(F)(F)F)o1
InChIKey: GORUONAPQBRBPQ-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Inhibition Assay: The enzyme preparation used in this assay is a membrane preparation from Sf9 cells overexpressing human (His)6DGAT1. During all steps samples were chilled to 4 C. Sf9 cells expressing human (His)6DGAT1 were thawed at room temperature and re-suspended at a 10:1 ratio (mL buffer/g of cells) in 50 mM HEPES, 1x Complete Protease Inhibitor, pH 7.5. The re-suspended pellet was homogenized for 1 min using a Brinkman PT 10/35 homogenizer with a 20 mm generator. Cells were lysed using Avestin Emulsiflex (chilled to 4 C.) at 10000-15000 psi. Lysate was centrifuged at 100,000xg for 1 h at 4 C. Supernatant was removed and pellets were re-suspended in 50 mM HEPES, 1x Complete Protease Inhibitor, pH 7.5 at 1/6 the volume of supernatant. Re-suspended pellets were pooled and homogenized with 10 strokes of a Glas-Col motor driven teflon pestle on setting 70. The protein concentration of the membrane preparation was quantified using BCA protein assay with 1% SDS.
Measurement: IC50 = 820 nM (pChEMBL 6.090000152587891)
Descriptors: MW 488.46, ALogP 5.66, HBA 5, HBD 2, PSA 101.66, QED 0.46
Compound (SMILES): O=C(O)CC1CCC(c2ccc(-c3ccc(Nc4nnc(C5CCC5)o4)cc3)cc2)OC1
InChIKey: HIHFZDGOQGUUEM-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Inhibition Assay: The enzyme preparation used in this assay is a membrane preparation from Sf9 cells overexpressing human (His)6DGAT1. During all steps samples were chilled to 4 C. Sf9 cells expressing human (His)6DGAT1 were thawed at room temperature and re-suspended at a 10:1 ratio (mL buffer/g of cells) in 50 mM HEPES, 1x Complete Protease Inhibitor, pH 7.5. The re-suspended pellet was homogenized for 1 min using a Brinkman PT 10/35 homogenizer with a 20 mm generator. Cells were lysed using Avestin Emulsiflex (chilled to 4 C.) at 10000-15000 psi. Lysate was centrifuged at 100,000xg for 1 h at 4 C. Supernatant was removed and pellets were re-suspended in 50 mM HEPES, 1x Complete Protease Inhibitor, pH 7.5 at 1/6 the volume of supernatant. Re-suspended pellets were pooled and homogenized with 10 strokes of a Glas-Col motor driven teflon pestle on setting 70. The protein concentration of the membrane preparation was quantified using BCA protein assay with 1% SDS.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 433.51, ALogP 5.69, HBA 6, HBD 2, PSA 97.48, QED 0.49
Compound (SMILES): C=CC(=O)N1CC[C@H](n2nc(C#Cc3cc(OC)cc(OC)c3)c3c(N)ncnc32)C1
InChIKey: KEIPNCCJPRMIAX-HNNXBMFYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition of human recombinant cytoplasmic domain FGFR2 (8 to 134 residues) incubated for 120 mins by mobility shift assay
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 418.46, ALogP 1.78, HBA 8, HBD 1, PSA 108.39, QED 0.51
Compound (SMILES): C#CC(=O)N1CC[C@H](n2nc(C#Cc3cc(OC)cc(OC)c3)c3c(N)ncnc32)C1
InChIKey: SENVOZNIDWYYED-HNNXBMFYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition Assay: Purified recombinant human FGFR3 protein was purchased from Carna Biosciences, Inc. When setting conditions for the measurement of the inhibitory effect of the compounds on FGFR3 kinase activity, a biotinylated peptide (biotin-EEPLYWSFPAKKK) was synthesized for use as a substrate by utilizing the amino acid sequence of FL-Peptide 22 (Caliper Life Sciences, Inc.) with biotin. The purified recombinant human FGFR3 protein used in the test was purchased from Carna Biosciences, Inc. In the measurement of the inhibitory effect of the compounds, first, a test compound was gradually diluted with dimethylsulfoxide (DMSO) to a concentration 20 times higher than the final concentration. Next, the purified human FGFR3 protein, substrate peptide (final concentration: 250 nM), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 190 .mu.M), and the test compound DMSO solution (final concentration of DMSO: 5%) were added to a reaction buffer.
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 416.44, ALogP 1.23, HBA 8, HBD 1, PSA 108.39, QED 0.64
Compound (SMILES): C=CC(=O)N1CC(n2nc(C#Cc3cc(OC)cc(OC)c3)c3c(N)ncnc32)C1
InChIKey: GYXODBZTJVUDOH-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of N-terminal GST tagged recombinant human VEGFR2 (aa805 to 1356 residues) expressed in baculovirus expression system using Poly- (Glu4:Tyr)-biotin as substrate incubated for 80 mins in presence of ATP by kinase glo method
Measurement: IC50 = 3029 nM (pChEMBL 5.519999980926514)
Descriptors: MW 404.43, ALogP 1.39, HBA 8, HBD 1, PSA 108.39, QED 0.52
Compound (SMILES): COc1cc(C#Cc2nn(C3CN(C(=O)/C=C/CN(C)C)C3)c3ncnc(N)c23)cc(OC)c1
InChIKey: HBIDSBZKIOZROC-AATRIKPKSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition Assay: When setting conditions for the measurement of the inhibitory effect of the compounds on FGFR2 kinase activity, FL-Peptide 22 (Caliper Life Sciences, Inc.) was used as a substrate. The purified recombinant human FGFR2 protein used in the test was purchased from Carna Biosciences, Inc. In the measurement of the inhibitory effect of the compounds, first, a test compound was gradually diluted with dimethylsulfoxide (DMSO) to a concentration that was 20 times higher than the final concentration. Next, the purified human FGFR2 protein, FL-Peptide 22 (final concentration: 1.5 .mu.M), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 75 .mu.M), and the test compound DMSO solution (final concentration of DMSO: 5%) were added to a reaction buffer (15 mM Tris-HCl pH 7.5, 0.01% Tween-20, 2 mM DTT), and the mixture was incubated at 25.degree. C. for 120 minutes to perform a kinase reaction. EDTA (final concentration: 30 mM) diluted with a separation buffer.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 461.53, ALogP 1.33, HBA 9, HBD 1, PSA 111.63, QED 0.43
Compound (SMILES): C=CC(=O)N1CC(Cn2nc(C#Cc3cc(OC)cc(OC)c3)c3c(N)ncnc32)C1
InChIKey: NLNSWQPNSJQAHU-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition Assay: When setting conditions for the measurement of the inhibitory effect of the compounds on FGFR2 kinase activity, FL-Peptide 22 (Caliper Life Sciences, Inc.) was used as a substrate. The purified recombinant human FGFR2 protein used in the test was purchased from Carna Biosciences, Inc. In the measurement of the inhibitory effect of the compounds, first, a test compound was gradually diluted with dimethylsulfoxide (DMSO) to a concentration that was 20 times higher than the final concentration. Next, the purified human FGFR2 protein, FL-Peptide 22 (final concentration: 1.5 .mu.M), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 75 .mu.M), and the test compound DMSO solution (final concentration of DMSO: 5%) were added to a reaction buffer (15 mM Tris-HCl pH 7.5, 0.01% Tween-20, 2 mM DTT), and the mixture was incubated at 25.degree. C. for 120 minutes to perform a kinase reaction. EDTA (final concentration: 30 mM) diluted with a separation buffer.
Measurement: IC50 = 9.3 nM (pChEMBL 8.029999732971191)
Descriptors: MW 418.46, ALogP 1.47, HBA 8, HBD 1, PSA 108.39, QED 0.49
Compound (SMILES): COc1cc(C#Cc2cn([C@H]3CCN(C(=O)C#CC(C)(C)O)C3)c3ncnc(N)c23)cc(OC)c1
InChIKey: NZYVQKUNDWKOTA-IBGZPJMESA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition Assay: When setting conditions for the measurement of the inhibitory effect of the compounds on FGFR2 kinase activity, FL-Peptide 22 (Caliper Life Sciences, Inc.) was used as a substrate. The purified recombinant human FGFR2 protein used in the test was purchased from Carna Biosciences, Inc. In the measurement of the inhibitory effect of the compounds, first, a test compound was gradually diluted with dimethylsulfoxide (DMSO) to a concentration that was 20 times higher than the final concentration. Next, the purified human FGFR2 protein, FL-Peptide 22 (final concentration: 1.5 .mu.M), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 75 .mu.M), and the test compound DMSO solution (final concentration of DMSO: 5%) were added to a reaction buffer (15 mM Tris-HCl pH 7.5, 0.01% Tween-20, 2 mM DTT), and the mixture was incubated at 25.degree. C. for 120 minutes to perform a kinase reaction. EDTA (final concentration: 30 mM) diluted with a separation buffer.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 473.53, ALogP 1.98, HBA 8, HBD 2, PSA 115.73, QED 0.56
Compound (SMILES): C=CC(=O)N1CC(N2C=C(C#Cc3cc(OC)cc(OC)c3)C3C(N)=NC=NC32)C1
InChIKey: CXJITJGGCTWHEG-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition Assay: When setting conditions for the measurement of the inhibitory effect of the compounds on FGFR2 kinase activity, FL-Peptide 22 (Caliper Life Sciences, Inc.) was used as a substrate. The purified recombinant human FGFR2 protein used in the test was purchased from Carna Biosciences, Inc. In the measurement of the inhibitory effect of the compounds, first, a test compound was gradually diluted with dimethylsulfoxide (DMSO) to a concentration that was 20 times higher than the final concentration. Next, the purified human FGFR2 protein, FL-Peptide 22 (final concentration: 1.5 .mu.M), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 75 .mu.M), and the test compound DMSO solution (final concentration of DMSO: 5%) were added to a reaction buffer (15 mM Tris-HCl pH 7.5, 0.01% Tween-20, 2 mM DTT), and the mixture was incubated at 25.degree. C. for 120 minutes to perform a kinase reaction. EDTA (final concentration: 30 mM) diluted with a separation buffer.
Measurement: IC50 < 0.3 nM (pChEMBL 9.52299976348877)
Descriptors: MW 405.46, ALogP 0.99, HBA 7, HBD 1, PSA 92.75, QED 0.6
Compound (SMILES): COc1cc(C#Cc2cn(C3CN(C(=O)/C=C/CN(C)C)C3)c3ncnc(N)c23)cc(OC)c1
InChIKey: NFYDPTASEPAEMT-AATRIKPKSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition Assay: When setting conditions for the measurement of the inhibitory effect of the compounds on FGFR2 kinase activity, FL-Peptide 22 (Caliper Life Sciences, Inc.) was used as a substrate. The purified recombinant human FGFR2 protein used in the test was purchased from Carna Biosciences, Inc. In the measurement of the inhibitory effect of the compounds, first, a test compound was gradually diluted with dimethylsulfoxide (DMSO) to a concentration that was 20 times higher than the final concentration. Next, the purified human FGFR2 protein, FL-Peptide 22 (final concentration: 1.5 .mu.M), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 75 .mu.M), and the test compound DMSO solution (final concentration of DMSO: 5%) were added to a reaction buffer (15 mM Tris-HCl pH 7.5, 0.01% Tween-20, 2 mM DTT), and the mixture was incubated at 25.degree. C. for 120 minutes to perform a kinase reaction. EDTA (final concentration: 30 mM) diluted with a separation buffer.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 460.54, ALogP 1.93, HBA 8, HBD 1, PSA 98.74, QED 0.44
Compound (SMILES): COc1cc(C#Cc2cn(C3CN(C(=O)/C=C/CN4CC[C@H](O)C4)C3)c3ncnc(N)c23)cc(OC)c1
InChIKey: YTDWYXQAKOWNAV-SHTLVRLNSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Inhibition Assay: Purified recombinant human FGFR4 protein was purchased from Carna Biosciences, Inc. When setting conditions for the measurement of the inhibitory effect of the compounds on FGFR4 kinase activity, a biotinylated peptide (biotin-EEPLYWSFPAKKK) was synthesized for use as a substrate by utilizing the amino acid sequence of FL-Peptide 22 (Caliper Life Sciences, Inc.) with biotin. The purified recombinant human FGFR4 protein used in the test was purchased from Carna Biosciences, Inc. In the measurement of the inhibitory effect of the compounds, first, a test compound was gradually diluted with dimethylsulfoxide (DMSO) to a concentration 20 times higher than the final concentration. Next, the purified human FGFR4 protein, substrate peptide (final concentration: 250 nM), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 190 .mu.M), and the test compound DMSO solution (final concentration of DMSO: 5%) were added to a reaction buffer.
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 502.58, ALogP 1.44, HBA 9, HBD 2, PSA 118.97, QED 0.38
Compound (SMILES): COc1cc(C#Cc2cn(C3CN(C(=O)/C=C/CN4CC[C@@H](O)C4)C3)c3ncnc(N)c23)cc(OC)c1
InChIKey: YTDWYXQAKOWNAV-YEFOHOTDSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Measurement of Inhibitory Effect on FGFR4 Kinase Activity: The inhibitory effect of the compounds on FGFR4 kinase activity was measured according to the method of Test Example 3. Purified recombinant human FGFR4 protein was purchased from Carna Biosciences, Inc. The final concentration of ATP was 200 μM.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 502.58, ALogP 1.44, HBA 9, HBD 2, PSA 118.97, QED 0.38
Compound (SMILES): CCC(=O)N1CC[C@H](n2nc(C#Cc3cc(OC)cc(OC)c3)c3c(N)ncnc32)C1
InChIKey: DZLNAZQOANZLHT-HNNXBMFYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human EGFR del19/T790M using biotinEEPLYWSFPAKKK-NH2 as substrate incubated for 120 mins by TR-FRET assay
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 420.47, ALogP 2.01, HBA 8, HBD 1, PSA 108.39, QED 0.64
Compound (SMILES): CC1(C)O[C@@H]2[C@H](O1)[C@@H](n1cnc3c(N)ncnc31)O[C@H]2Cn1cc(CCCCC[C@@H]2SC[C@@H]3NC(=O)N[C@@H]32)nn1
InChIKey: JOAMEQGLLLPUCO-YUBBLUDWSA-N
Target: Bifunctional ligase/repressor BirA (SINGLE PROTEIN), Escherichia coli (strain K12)
UniProt: P06709
Assay: Enzyme Inhibition Assay: Quantitation of BPL catalysed 3H-biotin incorporation into the biotin domain substrate was performed as previously described by Polyak et al, J. Biol. Chem (1999) 274(46) 32847-54. Briefly, the reaction mixture contained: 50 mM Tris HCl pH 8.0, 3 mM ATP, 4.5 uM biotin, 0.5 uM 3H-biotin, 5.5 mM MgCl2, 100 mM KCl, 0.1 mg/mL BSA and 10 uM biotin domain of S. aureus pyruvate carboxylase. The reaction was initiated by the addition of BPL to a final concentration of 4 nM. After 20 minutes at 37 C., 4 uL aliquots of the reaction were spotted onto Whatman paper pre-treated with biotin and trichloroacetic acid. The filters were washed twice with 10% v/v ice-cold trichloroacetic acid and once with ethanol before air-drying. Quantitation of protein-bound radiolabelled biotin was performed by liquid scintillation. One unit of enzyme activity was defined by the amount of BPL required to incorporate 1 nmol of biotin per minute. The IC50 value of each compound was determined.
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 570.68, ALogP 1.39, HBA 13, HBD 3, PSA 169.15, QED 0.25
Compound (SMILES): Cc1ccc2oc(=O)n(CCCCn3cc(CCCCC[C@@H]4SC[C@@H]5NC(=O)N[C@@H]54)nn3)c2c1
InChIKey: LIWDYWLHGFRYPL-NYVOZVTQSA-N
Target: Bifunctional ligase/repressor BirA (SINGLE PROTEIN), Escherichia coli (strain K12)
UniProt: P06709
Assay: Enzyme Inhibition Assay: Quantitation of BPL catalysed 3H-biotin incorporation into the biotin domain substrate was performed as previously described by Polyak et al, J. Biol. Chem (1999) 274(46) 32847-54. Briefly, the reaction mixture contained: 50 mM Tris HCl pH 8.0, 3 mM ATP, 4.5 uM biotin, 0.5 uM 3H-biotin, 5.5 mM MgCl2, 100 mM KCl, 0.1 mg/mL BSA and 10 uM biotin domain of S. aureus pyruvate carboxylase. The reaction was initiated by the addition of BPL to a final concentration of 4 nM. After 20 minutes at 37 C., 4 uL aliquots of the reaction were spotted onto Whatman paper pre-treated with biotin and trichloroacetic acid. The filters were washed twice with 10% v/v ice-cold trichloroacetic acid and once with ethanol before air-drying. Quantitation of protein-bound radiolabelled biotin was performed by liquid scintillation. One unit of enzyme activity was defined by the amount of BPL required to incorporate 1 nmol of biotin per minute. The IC50 value of each compound was determined.
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 484.63, ALogP 3.24, HBA 8, HBD 2, PSA 106.98, QED 0.32