lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#CCC1(n2ncc(-c3cc(-c4cn[nH]c4)cc4ncccc34)n2)CC(C#N)C1
InChIKey: RDPYCMBRCZLAJL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 2727 nM (pChEMBL 5.559999942779541)
Descriptors: MW 380.42, ALogP 3.43, HBA 7, HBD 1, PSA 119.86, QED 0.58
Compound (SMILES): COCc1ccc(NC(=O)c2cn(C(C)(C)C)c3ncnc(N)c23)cc1
InChIKey: PLDXFCFLWGXXPX-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Inhibitory Activity Against Resistance Mutation of RET (In Vitro): Regarding the conditions for measurement of in vitro inhibitory activity of compounds against RET (V804L) (i.e., RET with V804L mutation) kinase activity, the website of AnaSpec states that Srctide (GEEPLYWSFPAKKK) corresponds to the substrate peptide for reaction to measure RET kinase activity. Thus, the amino acid sequence was partly modified and biotinylated to prepare biotinylated peptides (biotin-EEPLYWSFPAKKK). The purified recombinant human RET (V804L) protein used in the test was purchased from Eurofins.To measure the inhibitory activity, first, the test compounds were individually diluted with dimethyl sulfoxide (DMSO) stepwise. Subsequently, RET (V804L) protein, the substrate peptides (the final concentration: 250 nM), magnesium chloride (the final concentration: 10 mM), ATP (the final concentration: 10 μM), and a solution of a test compound in DMSO (the final concentration of DMSO: 5%) were added to a buffer for kinase reaction (13.5 mM Tris, pH of 7.5, 2 mM dithiothreitol, 0.009% Tween-20). Each of the mixtures was incubated at 25° C. for 120 minutes to perform a kinase reaction. EDTA was then added thereto to give a final concentration of 40 mM so that the reaction was terminated. A detection solution containing Eu-labeled antiphosphotyrosine antibody PT66 (PerkinElmer) and SureLight APC-SA (PerkinElmer) was added thereto, and each mixture was allowed to stand at room temperature for 2 hours or more. Finally, the intensity of fluorescence under excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at the two wavelengths of 620 nm and 665 nm. The phosphorylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which phosphorylation was inhibited by 50% was defined as the IC50 value (nM).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 353.43, ALogP 3.17, HBA 6, HBD 2, PSA 95.06, QED 0.75
Compound (SMILES): O=C(O)C1CCC(C(=O)N2CC[C@@]3(S(=O)(=O)c4ccc(F)cc4)c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4CC[C@@H]23)C1
InChIKey: XRRXYBCURUVMRW-AZNPCEPYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 319 nM (pChEMBL 6.5)
Descriptors: MW 639.56, ALogP 5.83, HBA 4, HBD 1, PSA 91.75, QED 0.33
Compound (SMILES): COCc1ccc(NC(=O)c2cn(C(C)(C)C)c3ncnc(N)c23)cc1
InChIKey: PLDXFCFLWGXXPX-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Measurement of Inhibitory Activity Against RET (V804M): RET (V804M) (i.e., RET with V804M mutation) kinase activity was measured using purified recombinant human RET (V804M) protein purchased from Eurofins with the final concentration of ATP being 13 μM in the kinase reaction system, following procedure 1) for the other part.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 353.43, ALogP 3.17, HBA 6, HBD 2, PSA 95.06, QED 0.75
Compound (SMILES): O=C(O)C1CCC(C(=O)N2CC[C@@]3(S(=O)(=O)c4ccc(F)cc4)c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4CC[C@@H]23)C1
InChIKey: XRRXYBCURUVMRW-AZNPCEPYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 98 nM (pChEMBL 7.010000228881836)
Descriptors: MW 639.56, ALogP 5.83, HBA 4, HBD 1, PSA 91.75, QED 0.33
Compound (SMILES): Cc1cncc2ccc(NC(=O)[C@@H]3CC3c3ccc(S(=O)(=O)Nc4cnccn4)cc3)cc12
InChIKey: MQYWUAVOLVOSFD-FOIFJWKZSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: ROCK Kinase Inhibition Assays: All compounds were initially prepared as 10 mM stocks in anhydrous dimethylsulfoxide (DMSO). A 20 μl aliquot of the 10 mM solutions was transferred to individual wells in column 1 of a 96-well polypropylene microtiter plate (Corning #3363) and diluted with DMSO to give a final compound concentration of 4 mM. Test compounds were then serially diluted 1:5 in DMSO for an 11-point concentration response and further diluted in the assay buffer bringing all compound concentrations to a final range of 100 μM to 10 pM in 2.5% DMSO. The assay was performed in white 96-well, flat-bottom, half-area, non-binding assay plate (Corning #3642) in assay buffer consisting of 20 mM HEPES (pH 7.5), 10 mM MgCl2*6H2O, 100 μM sodium orthovanadate, 0.05% CHAPS and 0.1% bovine serum albumin. A 10 μL aliquot of compound from each well of the intermediate dilution plate and 20 μL of a 2× substrate/enzyme solution containing acceptor substrate (800 nM RSK2 peptide KRRRLSSLRA (SEQ ID NO: 1)), ROCK2 enzyme (10 nM), or ROCK1 enzyme, and 1,4-Dithiothreitol (DTT, 2 μM) were added to all wells. The reaction was initiated by the addition of 10 μL of 4× stock solution ATP (2 ∥M). Reactions were thoroughly mixed manually, covered and allowed to incubate at room temperature for 75 min. Protein kinase activity was quantitated using Promega's KINASE-GLO luminescent Kinase Assay Kit according to the manufacturer's directions. ATP concentrations remaining in Test wells following the termination of the enzymatic reaction were compared against control wells containing equivalent amounts of DMSO containing no inhibitor (CTRL). ATP concentrations in both Test wells and CTRL wells were normalized against background (BKG) ATP concentrations in wells containing concentrations of inhibitor that completely inhibited the protein kinase under investigation (i.e. a concentration that prevented any consumption of ATP over the course of the incubation). Percent of Control (POC) values were determined for each concentration of compound tested according to the equation: POC=((Test well value−BKG)/(CTRL−BKG))*100IC50 values were calculated using the following 4-parameter logistic curve-fitting algorithm: f(x)=(A+((B−A)/(1+((x/C){circumflex over ( )}D))))IC50 values were converted to Ki values using the Cheng-Prusoff Equation: Ki=IC50/(1+([ATP]/Km ATP])).
Measurement: IC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 459.53, ALogP 3.88, HBA 6, HBD 2, PSA 113.94, QED 0.45
Compound (SMILES): O=C(O)C1CCC(C(=O)N2CC[C@@]3(S(=O)(=O)c4ccc(F)cc4)c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4CC[C@@H]23)C1
InChIKey: XRRXYBCURUVMRW-AZNPCEPYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 398 nM (pChEMBL 6.400000095367432)
Descriptors: MW 639.56, ALogP 5.83, HBA 4, HBD 1, PSA 91.75, QED 0.33
Compound (SMILES): O=C(O)C1CCC(C(=O)N2CC[C@@]3(S(=O)(=O)c4ccc(F)cc4)c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4CC[C@@H]23)C1
InChIKey: XRRXYBCURUVMRW-AZNPCEPYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 225 nM (pChEMBL 6.650000095367432)
Descriptors: MW 639.56, ALogP 5.83, HBA 4, HBD 1, PSA 91.75, QED 0.33
Compound (SMILES): Cc1cncc2ccc(NC(=O)[C@@H]3CC3c3ccc(S(=O)(=O)Nc4cnccn4)cc3)cc12
InChIKey: MQYWUAVOLVOSFD-FOIFJWKZSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: ROCK Kinase Inhibition Assays: All compounds were initially prepared as 10 mM stocks in anhydrous dimethylsulfoxide (DMSO). A 20 μl aliquot of the 10 mM solutions was transferred to individual wells in column 1 of a 96-well polypropylene microtiter plate (Corning #3363) and diluted with DMSO to give a final compound concentration of 4 mM. Test compounds were then serially diluted 1:5 in DMSO for an 11-point concentration response and further diluted in the assay buffer bringing all compound concentrations to a final range of 100 μM to 10 pM in 2.5% DMSO. The assay was performed in white 96-well, flat-bottom, half-area, non-binding assay plate (Corning #3642) in assay buffer consisting of 20 mM HEPES (pH 7.5), 10 mM MgCl2*6H2O, 100 μM sodium orthovanadate, 0.05% CHAPS and 0.1% bovine serum albumin. A 10 μL aliquot of compound from each well of the intermediate dilution plate and 20 μL of a 2× substrate/enzyme solution containing acceptor substrate (800 nM RSK2 peptide KRRRLSSLRA (SEQ ID NO: 1)), ROCK2 enzyme (10 nM), or ROCK1 enzyme, and 1,4-Dithiothreitol (DTT, 2 μM) were added to all wells. The reaction was initiated by the addition of 10 μL of 4× stock solution ATP (2 ∥M). Reactions were thoroughly mixed manually, covered and allowed to incubate at room temperature for 75 min. Protein kinase activity was quantitated using Promega's KINASE-GLO luminescent Kinase Assay Kit according to the manufacturer's directions. ATP concentrations remaining in Test wells following the termination of the enzymatic reaction were compared against control wells containing equivalent amounts of DMSO containing no inhibitor (CTRL). ATP concentrations in both Test wells and CTRL wells were normalized against background (BKG) ATP concentrations in wells containing concentrations of inhibitor that completely inhibited the protein kinase under investigation (i.e. a concentration that prevented any consumption of ATP over the course of the incubation). Percent of Control (POC) values were determined for each concentration of compound tested according to the equation: POC=((Test well value−BKG)/(CTRL−BKG))*100IC50 values were calculated using the following 4-parameter logistic curve-fitting algorithm: f(x)=(A+((B−A)/(1+((x/C){circumflex over ( )}D))))IC50 values were converted to Ki values using the Cheng-Prusoff Equation: Ki=IC50/(1+([ATP]/Km ATP])).
Measurement: IC50 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 459.53, ALogP 3.88, HBA 6, HBD 2, PSA 113.94, QED 0.45
Compound (SMILES): Cc1cncc2ccc(NC(=O)[C@@H]3CC3c3ccc(S(=O)(=O)Nc4cnccn4)cc3)cc12
InChIKey: MQYWUAVOLVOSFD-FOIFJWKZSA-N
Target: Signal transducer and activator of transcription 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P42229
Assay: ROCK Kinase Inhibition Assays: All compounds were initially prepared as 10 mM stocks in anhydrous dimethylsulfoxide (DMSO). A 20 μl aliquot of the 10 mM solutions was transferred to individual wells in column 1 of a 96-well polypropylene microtiter plate (Corning #3363) and diluted with DMSO to give a final compound concentration of 4 mM. Test compounds were then serially diluted 1:5 in DMSO for an 11-point concentration response and further diluted in the assay buffer bringing all compound concentrations to a final range of 100 μM to 10 pM in 2.5% DMSO. The assay was performed in white 96-well, flat-bottom, half-area, non-binding assay plate (Corning #3642) in assay buffer consisting of 20 mM HEPES (pH 7.5), 10 mM MgCl2*6H2O, 100 μM sodium orthovanadate, 0.05% CHAPS and 0.1% bovine serum albumin. A 10 μL aliquot of compound from each well of the intermediate dilution plate and 20 μL of a 2× substrate/enzyme solution containing acceptor substrate (800 nM RSK2 peptide KRRRLSSLRA (SEQ ID NO: 1)), ROCK2 enzyme (10 nM), or ROCK1 enzyme, and 1,4-Dithiothreitol (DTT, 2 μM) were added to all wells. The reaction was initiated by the addition of 10 μL of 4× stock solution ATP (2 ∥M). Reactions were thoroughly mixed manually, covered and allowed to incubate at room temperature for 75 min. Protein kinase activity was quantitated using Promega's KINASE-GLO luminescent Kinase Assay Kit according to the manufacturer's directions. ATP concentrations remaining in Test wells following the termination of the enzymatic reaction were compared against control wells containing equivalent amounts of DMSO containing no inhibitor (CTRL). ATP concentrations in both Test wells and CTRL wells were normalized against background (BKG) ATP concentrations in wells containing concentrations of inhibitor that completely inhibited the protein kinase under investigation (i.e. a concentration that prevented any consumption of ATP over the course of the incubation). Percent of Control (POC) values were determined for each concentration of compound tested according to the equation: POC=((Test well value−BKG)/(CTRL−BKG))*100IC50 values were calculated using the following 4-parameter logistic curve-fitting algorithm: f(x)=(A+((B−A)/(1+((x/C){circumflex over ( )}D))))IC50 values were converted to Ki values using the Cheng-Prusoff Equation: Ki=IC50/(1+([ATP]/Km ATP])).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 459.53, ALogP 3.88, HBA 6, HBD 2, PSA 113.94, QED 0.45
Compound (SMILES): Cc1cncc2ccc(NC(=O)[C@@H]3CC3c3ccc(S(=O)(=O)Nc4cnccn4)cc3)cc12
InChIKey: MQYWUAVOLVOSFD-FOIFJWKZSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Kinase Assays: Compounds were prepared in the exact same manner as described in the ROCK Kinase Assay with the exception to the substrate and enzyme. The JAK 2× substrate/enzyme solution consisted of acceptor substrate (800 nM Abl peptide EAIYAAPFAKKK (SEQ ID NO: 2)), JAK2 or JAK3 enzyme (10 nM) and DTT (2 μM). All other steps and solutions remain identical to the ROCK Kinase Assay above.
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 459.53, ALogP 3.88, HBA 6, HBD 2, PSA 113.94, QED 0.45
Compound (SMILES): Cc1cncc2ccc(NC(=O)[C@@H]3CC3c3ccc(S(=O)(=O)Nc4cnccn4)cc3)cc12
InChIKey: MQYWUAVOLVOSFD-FOIFJWKZSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Kinase Assays: Compounds were prepared in the exact same manner as described in the ROCK Kinase Assay with the exception to the substrate and enzyme. The JAK 2× substrate/enzyme solution consisted of acceptor substrate (800 nM Abl peptide EAIYAAPFAKKK (SEQ ID NO: 2)), JAK2 or JAK3 enzyme (10 nM) and DTT (2 μM). All other steps and solutions remain identical to the ROCK Kinase Assay above.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 459.53, ALogP 3.88, HBA 6, HBD 2, PSA 113.94, QED 0.45
Compound (SMILES): Cc1cncc2ccc(NC(=O)[C@@H]3CC3c3ccc(S(=O)(=O)Nc4cnccn4)cc3)cc12
InChIKey: MQYWUAVOLVOSFD-FOIFJWKZSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Kinase Assays: Compounds were prepared in the exact same manner as described in the ROCK Kinase Assay with the exception to the substrate and enzyme. The JAK 2× substrate/enzyme solution consisted of acceptor substrate (800 nM Abl peptide EAIYAAPFAKKK (SEQ ID NO: 2)), JAK2 or JAK3 enzyme (10 nM) and DTT (2 μM). All other steps and solutions remain identical to the ROCK Kinase Assay above.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 459.53, ALogP 3.88, HBA 6, HBD 2, PSA 113.94, QED 0.45
Compound (SMILES): O=c1[nH]c2ncc(-c3ccc(F)cc3)cc2n1Cc1cccnc1
InChIKey: WLIGFRSFZDICAK-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 = 98 nM (pChEMBL 7.010000228881836)
Descriptors: MW 320.33, ALogP 2.97, HBA 4, HBD 1, PSA 63.57, QED 0.63
Compound (SMILES): CC(C)(O)c1cnc(OCc2cn(-c3cccc(OC(F)(F)F)c3)nn2)nc1
InChIKey: GYCBYHREXPLFEP-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 = 284 nM (pChEMBL 6.550000190734863)
Descriptors: MW 395.34, ALogP 2.76, HBA 8, HBD 1, PSA 95.18, QED 0.69
Compound (SMILES): Fc1ccc(-n2cc(CNc3ncc(Cl)cn3)nn2)cc1C(F)F
InChIKey: MDSVIRSWONSGIY-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 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 354.72, ALogP 3.4, HBA 6, HBD 1, PSA 68.52, QED 0.76
Compound (SMILES): O=C(O)[C@H](CCN(CCO)CCCCc1ccc2c(n1)NCCC2)Nc1ncnc2ccccc12
InChIKey: ZOONEWNJDYOTCN-QHCPKHFHSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Solid Phase Integrin alphav/beta6 Binding Assay (B-2): A third series of exemplary compounds was selected for testing in the solid phase integrin αvβ6 binding assay. The compounds tested were compound samples prepared according to procedures described in the Synthetic Examples section, with the stereochemical purity as indicated in the Examples. As in Example B1, microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 478.6, ALogP 2.96, HBA 8, HBD 4, PSA 123.5, QED 0.27
Compound (SMILES): COC[C@@H](F)CN(CCCCc1ccc2c(n1)NCCC2)CC[C@H](Nc1ncnc2c1cnn2C)C(=O)O
InChIKey: KCCSLLNAHMZLNL-UGKGYDQZSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Solid Phase Integrin alphav/beta6 Binding Assay (B-2): A third series of exemplary compounds was selected for testing in the solid phase integrin αvβ6 binding assay. The compounds tested were compound samples prepared according to procedures described in the Synthetic Examples section, with the stereochemical purity as indicated in the Examples. As in Example B1, microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 528.63, ALogP 2.68, HBA 10, HBD 3, PSA 130.32, QED 0.25
Compound (SMILES): Cc1cc2cnc(NC3CCN(S(=O)(=O)C4CC4)CC3)nc2n([C@@H]2CCC[C@@]2(C)O)c1=O
InChIKey: ZGBMIKQTQVROES-XMSQKQJNSA-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 = 1.24 nM (pChEMBL 8.90999984741211)
Descriptors: MW 461.59, ALogP 1.94, HBA 8, HBD 2, PSA 117.42, QED 0.7
Compound (SMILES): OCc1ccnc(OCc2cn(-c3cccc(C(F)F)c3)nn2)n1
InChIKey: RWMASHRJKARNFY-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 = 408 nM (pChEMBL 6.389999866485596)
Descriptors: MW 333.3, ALogP 2.07, HBA 7, HBD 1, PSA 85.95, QED 0.74
Compound (SMILES): CO[C@H](C)CN(CCCCc1ccc2c(n1)NCCC2)CC[C@H](Nc1nc(C)nc2cc(F)ccc12)C(=O)O
InChIKey: IOJXFJFHNHQUKS-CLOONOSVSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Solid Phase Integrin alphav/beta6 Binding Assay (B-1): Microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 538.67, ALogP 4.45, HBA 8, HBD 3, PSA 112.5, QED 0.26
Compound (SMILES): O=C(O)C(CCO[C@H]1C[C@H](CCc2ccc3c(n2)NCCC3)C1)NC(=O)[C@@H](CO)c1ccccc1
InChIKey: YDXCSYFVDZHHAN-KQSUZSRESA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Binding Assay: The biochemical potency of compounds was determined using a proximity-based assay (ALPHASCREEN®, Perkin Elmer, Waltham, Mass.) as described previously (Ullman E F et al., Luminescent oxygen channeling immunoassay: Measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5426-5430, June 1994). To gauge the potency of inhibitors of binding to human integrin αvβ6, inhibitor compounds and integrin were incubated together with TGF-b1 LAP and biotinylated anti-LAP antibody plus acceptor and donor beads, following the manufacture's recommendations. The donor beads were coated with streptavidin. The acceptor beads had a nitrilotriacetic acid Ni chelator, for binding to a 6×His Tag on human integrin αvβ6. All incubations occurred at room temperatures in 50 mM Tris-HCl, pH 7.5, 0.1% BSA supplemented with 1 mM each CaCl2 and MgCl2.The order of reagent addition was as follows:1. Alpha-v-beta-6 integrin, test inhibitor compound, LAP, biotinylated anti-LAP antibody and acceptor beads were all added together.2. After 2 hours, donor beads were added. After another 30 minute incubation, samples were then read.Integrin binding was evaluated by exciting donor beads at 680 nm, and measuring the fluorescent signal produced, between 520-620 nm, using a Biotek Instruments (Winooski, Vt., USA) SynergyNeo2 multimode plate reader.
Measurement: IC50 = 625 nM (pChEMBL 6.199999809265137)
Descriptors: MW 481.59, ALogP 2.9, HBA 6, HBD 4, PSA 120.78, QED 0.37
Compound (SMILES): CCc1cnc(Nc2cccc(NC(=O)c3ccc(NC(=O)/C=C/CN(C)C)cc3)c2)nc1-c1c[nH]c2ccccc12
InChIKey: NGJZZNXZCNMUCG-MDWZMJQESA-N
Target: Cyclin-dependent kinase 7/ cyclin H (PROTEIN COMPLEX), Homo sapiens
UniProt: P51946
Assay: In Vitro Kinase Assay: In vitro kinase assays with immunoprecipitated proteins (or recombinant CAK complexes) were performed as follows. Kinase reactions were performed in 25 μL of final volume of reaction buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5% glycerol, 25 μM ATP, and 10 μCi of [γ-32P]ATP). Each reaction contained 1 μg of RNAP II. Incubation was at 30° C. for 30-45 min. Reactions were terminated by addition of 5 μL of 5×SDS-PAGE sample buffer and incubated at 95° C. for 5 min. Reactions were analyzed by SDS-PAGE, and the dried gel was exposed to film or PhosphorImager plate.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 559.67, ALogP 6.24, HBA 6, HBD 4, PSA 115.04, QED 0.15
Compound (SMILES): CC(C)c1cnc2ccc3cnc(Nc4ccc(N5CCC6(CNC6)C5)cn4)nc3n12
InChIKey: IOQSBRPAWCWDTI-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 414.52, ALogP 3.34, HBA 8, HBD 2, PSA 83.27, QED 0.53
Compound (SMILES): O=C(Cn1c(=O)[nH]c2ncc(-c3ccc(C(F)(F)F)s3)cc21)N1CCC1
InChIKey: INTCEBHIEDIHMH-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 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 382.37, ALogP 2.7, HBA 5, HBD 1, PSA 70.99, QED 0.76
Compound (SMILES): Cc1cc(Oc2cc(C(=O)NCc3ccc(C)nn3)cc(-c3ncc(C)s3)c2)ccn1
InChIKey: XCSQCNBYINKWNK-UHFFFAOYSA-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 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 431.52, ALogP 4.64, HBA 7, HBD 1, PSA 89.89, QED 0.48
Compound (SMILES): COc1ccccc1-c1cc(Nc2ccc(N(C)C)c(Cn3cnc4ccccc43)c2)ncn1
InChIKey: TVLKGSNODYWKDT-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 450.55, ALogP 5.36, HBA 7, HBD 1, PSA 68.1, QED 0.36
Compound (SMILES): C[C@@H]1Cn2c(c(-c3ccc(Cl)cc3)c3ncccc32)C(=O)N1C1CC1
InChIKey: WAYXCSKMOOKIDO-GFCCVEGCSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4A (SINGLE PROTEIN), Homo sapiens
UniProt: P27815
Assay: Scintillation Proximity Assay: The PDE4A3, PDE4B1, PDE4C1 and PDE4D3 assays use the Scintillation Proximity Assay (SPA) technology to measure the inhibition of human recombinant PDE4A1, PDE4B3, PDE4C1, and PDE4D3 enzyme activity by compounds in vitro. The PDE4A1, PDE4B3, PDE4C1, and PDE4D3 assays are run in parallel using identical parameters, except for the concentration of enzyme (80 μM PDE4A3, 40 μM PDE4B3, 40 μM PDE4C1 and 10 μM PDE4D). The assays are performed in a 384-well format with 50 uL assay buffer (50 mM TRIS pH7.5; 1.3 mM MgCl2; 0.01% Brij) containing enough PDE4A3, PDE4B1, PDE4C1, and PDE4D to convert 20% of substrate (1 μM cAMP consisting of 20 nM 3H-cAMP+980 uM cold cAMP) and a range of inhibitors. Reactions are incubated for 30 min at 25° C. The addition of 20 uL of 8 mg/ml yitrium silicate SPA beads (Perkin Elmer) stops the reaction. The plates are sealed (TopSeal, Perkin Elmer) and the beads are allowed to settle for 8 hrs, after which they are read on the Trilux Microbeta overnight.
Measurement: IC50 = 15.5 nM (pChEMBL 7.809999942779541)
Descriptors: MW 351.84, ALogP 4.36, HBA 3, HBD 0, PSA 38.13, QED 0.69