lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C)Cc1nc(Nc2ccc(-c3cnc4cc(F)ccn34)c3c2C(=O)NC3)ccc1C1CCOCC1
InChIKey: DRNGJPGUKLOJKE-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of HERG expressed in HEK293 cells at -80mV holding potential incubated for 24 to 48 hrs by whole-cell patch clamp method
Measurement: IC50 = 2850 nM (pChEMBL 5.539999961853027)
Descriptors: MW 500.58, ALogP 4.48, HBA 7, HBD 2, PSA 83.79, QED 0.41
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C[C@H]1SSC[C@H]1O
InChIKey: UORAPQCBWYIISR-IAGOWNOFSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Radioisotope Filter Binding Assay: IC50 Table 13-20: The protocol calls for test compound of the invention to be incubated with kinase, substrate, cofactors, and radio-isotope-labeled ATP (33P-gamma-ATP). The reaction mixtures are then spotted onto filter papers which bind the radioisotope labeled catalytic product. Unreacted phosphate is removed via washing. The reagents used include base reaction 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. The reaction procedure include the following steps: (1) prepare indicated substrate in freshly prepared base reaction buffer, (2) deliver any required cofactors to the substrate solution above, (3) deliver indicated kinase into the substrate solution and gently mix, (4) deliver compounds in DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 minutes at room temperature, (5) deliver 33P-ATP (specific activity 10 μCi/μL) into the reaction mixture to initiate the reaction, (6) incubate kinase reaction for 2 hours at room temperature, (7) reactions are spotted onto P81 ion exchange paper, (8) detect kinase activity by filter-binding method.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 437.55, ALogP 3.99, HBA 9, HBD 2, PSA 99.08, QED 0.45
Compound (SMILES): CN(C)Cc1nc(Nc2ccc(-c3cnc4cc(F)ccn34)c3c2C(=O)NC3)ccc1C1CCOCC1
InChIKey: DRNGJPGUKLOJKE-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 M), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 l/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 500.58, ALogP 4.48, HBA 7, HBD 2, PSA 83.79, QED 0.41
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C[C@H]1SSC[C@H]1O
InChIKey: UORAPQCBWYIISR-IAGOWNOFSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biological Activity Assay: IC50: Methods for In Vitro Evaluation Assays known in the art for testing compounds are used to test compounds of this invention and to assess the biological activities. In order to support that this invention described herein, the following biological assays are set forth. Examples are for illustrative purposes only and are not met to be limiting. Please see paper for the assay method.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 437.55, ALogP 3.99, HBA 9, HBD 2, PSA 99.08, QED 0.45
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C[C@H]1SSC[C@H]1O
InChIKey: UORAPQCBWYIISR-IAGOWNOFSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Radioisotope Filter Binding Assay: IC50 Table 13-20: The protocol calls for test compound of the invention to be incubated with kinase, substrate, cofactors, and radio-isotope-labeled ATP (33P-gamma-ATP). The reaction mixtures are then spotted onto filter papers which bind the radioisotope labeled catalytic product. Unreacted phosphate is removed via washing. The reagents used include base reaction 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. The reaction procedure include the following steps: (1) prepare indicated substrate in freshly prepared base reaction buffer, (2) deliver any required cofactors to the substrate solution above, (3) deliver indicated kinase into the substrate solution and gently mix, (4) deliver compounds in DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 minutes at room temperature, (5) deliver 33P-ATP (specific activity 10 μCi/μL) into the reaction mixture to initiate the reaction, (6) incubate kinase reaction for 2 hours at room temperature, (7) reactions are spotted onto P81 ion exchange paper, (8) detect kinase activity by filter-binding method.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 437.55, ALogP 3.99, HBA 9, HBD 2, PSA 99.08, QED 0.45
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C[C@H]1SSC[C@H]1O
InChIKey: UORAPQCBWYIISR-IAGOWNOFSA-N
Target: Receptor tyrosine-protein kinase erbB-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15303
Assay: Radioisotope Filter Binding Assay: IC50 Table 13-20: The protocol calls for test compound of the invention to be incubated with kinase, substrate, cofactors, and radio-isotope-labeled ATP (33P-gamma-ATP). The reaction mixtures are then spotted onto filter papers which bind the radioisotope labeled catalytic product. Unreacted phosphate is removed via washing. The reagents used include base reaction 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. The reaction procedure include the following steps: (1) prepare indicated substrate in freshly prepared base reaction buffer, (2) deliver any required cofactors to the substrate solution above, (3) deliver indicated kinase into the substrate solution and gently mix, (4) deliver compounds in DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 minutes at room temperature, (5) deliver 33P-ATP (specific activity 10 μCi/μL) into the reaction mixture to initiate the reaction, (6) incubate kinase reaction for 2 hours at room temperature, (7) reactions are spotted onto P81 ion exchange paper, (8) detect kinase activity by filter-binding method.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 437.55, ALogP 3.99, HBA 9, HBD 2, PSA 99.08, QED 0.45
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C[C@H]1SSC[C@H]1O
InChIKey: UORAPQCBWYIISR-IAGOWNOFSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13163
Assay: Radioisotope Filter Binding Assay: IC50 Table 13-20: The protocol calls for test compound of the invention to be incubated with kinase, substrate, cofactors, and radio-isotope-labeled ATP (33P-gamma-ATP). The reaction mixtures are then spotted onto filter papers which bind the radioisotope labeled catalytic product. Unreacted phosphate is removed via washing. The reagents used include base reaction 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. The reaction procedure include the following steps: (1) prepare indicated substrate in freshly prepared base reaction buffer, (2) deliver any required cofactors to the substrate solution above, (3) deliver indicated kinase into the substrate solution and gently mix, (4) deliver compounds in DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 minutes at room temperature, (5) deliver 33P-ATP (specific activity 10 μCi/μL) into the reaction mixture to initiate the reaction, (6) incubate kinase reaction for 2 hours at room temperature, (7) reactions are spotted onto P81 ion exchange paper, (8) detect kinase activity by filter-binding method.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 437.55, ALogP 3.99, HBA 9, HBD 2, PSA 99.08, QED 0.45
Compound (SMILES): C[C@@H]1CCC[C@@H]1n1c(=O)c(CC(N)=O)cc2cnc(NC3CCN(S(C)(=O)=O)CC3)nc21
InChIKey: KQWXMJOPENRODE-DYVFJYSZSA-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 = 6.71 nM (pChEMBL 8.170000076293945)
Descriptors: MW 462.58, ALogP 1.02, HBA 8, HBD 2, PSA 140.28, QED 0.65
Compound (SMILES): Cc1cnc(-c2cc(OC[C@@H]3CN(C)CCO3)cc(C(=O)N[C@@H](C)c3ccc(C(F)(F)F)nn3)c2)s1
InChIKey: IJFKDPNABTXGCF-KXBFYZLASA-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 = 10000 nM (pChEMBL 5)
Descriptors: MW 521.57, ALogP 4.13, HBA 8, HBD 1, PSA 89.47, QED 0.5
Compound (SMILES): CC(=O)c1cnc(OCc2cn(-c3ccc(C)c(OC(F)F)c3)nn2)nc1
InChIKey: YOHSOKUXLWWCLC-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 = 93 nM (pChEMBL 7.03000020980835)
Descriptors: MW 375.33, ALogP 2.75, HBA 8, HBD 0, PSA 92.02, QED 0.59
Compound (SMILES): CC(C)c1cnc2ccc3cnc(Nc4ccc(N5CCCCC5)cn4)nc3n12
InChIKey: GKCCTOCVAIDPIB-UHFFFAOYSA-N
Target: CDK6/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: Biochemical Kinase Inhibition Assay: Potency determination of biochemical kinase inhibition test. Kinase activity test and IC50 determination. Firstly, 10 ng of recombinant CDK4/Cyclin D1 (Life Technologies PV4204) was diluted in kinase buffer (20 mM Tris pH 7.5, 10 mM MgCl2, 0.01% NP-40, 2 mM DTT), and was incubated with inhibitors at the indicated concentration at room temperature for 30 minutes. The kinase reaction was initiated by adding 1 μg (1.5 μM) of recombinant retinoblastoma protein, 5 μM ATP and 10μ Ci γ-32P-ATP. The reaction was incubated at 30° C. for 20 minutes, and was terminated by adding 2×Laemmli sample buffer, heated at 95° C. for three minutes, and the resulting solution was dissolved with 12% acrylamide SDS-PAGE and autoradiographed. A densitometer (Bio-Rad) was used to quantify the corresponding phosphorylated substrate protein bands. The resulting density values were plotted as a function of the log drug concentration using Prism 4 Graphpad software, IC50 values were determined by plotting a non-linear regression curve with a variable slope.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 387.49, ALogP 4.53, HBA 7, HBD 1, PSA 71.24, QED 0.55
Compound (SMILES): CC(NC(=O)c1ccccc1NC(=O)CN1C(=O)C(=O)N(Cc2ccccc2)C1=O)c1ccccc1
InChIKey: PWDOMNUZQXPHIM-UHFFFAOYSA-N
Target: Integrin alpha-4/beta-7 (PROTEIN COMPLEX), Homo sapiens
UniProt: P13612
Assay: ELISA assay : The studies began by screening for the optimal protein concentrations for the method. The studies were conducted in goat anti-mouse IgG coated black React-Bind 96 welled-plates (R&D Biosystems), referred to herein as the anti-mouse IgG plate. Twelve stock solutions were prepared containing a 1:1 stoichiometric mixture of gp120 and α4β7 integrin in PBS at pH 7.2 as given by 0 μM or control, 0.001 μM, 0.01 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM. 1 μM, 2.5 μM, 5 μM, 10 μM and 25 μM in protein (step 1, FIG. 1). A 200 μL aliquot of each stock solutions was then loaded across a 96 welled plate and treated either with 20 μL of PBS pH 7.2 (control) or 20 μL of a 100 μM stock solution of repandusinic acid (RA: compound whose anti-HIV activity is to be tested) in PBS pH 7.2 containing 1% DMSO. Three repetitions were run for both the control and positive or repandusinic acid treated experiments. The final concentration of repandusinic acid in each positive well was 10 μM. The plate was incubated for 4 h at 4° C. on a plate mixer at a speed that created a vortex in each well.
Measurement: IC50 = 0.61 nM (pChEMBL 9.210000038146973)
Descriptors: MW 484.51, ALogP 3.11, HBA 5, HBD 2, PSA 115.89, QED 0.38
Compound (SMILES): CN(C)C(=O)c1ccc(Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1
InChIKey: FCLBVILBAPMKST-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 419.49, ALogP 2.99, HBA 7, HBD 3, PSA 104.18, QED 0.6
Compound (SMILES): Cc1cc(=O)c2c(O)cc(O)c(C3CCN(C)CC3O)c2o1
InChIKey: MOCVYVBNJQIVOV-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60563
Assay: Inhibition Assay: CDK-9/Cyclin T1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1% mercaptoethanol) was assayed against a substrate peptide (YSPTSPSYSPTSPSYSPTSPKKK) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EDTA, 10 mM DTT, 1 mg/ml BSA, 0.3 mM YSPTSPSYSPTSPSYSPTSPKKK, 10 mM magnesium acetate and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated for 30 mM at room temperature. Assays were stopped by addition of 5 μl of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 305.33, ALogP 1.29, HBA 6, HBD 3, PSA 94.14, QED 0.73
Compound (SMILES): O=c1[nH]c2ncc(-c3cccc(C(F)(F)F)c3)cc2n1CCN1CCC(F)CC1
InChIKey: AXLCXEXFZROUAT-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 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 408.4, ALogP 3.84, HBA 4, HBD 1, PSA 53.92, QED 0.67
Compound (SMILES): COc1ccc(-c2cc(Nc3ccc(OC)c([N+](=O)[O-])c3)ncn2)cc1
InChIKey: ZMFVIJUBHZVNCX-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 > 10000 nM (pChEMBL 5)
Descriptors: MW 352.35, ALogP 3.81, HBA 7, HBD 1, PSA 99.41, QED 0.53
Compound (SMILES): CCOc1ccc(F)cc1-c1cnc2[nH]c(=O)n(Cc3cccc(F)c3)c2c1
InChIKey: ZCBPEYRXRJNYBX-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 = 5643 nM (pChEMBL 5.25)
Descriptors: MW 381.38, ALogP 4.12, HBA 4, HBD 1, PSA 59.91, QED 0.57
Compound (SMILES): CC(CO)CNC(=O)O[C@@H]1CC[C@H](c2cc(NC(=O)Cc3cc(F)cc(F)c3)n[nH]2)C1
InChIKey: GZODEJHBBBCAFW-CRLJJSMGSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). 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. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 2.35 nM (pChEMBL 8.630000114440918)
Descriptors: MW 436.46, ALogP 2.86, HBA 5, HBD 4, PSA 116.34, QED 0.51
Compound (SMILES): CC(C)c1cnc2ccc3cnc(Nc4ccc(N5CCN(C)CC5)cn4)nc3n12
InChIKey: VPDDPNCCVMBGNF-UHFFFAOYSA-N
Target: CDK6/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: Biochemical Kinase Inhibition Assay: Potency determination of biochemical kinase inhibition test. Kinase activity test and IC50 determination. Firstly, 10 ng of recombinant CDK4/Cyclin D1 (Life Technologies PV4204) was diluted in kinase buffer (20 mM Tris pH 7.5, 10 mM MgCl2, 0.01% NP-40, 2 mM DTT), and was incubated with inhibitors at the indicated concentration at room temperature for 30 minutes. The kinase reaction was initiated by adding 1 μg (1.5 μM) of recombinant retinoblastoma protein, 5 μM ATP and 10μ Ci γ-32P-ATP. The reaction was incubated at 30° C. for 20 minutes, and was terminated by adding 2×Laemmli sample buffer, heated at 95° C. for three minutes, and the resulting solution was dissolved with 12% acrylamide SDS-PAGE and autoradiographed. A densitometer (Bio-Rad) was used to quantify the corresponding phosphorylated substrate protein bands. The resulting density values were plotted as a function of the log drug concentration using Prism 4 Graphpad software, IC50 values were determined by plotting a non-linear regression curve with a variable slope.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 402.51, ALogP 3.29, HBA 8, HBD 1, PSA 74.48, QED 0.56
Compound (SMILES): Cc1cnc(N[C@@H]2CCN(S(C)(=O)=O)C[C@H]2O)nc1-c1cc(F)c2nc(C)n(C(C)C)c2c1
InChIKey: VSVLGWWGGFOSTP-IEBWSBKVSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: Biological Assay: CDK6/Cyclin D3: The purpose of the CDK6/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. CDK6/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:1). 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), 2% glycerol, 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 4 nM (active sites) activated CDK6/Cyclin D3 in 40 mM HEPES buffer at pH 7.5.
Measurement: Ki = 88.5 nM (pChEMBL 7.050000190734863)
Descriptors: MW 476.58, ALogP 2.64, HBA 8, HBD 2, PSA 113.24, QED 0.58
Compound (SMILES): C[C@@H](O[Si](C)(C)C(C)(C)C)c1nc2c(F)cc(-c3nc(N[C@@H]4CCOC[C@H]4O)ncc3Cl)cc2n1C(C)(C)C
InChIKey: IEYXXFUMUXPXFO-BSLALVQMSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: Biological Assay: CDK6/Cyclin D3: The purpose of the CDK6/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. CDK6/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:1). 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), 2% glycerol, 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 4 nM (active sites) activated CDK6/Cyclin D3 in 40 mM HEPES buffer at pH 7.5.
Measurement: Ki = 33.6 nM (pChEMBL 7.46999979019165)
Descriptors: MW 578.21
Compound (SMILES): Cc1cccnc1Cn1c(=O)[nH]c2ncc(-c3ccc(F)c(F)c3)cc21
InChIKey: IQLQBWWCLVVCTR-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 > 10000 nM (pChEMBL 5)
Descriptors: MW 352.34, ALogP 3.42, HBA 4, HBD 1, PSA 63.57, QED 0.61
Compound (SMILES): O=C(Cn1c(=O)n(CCF)c2ncc(-c3ccc(C(F)(F)F)s3)cc21)N1CCC1
InChIKey: KWPJJMAUEQTPMK-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 = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 428.41, ALogP 3.15, HBA 6, HBD 0, PSA 60.13, QED 0.59
Compound (SMILES): Cc1cnc(OCc2cn(-c3ccc(Cl)c(C(C)(F)F)c3)nn2)nc1C
InChIKey: BLRWUCNCAIAODI-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 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 379.8, ALogP 4.02, HBA 6, HBD 0, PSA 65.72, QED 0.67
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc3ccc(=O)n([C@H]4C[C@H](O)CC[C@@H]4F)c3n2)CC1
InChIKey: FTFMKZBZJAZXTH-PMPSAXMXSA-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 439.51, ALogP 1.05, HBA 8, HBD 2, PSA 117.42, QED 0.73
Compound (SMILES): CN(C)C(=O)c1ccc(Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1
InChIKey: FCLBVILBAPMKST-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 82 nM (pChEMBL 7.090000152587891)
Descriptors: MW 419.49, ALogP 2.99, HBA 7, HBD 3, PSA 104.18, QED 0.6
Compound (SMILES): Cc1cnc(-c2cc(OC[C@H]3CCOC3)cc(C(=O)N[C@H](C)c3ccc(C(F)(F)F)nn3)c2)s1
InChIKey: BICIIZABCQYPPY-CABCVRRESA-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 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 492.52, ALogP 4.83, HBA 7, HBD 1, PSA 86.23, QED 0.51
Compound (SMILES): CN(C)C(=O)c1ccc(Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1
InChIKey: FCLBVILBAPMKST-UHFFFAOYSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 216 nM (pChEMBL 6.670000076293945)
Descriptors: MW 419.49, ALogP 2.99, HBA 7, HBD 3, PSA 104.18, QED 0.6
Compound (SMILES): CN(C)C(=O)c1ccc(Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1
InChIKey: FCLBVILBAPMKST-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: O60563
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 398 nM (pChEMBL 6.400000095367432)
Descriptors: MW 419.49, ALogP 2.99, HBA 7, HBD 3, PSA 104.18, QED 0.6
Compound (SMILES): CN(C)Cc1nc(Nc2ccc(-c3cnc4cc(F)ccn34)c3c2C(=O)NC3)ccc1C1CCOCC1
InChIKey: DRNGJPGUKLOJKE-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24941
Assay: Inhibition of human CDK2/Cyclin E1 preincubated for 10 mins followed by ULight-4E-BP1 (Thr37/46) and ATP addition for 30 mins by plate reader assay
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 500.58, ALogP 4.48, HBA 7, HBD 2, PSA 83.79, QED 0.41
Compound (SMILES): CCN1C[C@@H](NC(=O)Nc2c(C)c(OCCCN(C)C)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)O1
InChIKey: ZYNPOOPUQLHCGU-RPWUZVMVSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 528.6, ALogP 4.29, HBA 7, HBD 2, PSA 83.89, QED 0.38