lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCC(C)(C)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: RFDKAAFEUKRTQE-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 148 nM (pChEMBL 6.829999923706055)
Descriptors: MW 561.55, ALogP 4.66, HBA 9, HBD 3, PSA 136.05, QED 0.24
Compound (SMILES): COCC(C)(C)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: RFDKAAFEUKRTQE-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 174 nM (pChEMBL 6.760000228881836)
Descriptors: MW 561.55, ALogP 4.66, HBA 9, HBD 3, PSA 136.05, QED 0.24
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3)ncnc2cc1O[C@H]1CO[C@H]2[C@@H]1OCC21OCCO1
InChIKey: ZDOPWPQLWDULEV-HBMCJLEFSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 550.79, ALogP 4.09, HBA 9, HBD 1, PSA 93.19, QED 0.5
Compound (SMILES): COCC(C)(C)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: RFDKAAFEUKRTQE-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 123 nM (pChEMBL 6.909999847412109)
Descriptors: MW 561.55, ALogP 4.66, HBA 9, HBD 3, PSA 136.05, QED 0.24
Compound (SMILES): Cc1cc(C)cc(-c2cnc3[nH]c(=O)n(Cc4cc(C)on4)c3c2)c1
InChIKey: OERDWUAFNZIISI-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: Biological Assay (standard): NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ~EC40 used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 491 nM (pChEMBL 6.309999942779541)
Descriptors: MW 334.38, ALogP 3.35, HBA 5, HBD 1, PSA 76.71, QED 0.62
Compound (SMILES): C[C@]1(O)CCC[C@@H]1n1c(=O)ccc2cnc(NC3CCN(S(=O)(=O)CC(F)(F)F)CC3)nc21
InChIKey: FCFWUBWRSPVMRL-KXBFYZLASA-N
Target: CDK6/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: CDK6/Cyclin D1 Mobility Shift Assay: The purpose of the CDK6/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK6/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:2). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Typical reaction solutions contained 2% DMSO (± inhibitor), 10 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 3 nM (active sites) CDK6/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.Inhibitor Ki determinations for non-phosphorylated CDK6/CyclinD1 (LJIC-2003A2/1865) were initiated with the addition of ATP (50 μL final reaction volume), following a twelve minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. The reaction was stopped after 35 minutes by the addition of 50 μL of 25 mM EDTA. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 8.23 nM (pChEMBL 8.09000015258789)
Descriptors: MW 489.52, ALogP 2.04, HBA 8, HBD 2, PSA 117.42, QED 0.66
Compound (SMILES): Cc1nc(OCc2cn(-c3ccc(C(F)(F)F)s3)nn2)ncc1F
InChIKey: IVZYOHJJVZVNLL-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 359.31, ALogP 3.16, HBA 7, HBD 0, PSA 65.72, QED 0.67
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc3ccc(=O)n([C@@H]4CCC[C@@H](O)C4)c3n2)CC1
InChIKey: VXSBVQGQHGROBA-HZPDHXFCSA-N
Target: CDK6/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: CDK6/Cyclin D1 Mobility Shift Assay: The purpose of the CDK6/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK6/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:2). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Typical reaction solutions contained 2% DMSO (± inhibitor), 10 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 3 nM (active sites) CDK6/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.Inhibitor Ki determinations for non-phosphorylated CDK6/CyclinD1 (LJIC-2003A2/1865) were initiated with the addition of ATP (50 μL final reaction volume), following a twelve minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. The reaction was stopped after 35 minutes by the addition of 50 μL of 25 mM EDTA. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 1.17 nM (pChEMBL 8.930000305175781)
Descriptors: MW 421.52, ALogP 1.1, HBA 8, HBD 2, PSA 117.42, QED 0.76
Compound (SMILES): CCOc1ccc(F)cc1-c1cnc2[nH]c(=O)n(Cc3c(C)noc3C)c2c1
InChIKey: RRERTRUHVYIKMV-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: Biological Assay (HTS): NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device. Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (150 mM, 3 mM, 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 > 29000 nM (pChEMBL 4.538000106811523)
Descriptors: MW 382.4, ALogP 3.58, HBA 6, HBD 1, PSA 85.94, QED 0.57
Compound (SMILES): COc1ccc(Nc2cc(-c3ccccc3OC(C)C)ncn2)cc1N
InChIKey: NJCDGFOWBZHIDI-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60563
Assay: In Vitro CDK9/CyclinT Assay: The activity of the compounds described in the present invention can be determined by measuring the phosphorylation of a fluorescently-labeled peptide by human CDK9/CyclinT kinase complex by fluorescent polarization using a commercially available IMAP Screening Express Assay Kit (Molecular devices).Test compounds were diluted in 100% DMSO to 5 mM stock concentration, then further dilutions were made in H2O or 100% DMSO to desirable concentrations.Each reaction consisted of 5 nM enzyme: CDK9/CyclinT (Proqinase cat#0371-0345-1), 400 nM TAMRA-Rbtide (synthetic 15-mer peptide derived from human retinoblastoma tumor suppressor protein labeled with TAMRA dye, Genecust Europe), 12 μM ATP (=Kmapp, Sigma-Aldrich) and kinase buffer: 20 mM MOPS pH 7 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 0.01% Tween 20 (Sigma-Aldrich).For each reaction, 4 or 6 μl containing TAMRA-Rbtide, ATP and kinase buffer were combined with 2 μl diluted compound in H2O or 0.028 μl compound in 100% DMSO. The kinase reaction was started by the addition of 2 μl diluted enzyme. The reaction was allowed to run for 1 hour at room temperature. The reaction was stopped by adding 15 μl IMAP beads (1:400 beads in progressive (100% buffer A) 1× buffer). After an additional 1 hour, fluorescent polarization (Ex: 550-10 nm, Em: 590-10 nm, Dich: 561 nm) was measured using an Analyst GT (Molecular devices).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 350.42, ALogP 4.27, HBA 6, HBD 2, PSA 82.29, QED 0.65
Compound (SMILES): O=c1[nH]c2ncc(-c3cccc(C(F)(F)F)c3)cc2n1Cc1nn[nH]n1
InChIKey: QTRIXDQRYLDLJZ-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: Biological Assay (standard): NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ~EC40 used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 361.29, ALogP 1.97, HBA 6, HBD 2, PSA 105.14, QED 0.58
Compound (SMILES): C.C.CC(=O)c1c(C)c2cnc(Nc3ccc4c(n3)CCNC4)nc2n(C2CCCC2)c1=O
InChIKey: WMTXUDXOUDCMAX-UHFFFAOYSA-N
Target: CDK4/Cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: Kinase Assay: In vitro enzymatic activity of the CDK isoforms CDK2/CycA2, CDK4/CycD3 and CDK6/cycD3 were measured using Mobility Shift Assay that monitors phosphorylation ratio of FAM labelled peptide (Peptide 18 for CDK2/CycA2, Peptide 8 for CDK4/CycD3 and CDK6/cycD3). CDK2/CycA2 and CDK6/cycD3 were assayed under buffer conditions in the presence of 50 mM HEPES (pH 7.5), 10 mM MgCl2, 0.0015% Brij-35, and 2 mM dithiothreitol, CDK4/CycD3 with buffer condition of 20 mM HEPES (pH 7.5), 10 mM MgCl2, 0.01% Triton X-100, and 2 mM dithiothreitol. Prepare compounds to 50× of the final desired highest inhibitor concentration by 100% DMSO and serial dilution in 3-fold for total of 10 concentrations. For each isoform, dosage of enzyme and substrate are CDK2/CycA2 12 nM, ATP Km 39 μM; CDK4/CycD3 10 nM, ATP Km 221 μM; CDK6/cycD3 15 nM, ATP Km 800 μM. After assay for 60 min, 180 min, 60 min respectively at 28° C., reactions were terminated with stop solution (50 mM EDTA, 0.015% Brij-35, 0.2% Coating Reagent #3 and 100 mM HEPES (pH 7.5)). Collect conversion on Caliper EZ Reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 450.59
Compound (SMILES): Cn1cc(Cn2c(=O)n(C)c3ncc(-c4ccc(F)c(F)c4)cc32)cn1
InChIKey: ASOSWPLBVZAQSI-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: Biological Assay (standard): NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ~EC40 used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 697 nM (pChEMBL 6.159999847412109)
Descriptors: MW 355.35, ALogP 2.46, HBA 6, HBD 0, PSA 57.64, QED 0.57
Compound (SMILES): CC#Cc1cccc2nc([C@H](CC)Nc3ncnc(N)c3-c3nnc(C)o3)n(-c3ccccc3)c(=O)c12
InChIKey: NLNPFTGLMMZBJA-IBGZPJMESA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: PI3K (p110beta/p85alpha) (h) [Non-Radioactive Assay]: PI3K (p110β/p85α) (h) is incubated in assay buffer containing 10 μM phosphatidylinositol-4,5-bisphosphate and MgATP (concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 30 minutes at room temperature, the reaction is stopped by the addition of stop solution containing EDTA and biotinylated phosphatidylinositol-3,4,5-trisphosphate. Finally, detection buffer is added, which contains europium-labelled anti-GST monoclonal antibody, GST-tagged GRP1 PH domain and streptavidin-allophycocyanin. The plate is then read in timeresolved fluorescence mode and the homogenous time-resolved fluorescence (HTRF) signal is determined according to the formula HTRF=10000×(Em665 nm/Em620 nm).
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 492.54, ALogP 4.05, HBA 10, HBD 2, PSA 137.64, QED 0.34
Compound (SMILES): CC#Cc1cccc2cc([C@H](C)Nc3ncnc(N)c3-c3nc(C)no3)n(-c3ccccc3)c(=O)c12
InChIKey: DWSBFBNKOHQARX-INIZCTEOSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: PI3K (p110beta/p85alpha) (h) [Non-Radioactive Assay]: PI3K (p110β/p85α) (h) is incubated in assay buffer containing 10 μM phosphatidylinositol-4,5-bisphosphate and MgATP (concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 30 minutes at room temperature, the reaction is stopped by the addition of stop solution containing EDTA and biotinylated phosphatidylinositol-3,4,5-trisphosphate. Finally, detection buffer is added, which contains europium-labelled anti-GST monoclonal antibody, GST-tagged GRP1 PH domain and streptavidin-allophycocyanin. The plate is then read in timeresolved fluorescence mode and the homogenous time-resolved fluorescence (HTRF) signal is determined according to the formula HTRF=10000×(Em665 nm/Em620 nm).
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 477.53, ALogP 4.27, HBA 9, HBD 2, PSA 124.75, QED 0.36
Compound (SMILES): Cc1c(-c2cnc3c(c2)n(CC(O)CCO)c(=O)n3C)cccc1C(F)(F)F
InChIKey: CRQSXZFRJUEKFM-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13224
Assay: Biological Assay (standard): NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl, 4 mM KCl, 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ~EC40 used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 5100 nM (pChEMBL 5.289999961853027)
Descriptors: MW 395.38, ALogP 2.47, HBA 6, HBD 2, PSA 80.28, QED 0.7
Compound (SMILES): Cc1cnc(-c2cc(OC3CN(C)C3)cc(C(=O)N[C@H](C)c3cnc(C(F)(F)F)nc3)c2)s1
InChIKey: NFRXFSQBXDHWHW-CYBMUJFWSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity : A fluorescent imaging plate reader (FLEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Fluo-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C. The final assay DMSO concentration is 1%.The agonist, α,β-Me-ATP, was added in a volume of 20 μl at a concentration representing the EC80 value. The fluorescence was measured for an interval of 90 sec at 2 sec intervals and analyzed based on the increase in peak relative fluorescence units (RFU) compared to the basal fluorescence. Peak fluorescence was used to determine the response to agonist obtained at each concentration of test compound by the following equation: % Response=100*(RFU(test compound)−RFU(control))/(RFU(DMSO)−RFU(control)).
Measurement: IC50 = 3047 nM (pChEMBL 5.519999980926514)
Descriptors: MW 477.51, ALogP 4.11, HBA 7, HBD 1, PSA 80.24, QED 0.58
Compound (SMILES): Cc1cnc(-c2cc(O[C@@H]3CCOC3)cc(C(=O)NCc3cnc(C(F)(F)F)nc3)c2)s1
InChIKey: MLKRPMMAEMGGNI-MRXNPFEDSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Biological Assay: A fluorescent imaging plate reader (FILEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Rim-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C.
Measurement: IC50 = 461 nM (pChEMBL 6.340000152587891)
Descriptors: MW 464.47, ALogP 4.03, HBA 7, HBD 1, PSA 86.23, QED 0.59
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc3ccc(=O)n([C@@H]4CCC[C@@H](O)C4)c3n2)CC1
InChIKey: VXSBVQGQHGROBA-HZPDHXFCSA-N
Target: CDK4/Cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: CDK4/Cyclin D3 Mobility Shift Assay: The purpose CDK4/Cyclin D3 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK4/Cyclin D3 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:2). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % Conversion of substrate to product by the LabChip EZ Reader. Typical reaction solutions contained 2% DMSO (± inhibitor), 10 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.005% TW-20, 3 μM 5-FAM-Dyrktide, 2 nM (active sites) CDK4/Cyclin D3 in 40 mM HEPES buffer at pH 7.5.Inhibitor Ki determinations for non-phosphorylated CDK4/Cyclin D3 (LJIC-2007/2010) were initiated with the addition of ATP (50 μL final reaction volume), following a twelve minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. The reaction was stopped after 35 minutes by the addition of 50 μL of 25 mM EDTA. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 3.43 nM (pChEMBL 8.460000038146973)
Descriptors: MW 421.52, ALogP 1.1, HBA 8, HBD 2, PSA 117.42, QED 0.76
Compound (SMILES): Cc1cnc(-c2cc(O[C@@H]3CCOC3)cc(C(=O)N[C@H](C)c3ccc(Cl)cn3)c2)s1
InChIKey: DURBVTDNVKUTOV-RDTXWAMCSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: P56373
Assay: Biological Assay: A fluorescent imaging plate reader (FILEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Rim-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C.
Measurement: IC50 = 147 nM (pChEMBL 6.829999923706055)
Descriptors: MW 443.96, ALogP 4.83, HBA 6, HBD 1, PSA 73.34, QED 0.59
Compound (SMILES): C.C.CC(=O)c1c(C)c2cnc(Nc3ccc4c(n3)CCNC4)nc2n(C2CCCC2)c1=O
InChIKey: WMTXUDXOUDCMAX-UHFFFAOYSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: Kinase Assay: In vitro enzymatic activity of the CDK isoforms CDK2/CycA2, CDK4/CycD3 and CDK6/cycD3 were measured using Mobility Shift Assay that monitors phosphorylation ratio of FAM labelled peptide (Peptide 18 for CDK2/CycA2, Peptide 8 for CDK4/CycD3 and CDK6/cycD3). CDK2/CycA2 and CDK6/cycD3 were assayed under buffer conditions in the presence of 50 mM HEPES (pH 7.5), 10 mM MgCl2, 0.0015% Brij-35, and 2 mM dithiothreitol, CDK4/CycD3 with buffer condition of 20 mM HEPES (pH 7.5), 10 mM MgCl2, 0.01% Triton X-100, and 2 mM dithiothreitol. Prepare compounds to 50× of the final desired highest inhibitor concentration by 100% DMSO and serial dilution in 3-fold for total of 10 concentrations. For each isoform, dosage of enzyme and substrate are CDK2/CycA2 12 nM, ATP Km 39 μM; CDK4/CycD3 10 nM, ATP Km 221 μM; CDK6/cycD3 15 nM, ATP Km 800 μM. After assay for 60 min, 180 min, 60 min respectively at 28° C., reactions were terminated with stop solution (50 mM EDTA, 0.015% Brij-35, 0.2% Coating Reagent #3 and 100 mM HEPES (pH 7.5)). Collect conversion on Caliper EZ Reader.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 450.59
Compound (SMILES): CC#Cc1cccc2nc([C@H](CC)Nc3ncnc(N)c3-c3nnc(C)o3)n(-c3ccccc3)c(=O)c12
InChIKey: NLNPFTGLMMZBJA-IBGZPJMESA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: PI3K (p110delta/p85alpha) (h) [Non-Radioactive Assay]: PI3K (p110δ/p85α) (h) is incubated in assay buffer containing 10 μM phosphatidylinositol-4, 5-bisphosphate and MgATP (concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 30 minutes at room temperature, the reaction is stopped by the addition of stop solution containing EDTA and biotinylated phosphatidylinositol-3,4,5-trisphosphate. Finally, detection buffer is added, which contains europium-labelled anti-GST monoclonal antibody, GST-tagged GRP1 PH domain and streptavidin-allophycocyanin. The plate is then read in timeresolved fluorescence mode and the homogenous time-resolved fluorescence (HTRF) signal is determined according to the formula HTRF=10000×(Em665 nm/Em620 nm).
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 492.54, ALogP 4.05, HBA 10, HBD 2, PSA 137.64, QED 0.34
Compound (SMILES): CC#Cc1cccc2cc([C@H](C)Nc3ncnc(N)c3-c3nc(C)no3)n(-c3ccccc3)c(=O)c12
InChIKey: DWSBFBNKOHQARX-INIZCTEOSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: PI3K (p110delta/p85alpha) (h) [Non-Radioactive Assay]: PI3K (p110δ/p85α) (h) is incubated in assay buffer containing 10 μM phosphatidylinositol-4, 5-bisphosphate and MgATP (concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 30 minutes at room temperature, the reaction is stopped by the addition of stop solution containing EDTA and biotinylated phosphatidylinositol-3,4,5-trisphosphate. Finally, detection buffer is added, which contains europium-labelled anti-GST monoclonal antibody, GST-tagged GRP1 PH domain and streptavidin-allophycocyanin. The plate is then read in timeresolved fluorescence mode and the homogenous time-resolved fluorescence (HTRF) signal is determined according to the formula HTRF=10000×(Em665 nm/Em620 nm).
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 477.53, ALogP 4.27, HBA 9, HBD 2, PSA 124.75, QED 0.36
Compound (SMILES): O=C(O)c1cc2cc(NC(=O)[C@H]3CCc4cc(-c5cc(Cl)ccc5-n5cnnn5)cc(=O)n43)ccc2[nH]1
InChIKey: WZZLEVNLUGKSFY-JOCHJYFZSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: In Vitro Inhibitory Effect of the Compounds of the Present Invention on the Enzymatic Activity of Blood-Coagulation Factor XIa: The following method was used to test the in vitro inhibitory effect of the compounds of the present invention on the activity of human blood-coagulation factor XIa, expressed by the Inhibition constant Ki.Solution preparation: reaction buffer: 0.03M HEPES acid, 0.145M NaCl, 0.005 M KCl, 0.1% PEG-8000, pH=7.5;HEPES 8.499 g, NaCl 8.47 g, KCl 0.3725, PEG 8000 1 g, plus ddH2O 800 ml; the pH was adjusted to 7.4 with HCl, and the volume was metered to 1 L.S2366 substrate stock solution (2 mM): a volume of substrate (25 ml) was dissolved in 23 ml sterile deionized water, aliquoted, and stored at 4° C. in darkness.S2366 substrate working solution: the stock solution was diluted by 4 folds with the reaction buffer before use.FXIa working solution: 1 μl FXIa stock solution was added to 10 ml reaction buffer and thoroughly mixed before use.Method: 15 μl test sample working solution (15 μl DMSO for the control group) and 75 μl FXIa working solution were added to a 96-well plate, and incubated at room temperature for 15 min. Then 60 μl S2366 substrate working solution was added to initiate the reaction. The absorbance at 405 nm for the test compounds was continually assayed once every 3 minutes, and a ΔA-time curve was plotted to calculate the slope as the reaction rate. In accordance with the following equation, IC50 of each test sample when the substrate concentration was 200 μM was calculated with spss 16.0. Inhibition (I %) and Ki of the test samples were calculated in accordance with the follow equations, and the results are shown in Table 1. I %=(V0−Vi)/V0×100 where V0 is the reaction rate in the control wells, and Vi is the reaction rate in the test sample wells; IC50=Ki(1+[S]/Km) where Km=0.2 mM, and [S] is the substrate concentration=200 μM.
Measurement: Ki = 1030 nM (pChEMBL 5.989999771118164)
Descriptors: MW 515.92, ALogP 3.45, HBA 8, HBD 3, PSA 147.79, QED 0.32
Compound (SMILES): N[C@H]1CCCC[C@H]1Nc1ncc2ccc(C(=O)Nc3ccccc3C(F)(F)F)n2n1
InChIKey: PWJQBIGGUUBLFR-GOEBONIOSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 392 nM (pChEMBL 6.409999847412109)
Descriptors: MW 418.42, ALogP 3.68, HBA 6, HBD 3, PSA 97.34, QED 0.6
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cn(C)nc4C)cc3N(Cc3ccc(F)c(F)c3)C2=O)c1F
InChIKey: LYFIGUDTINIDEL-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 541.51, ALogP 5.51, HBA 6, HBD 0, PSA 72.72, QED 0.3
Compound (SMILES): CCNC(=O)[C@@H](CCCNC(=N)N)NC(=O)[C@@H]1C[C@@H](O)CN1C(=O)[C@@H]1CCSCCC(=O)N[C@@H](Cc2ccc(Cl)cc2)C(=O)N[C@@H](Cc2ccccc2)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N1
InChIKey: SSROCUAHJLQMAD-SKGYAMTKSA-N
Target: Vasopressin V2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30518
Assay: In Vitro V2 Receptor Assay: Agonist activity of compounds on the human V2 receptor (h V2R) was determined in a transcriptional reporter gene assay by transiently transfecting an h V2 receptor expression DNA into HEK-293 (human embryonic kidney 293 cell line) cells in concert with a reporter DNA containing intracellular calcium responsive promoter elements regulating expression of firefly luciferase. See Boss, V., Talpade, D. J., Murphy, T. J. J. Biol. Chem. 1996, May 3; 271(18), 10429-10432 for further guidance on this assay. Cells were exposed to serial dilutions of compounds diluted 10-fold per dose for 5 h, followed by lysis of cells, determination of luciferase activity, and determination of compound efficacies and EC50 values through non-linear regression. Desmopressin (dDAVP) was used as an internal control in each experiment.
Measurement: EC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 1027.65
Compound (SMILES): N#CCCCS(=O)(=O)Nc1cc(F)c(-c2cc(NC(=O)C3CC3)nc3[nH]ccc23)cc1F
InChIKey: NHUMZAKDJOXISH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 459.48, ALogP 3.9, HBA 5, HBD 3, PSA 127.74, QED 0.44
Compound (SMILES): C=CC(=O)N1CCC[C@@H]1Cn1c(NC(=O)c2ccc(Cl)cc2)nc2cc(CN[C@@H](C)C(C)(C)C)ccc21
InChIKey: BEDWKJCGDZYRIO-WMZHIEFXSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Enzymatic Activity Assay: The ability of the compounds of the present disclosure to inhibit ITK was measured using the Caliper assay format, which is an electrophoretic separation of a phosphorylated peptide substrate from unphosphorylated peptide. The enzymatic reaction occurred in a buffer of 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Three-fold dilutions of compounds were prepared in DMSO. Compounds were added to enzyme and pre-incubated for 15 minutes prior to reaction. The enzymatic reaction was initiated by addition of phospho-acceptor peptide FAM-GEEPLYWSFPAKKK-NH2 (also known as SRCtide) to 1 uM and ATP to its Km value (ITK: 10 uM). The reaction proceeded for 3 hours and was terminated by addition of EDTA. The assay employed an enzyme concentration of 1 nM. The top compound concentration was 5 uM. The amount of phosphorylated substrate was determined by the Caliper instrumentation, and dose-response curves were fit using standard methods to determine the IC50 values.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 522.09, ALogP 5.64, HBA 5, HBD 2, PSA 79.26, QED 0.38
Compound (SMILES): Nc1ncnc2c1c(-c1cc(F)c(NC(=O)Nc3cc(C4(C(F)(F)F)CC4)no3)c(F)c1)cn2C1CC1
InChIKey: RVRUVEXLJYVGEZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Nek7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDX7
Assay: Enzymatic Assay: Casein substrate (from bovine milk, hydrolyzed and partially dephosphorylated mixture of α, β and a caseins, obtained from Sigma Aldrich, catalogue #C4765, diluted in distilled water to a final concentration of 1 mg/mL) and full-length recombinant human NEK7 (expressed by baculovirus in Sf9 insect cells using a N-terminal GST tag, obtained from SignalChem, catalogue #N09-10G, 0.1 μg/μL) were mixed in assay buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO). Compounds of interest (serial 3-fold dilution in DMSO from 10 μM to 0.5 nM) or vehicle (1% DMSO) were dispensed into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range). After incubation at room temperature for 20 minutes, the kinase reaction was initiated by addition of [33P]-ATP (specific activity 10 ρCi/μl) and the mixture was incubated at room temperature for 2 hours. The reaction was then stopped by spotting the reaction mixture on strips of phosphocellulose P81 paper. Following washing, the radioactivity of the P81 paper was measured and kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle reactions.
Measurement: IC50 > 1500 nM (pChEMBL 5.823999881744385)
Descriptors: MW 519.43, ALogP 5.52, HBA 7, HBD 3, PSA 123.89, QED 0.3