lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(N)c1nc2c(F)cc(-c3nc(N[C@@H]4CCN(S(C)(=O)=O)C[C@H]4O)ncc3Cl)cc2n1C(C)C
InChIKey: YKKFCIYOTDLCGD-QMWRDMRBSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Biological Assay: CDK4/Cyclin D1: The purpose CDK4/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. CDK4/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: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), 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% TW-20, 3 μM 5-FAM-Dyrktide, 3 nM (active sites) activated CDK4/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.
Measurement: Ki = 0.1 nM (pChEMBL 10)
Descriptors: MW 526.04, ALogP 2.69, HBA 9, HBD 3, PSA 139.26, QED 0.45
Compound (SMILES): N#CN1CCCC(n2nc(-c3cccc(Oc4ccccc4)c3)c(C(N)=O)c2N)C1
InChIKey: WYCJNYRHIZFKRG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Human BTK LanthaScreen Assay: TR-FRET LanthaScreen assays were performed by incubating a dilution series of inhibitor concentrations with 50 μM ATP, 100 nM FAM-Srctide peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2, SEQ ID NO: 1, Molecular Devices, RP7595) and 70 μM of human full-length BTK Kinase (expressed in Sf9 insect cells and purified in-house). The assays were performed with and without pre-incubating the inhibitors with the enzyme for 60 minutes before starting the kinase reaction by adding ATP and the peptide substrate. Samples containing enzyme but no inhibitor were included to determine the maximal extent of reaction. Samples containing no enzyme served as the negative control. The kinase reaction mixtures were incubated at room temperature for 60 minutes before stopping the kinase activity by the addition of 15 mM EDTA. The extent of peptide phosphorylation by BTK was detected using a Terbium-conjugated anti-phospho-Tyrosine antibody (Tb-PT66 antibody, invitrogen # PV3557). Phosphorylation of peptide substrate was measured by determining the ratio of 520/495 nm on an Envision Multi-label Reader (Perkin Elmer) and IC50 values were calculated by fitting the data to a four-parameter equation using XLFit4 (IDBS).
Measurement: IC50 = 154 nM (pChEMBL 6.809999942779541)
Descriptors: MW 402.46, ALogP 3.14, HBA 7, HBD 2, PSA 123.19, QED 0.63
Compound (SMILES): O=C(Nc1ccc(N2CCCC2=O)cc1)c1cccc(C2CC2NCC2CC2)c1
InChIKey: WFPXRXCLXFWFHJ-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: MAO-B Inhibition Assay: The MAO-B inhibitory activity evaluation described below followed the protocol of MAO-Glo (registered trademark) Assay of Promega KK.A test compound dissolved in 4% DMSO was added by 12.5 μL to 25 μL reaction solution (100 mM HEPES (pH 7.5), 5% glycerol, 10% DMSO) containing 400 ng of MAO-B enzyme (Sigma-Aldrich Co. LLC.), and the mixture was reacted at room temperature for 10 min. MAO substrate (Promega KK) (16 μM) was added by 12.5 μL to start the reaction. After reaction at room temperature for 60 min, Luciferine detection reagent (Promega KK) (50 μL) was added to terminate the reaction. After reaction at room temperature for 20 min with stirring, the luminescence was measured by Envision (PerkinElmer). The MAO-B inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate(%)=(1−(test compound count−blank)÷(control−blank))×100
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 389.5, ALogP 3.92, HBA 3, HBD 2, PSA 61.44, QED 0.75
Compound (SMILES): COCc1ccc(NC(=O)C2c3c(N)ncnc3N(C3(C)CC3)C2C#Cc2cccc(F)c2)cc1
InChIKey: MBDKDIZQBUSNMG-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Measurement of Inhibitory Activity Against RET (V804L, V804M): Regarding the conditions for measurement of in vitro inhibitory activity of compounds against RET (V804L or V804M) (i.e., RET with V804L or V804M 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 or V804M) 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 or V804M) 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 = 13.5 nM (pChEMBL 7.869999885559082)
Descriptors: MW 471.54, ALogP 3.86, HBA 6, HBD 2, PSA 93.37, QED 0.55
Compound (SMILES): COc1cc2nccc(Oc3ccc(NC(=O)C4(C(=O)Nc5ccc(F)cc5)CC4)cc3)c2cc1-c1cccn1C(=O)OC(C)(C)C
InChIKey: XDXOWJKRCDMDIP-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Met Kinase Assay: Human Met (residues R974-S1390 with A1209G and V1290L, 3.4 nM) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM KKKGQEEEYVFIE (SEO ID NO. 45), 1 mM sodium orthovanadate, 5 mM sodium-6-glycerophosphate, 10 mM magnesium acetate, and 10 μM [γ-33P-ATP]. The reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of phosphoric acid to a concentration of 0.5%. A reaction aliquot of 10 sL was then spotted onto a P30 filtermat and washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting. Incorporated 33P was measured using the Wallac Microbeta scintillation counter (Perkin Elmer).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 636.68, ALogP 7.78, HBA 8, HBD 2, PSA 120.78, QED 0.17
Compound (SMILES): CCOc1c(C(C)NC(=O)c2c(N)nc3ccc(-c4cncc(CO)c4)nn23)cc(Cl)c2cn[nH]c12
InChIKey: QGTLHHVCNFBGDH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: THP-1 RPS6 ELISA Assay: To measure the Phosphorylated Ribosomal Protein S6 (RPS6) in cell lysates, THP-1 cells (Human Acute Monocytic Leukemia) are purchased from ATCC (Manassas, Va.) and maintained in RPMI with 10% FBS (Gibco/Life Technologies, Carlsbad, Calif.). For the assay, THP-1 cells are serum starved overnight in RPMI, then plated in RPMI (2×105 cells/well in 90 μL) into 96-well flat-bottom tissue culture treated plates (Corning, Corning, N.Y.), in the presence or absence of a concentration range of test compounds. Covered plates are incubated for 2 hours at 37° C., 5% CO2 then treated with or without 10 nM MCP-1(MYBioSource, San Diego, Calif.) for 15 minutes at 37° C., 5% CO2. Plates are centrifuged at 1600 RPM and supernatants are removed. Cells are lysed in Lysis Buffer (Cell Signaling, Danvers, Mass.) with Protease Inhibitor (Calbiochem/EMD, Germany), PMSF (Sigma, St Louis Mo.), HALTS (Thermo Fisher, Rockford, Ill.) for 30 min on wet ice. Cell lysates are frozen at −80° C. before testing. The lysates are tested in the Human/Mouse/Rat Phospho-RPS6 ELISA (R&D Systems, Inc. Minn, MN). The plate is measured using a microplate reader (SpectraMax M5-Molecular Devices, LLC Sunnyvale, Calif.) set to 450 nm with a wavelength correction of 540. IC50 determination is performed by fitting the curve of inhibitor percent inhibition versus the log of the inhibitor concentration using the GraphPad Prism 5.0 software.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 506.95, ALogP 3.29, HBA 9, HBD 4, PSA 156.34, QED 0.26
Compound (SMILES): N#Cc1cccc(N(C(=O)[C@@H]2CCC(=O)N2c2nccc(C#N)n2)[C@H](C(=O)NC2CCC(F)(F)CC2)c2ccccc2Cl)c1
InChIKey: IFPDRGUZVMVZSF-BDYUSTAISA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: In Vitro Assay: A test compound is prepared as 10 mM stock in DMSO and diluted to 50× final concentration in DMSO, for a 50 μl reaction mixture. IDH enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutaric acid is measured using a NADPH depletion assay. In the assay the remaining cofactor is measured at the end of the reaction with the addition of a catalytic excess of diaphorase and resazurin, to generate a fluorescent signal in proportion to the amount of NADPH remaining. IDH1-R132 homodimer enzyme is diluted to 0.125 μg/ml in 40 μl of Assay Buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 0.05% BSA, 2 mM b-mercaptoethanol); 1 μl of test compound dilution in DMSO is added and the mixture is incubated for 60 minutes at room temperature. The reaction is started with the addition of 10 μl of Substrate Mix (20 μl NADPH, 5 mM alpha-ketoglutarate, in Assay Buffer) and the mixture is incubated for 90 minutes at room temperature. The reaction is terminated with the addition of 25 μl of Detection Buffer (36 μg/ml diaphorase, 30 mM resazurin, in 1× Assay Buffer), and is incubated for 1 minute before reading on a SpectraMax platereader at Ex544/Em590.Compounds are assayed for their activity against IDH1 R132C following the same assay as above with the following modifications: Assay Buffer is (50 mM potassium phosphate, pH 6.5; 40 mM sodium carbonate, 5 mM MgCl2, 10% glycerol, 2 mM b-mercaptoethanol, and 0.03% BSA). The concentration of NADPH and alpha-ketoglutarate in the Substrate Buffer is 20 μM and 1 mM, respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 618.04, ALogP 4.84, HBA 7, HBD 1, PSA 143.08, QED 0.4
Compound (SMILES): CN(C)C(=O)CCc1ccc2c(nc(Nc3ccc(OC(F)(F)F)cc3)n2C2CC(C)(C)CC(C)(C)C2)c1F
InChIKey: ZIFXWRWUIMRLQI-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μL and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μL of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/mL FMN, 2 μL/ml decanal/ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 548.63, ALogP 7.62, HBA 5, HBD 1, PSA 59.39, QED 0.31
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CNS(C)(=O)=O)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: MDOYFSFRZMYPOP-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 44356 nM (pChEMBL 4.349999904632568)
Descriptors: MW 508.65, ALogP 3.56, HBA 6, HBD 2, PSA 112.46, QED 0.41
Compound (SMILES): CN1C(=O)COc2ccc(CCN3CCN(c4cccc5sccc45)CC3)cc21
InChIKey: FKHXVLVBZSVIRU-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: 5-HT2A Receptor Antagonism Activity Test: The 5-HT2A receptor antagonism activity test (The antagonism activity of test compounds on 5-HT2A receptor expressing human recombinant 5-HT2A receptor in CHO-K1 cells) was performed using FLIPR Calcium 5 Assay Kit (Product of Molecular Devices USA Inc.) according to the method documented in the kit instructions. The concentration of the test compounds was 0.1 nM-10000 nM and risperidone was used as a positive control. Test method is as follows: On the first day the seed cells are placed in T-175 flask containing 25 ml growth medium (F-12 nutrient mixture+10% FBS+1% penicillin/streptomycin+1.2% 50 mg/ml Geneticin) at a density of 14 million per bottle, cells were cultured under 37° C./5% CO2/humidified conditions for 24 hours; On the next day the seed cells were inoculated to the 384-well cell culture plate (each well containing 20,000 cells), the growth medium was replaced by 50 μL detecting medium (F-12 nutrient mixture+1.5% activated carbon-treated FBS), the cells were cultured under 37° C./5% CO2/humidified conditions for 16 hours; On the third day the medium was removed, each well of the cells plates were added with 24 μL freshly prepared loading dye solution (formulated according to the instructions), the plates were placed in an incubator and incubated under 37° C./5% CO2/humidified conditions for 120 minutes; transfer 6 μL test compound solution to the assay plate and gently shaking for one minute, incubated under 37° C./5% CO2/humidified conditions for 30 minutes; each well of the assay plate was added with 10 μL freshly prepared α-methyl-5-hydroxytryptamine solution (1.2 M, the final concentration of α-methyl-5-hydroxytryptamine is 300 nM), data were detected and analyzed by FLIPR (Product of US Molecular Devices Corporation). Inhibition rate of the compounds at different concentrations was calculated, IC50 value was calculated by the Excelfit software
Measurement: IC50 = 461 nM (pChEMBL 6.340000152587891)
Descriptors: MW 407.54, ALogP 3.62, HBA 5, HBD 0, PSA 36.02, QED 0.66
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CN(C(=O)CN(C)C)CCO4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: JEDHPNXCKMDORP-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 475.6, ALogP 3.47, HBA 7, HBD 1, PSA 91.65, QED 0.48
Compound (SMILES): COc1c(C)c(-c2[nH]c3ccc(C4CCN(CC(=O)NCC5(C)COC5)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: YOTKWLZJSYDJER-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 10992 nM (pChEMBL 4.960000038146973)
Descriptors: MW 544.7, ALogP 4.65, HBA 7, HBD 2, PSA 96.78, QED 0.34
Compound (SMILES): Cc1ccc(S(=O)(=O)c2cc(-c3cnc(N)nc3)cc(N3CCOCC3)c2)cc1
InChIKey: KOGZQRMLAGNKRT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTor Assay Protocol: The substrate was prepared in 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), and required cofactors were added to the substrate solution. The mTor kinase was delivered into the substrate solution, and the solution was gently mixed. Testing compounds were dissolved in 100% DMSO to specific concentration. The serial dilution was conducted by Integra Viaflo Assist in DMSO. The compounds were delivered into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), and the reaction mixture was incubated for 20 min at room temperature. Then, 33P-ATP (Specific activity 10 μCi/μl) was delivered into the reaction mixture to initiate the reaction. The reaction mixture was incubated for 2 hours at room temperature. Radioactivity was detected by a filter-binding method. Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC50 values and curve fits were obtained using Prism (GraphPad Software).
Measurement: IC50 = 9390 nM (pChEMBL 5.03000020980835)
Descriptors: MW 410.5, ALogP 2.7, HBA 7, HBD 1, PSA 98.41, QED 0.71
Compound (SMILES): CCN(C)S(=O)(=O)C[C@H]1C[C@@H](N(C)c2ncnc3[nH]ccc23)C1
InChIKey: IGQKRRODHIRPHD-TXEJJXNPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (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 JAK3 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%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 337.45, ALogP 1.45, HBA 5, HBD 1, PSA 82.19, QED 0.86
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(-c4nnc(C5CN6CCC5CC6)o4)cc3c2C(C)C)cc(C)n1
InChIKey: IMLFPXURQXDBEI-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κc Band AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Bue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 113 nM (pChEMBL 6.949999809265137)
Descriptors: MW 441.58, ALogP 5.83, HBA 5, HBD 1, PSA 70.84, QED 0.43
Compound (SMILES): CCNc1cc2c(cn1)c(-c1ccc(CN3CC[C@@H](O)C3)cc1)nn2C1CCC(Oc2ccc(=O)n(C)n2)CC1
InChIKey: NHQHROKXXUCMLE-NSQNTRMNSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzyme Assay: TYRO3: Compounds of Formula I were screened for their ability to inhibit TYRO3 kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. GST-tagged recombinant human TYRO3 kinase domain from Carna (5 nM; Cat. No. PR7480A) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A) and 2 nM Europium-anti-GST (Cat. No. A15116) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds are typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values were fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 543.67, ALogP 3.75, HBA 10, HBD 2, PSA 110.33, QED 0.35
Compound (SMILES): C[C@@H](NC(=O)c1cc(=O)[nH]c(C2CC2)n1)c1ccc(C2CC2)cc1
InChIKey: DATHLNFSXQAOBE-LLVKDONJSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 323.4, ALogP 3.02, HBA 3, HBD 2, PSA 74.85, QED 0.89
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)CC3CCCN(S(=O)(=O)C4CC4)C3)c2)c1
InChIKey: HZZDPRICRDWSEQ-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95251
Assay: KAT-7 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-7 enzyme (6 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 2 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 120 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 534.04, ALogP 1.65, HBA 7, HBD 2, PSA 130.47, QED 0.5
Compound (SMILES): COc1cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(P(C)(C)=O)c4OC)c4ccccc34)ccn2)cc(S(=O)(=O)N(C)CCN2CCN(C)CC2)c1
InChIKey: QGDUHRXRHUCXIQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Enzyme Binding Assay: Method 2: The inhibitory activities of compounds of the invention against p38MAPKγ (MAPK12: Invitrogen), are evaluated in a similar fashion to that described hereinabove. The enzyme (800 ng/mL, 2.5 μL) is incubated with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solution (2.5 μL, 400 μM) is then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, Thermo Scientific).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 858.01, ALogP 7.85, HBA 11, HBD 3, PSA 154.67, QED 0.09
Compound (SMILES): COCCCc1cnc2c(C(=O)Nc3cnccc3N3C[C@H](C)C[C@H](N)C3)c(N)oc2c1
InChIKey: DVJHYLXFXFXKDO-ZBFHGGJFSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μm Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was pre-incubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.Pim-2 kinase assay-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μm Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 438.53, ALogP 2.81, HBA 8, HBD 3, PSA 132.53, QED 0.48
Compound (SMILES): CCCCNc1ncc(-c2ccnc(N)n2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: AAUNIEBMIUGNTA-JOCQHMNTSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 357.46, ALogP 2.44, HBA 8, HBD 4, PSA 121.87, QED 0.56
Compound (SMILES): C=CC(=O)N1CCN(CCCn2c(=O)c(-c3cc(OC)cc(OC)c3Cl)cc3cnc(N[C@@H]4CCOC4)nc32)CC1
InChIKey: XFGMNTTVFYLJIS-HXUWFJFHSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Caliper-Based Kinase Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of FGFR family (FGFR1, FGFR2, FGFR3, FGFR4) kinase activity of a compound of Formula (III). Serial dilutions of test compounds were incubated with either human recombinant FGFR1 (0.5 nM), FGFR2 (0.1 nM, FGFR3 (0.9 nM), or FGFR4 (2 nM), ATP (FGFR1: 100 μM; FGFR2: 75 μM; FGFR3: 120 μM; FGFR4: 250 μM) and a phosphoacceptor peptide substrate FAM-KKKKEEIYFFF-CONH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper LabChip 3000. Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 583.09, ALogP 3.05, HBA 10, HBD 1, PSA 111.05, QED 0.36
Compound (SMILES): CCN(CC)CC(=O)N1CCN(c2ccc3[nH]c(-c4cn5ncnc5c(C)c4C)c(C(C)C)c3n2)CC1
InChIKey: PLYQMNCWJFJIMC-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 502.67, ALogP 4, HBA 7, HBD 1, PSA 85.66, QED 0.41
Compound (SMILES): CCCCNc1ncc(-c2ccc(CNCC34CC5CC(CC(C5)C3)C4)cn2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: GMAFXYZWTSGYMU-LCKWQUASSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 0.42 nM (pChEMBL 9.380000114440918)
Descriptors: MW 518.75, ALogP 5.77, HBA 7, HBD 4, PSA 94.99, QED 0.28
Compound (SMILES): Cc1csc(-c2cncc(-c3ccnc(Nc4cc(NC(=O)c5ccc(CN6CCN(C)CC6)cc5)ccc4C)n3)c2)n1
InChIKey: UUUZEMLVABCSCR-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kinase Assay: Kinase base buffer (50 mM HEPES, pH 7.5 0.0015% Brij-35; 10 mM MgCl2 2 mM DTT) and Stop buffer, (100 mM HEPES, pH 7.5 0.015% Brij-35; 0.2% Coating Reagent (50 mM EDTA) are prepared. Test compound is diluted in 100% DMSO to 50-times the desired final inhibitor concentration (the Stock Solution) and serially diluted in half-log increments resulting in final concentrations 250 μM to 75 μM, 25 μM, 7.5 μM, 2.5 μM, 0.75 μM, 0.25 μM, 75 nM, 25 nM, 7.5 nM in DMSO. 10 μl of each compound is placed in a 96-well plate as the intermediate plate. 90 μl of Kinase Buffer is added to to each well to prepare the intermediate plate.Figure US10906896-20210202-P00001Mix the compounds in intermediate plate for 10 min on shaker. For the assay of enzyme inhibitions, 5 μl of each well from the intermediate plate is transferred to a 384-well plate in duplicates, 10. Then 10 μl of 2.5× enzyme solution is added to each well of the 384-well assay plate and incubated for 10 min. Then enzyme substrate is added as 10 μl of 2.5×FAM-labeled peptide+ATP solution to each well of the 384-well assay plate. The reaction is allowed to proceed at 28° C. and quenched with the addition of 25 μl of stop buffer.
Measurement: Ki = 5.7 nM (pChEMBL 8.239999771118164)
Descriptors: MW 590.76, ALogP 6.02, HBA 9, HBD 2, PSA 99.17, QED 0.23
Compound (SMILES): CCC(n1cc(-c2nc(-c3cnn(C[C@@H](O)CO)c3)cn3nccc23)cn1)C(F)(F)F
InChIKey: BNGZGHJLRLYZLX-XPCCGILXSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK2 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 10 μM Omnia® Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 4 nM JAK2 in a total volume of 25 μL. JAK2 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4288).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 435.41, ALogP 2.32, HBA 9, HBD 2, PSA 106.29, QED 0.46
Compound (SMILES): COc1cc2ncnc(Nc3ccc(NC(=O)Nc4ccc(Cl)c(C(F)(F)F)c4)c(Cl)c3)c2c2c1OCCO2
InChIKey: CJFHQQDWGGWHLL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Test of Small Molecule Compounds for Inhibiting the Activity of C-RAF and B-RAF Kinases: 1. Preparation of test compounds: according to the molecular weight of the compounds, an appropriate volume of DMSO was directly added to dissolve the test compounds. For storing the compound, the concentration of DMSO is 100%, and the final concentration of DMSO in the experimental system is 1%. The compounds were 3-fold serially diluted with DMSO to obtain a total of 8 dilutions, with a maximum concentration of 1000 nM and a minimum concentration of 0.46 nM.2. Preparation of the sorafenib positive control: sorafenib, a selective inhibitor of BRAF and RAF1, was used as the positive control of this experiment, and the dilution method thereof was the same as that of the above test compounds.3. Test Conditions:Enzyme: B-RAF: 0.1 ng/l (the final concentration in the reaction system); C-RAF: 0.1 ng/μl (the final concentration in the reaction system)Substrate and ATP: inactive MEKI: 2 ng/μl (the final concentration in the reaction system); ATP: 35 μM (the final concentration in the reaction system)HPE: the reaction without enzyme (1% DMSO)ZPE: the reaction with enzyme but without compound (1% DMSO)4. Test procedure:a) 1 ul of 10-fold diluted compound or 10% DMSO was added to a 384-well assay plate,b) 4 ul enzyme solution or assay buffer was added to the wells of the assay plate,c) the plate was centrifuged at 1000 rpm for 1 minute to homogeneous,d) 5 ul of ATP-substrate mixture was added to the wells of the assay plate,e) the plate was shaked for mixing for 2 minutes,f) the plate was incubated at 30° C. for 1 hour,g) 10 ul of ADP-Glo reagent was added to the wells of the assay plate, and the plate was incubated for 40 minutes at 27° C.,h) 20 ul of kinase assay solution was added to the wells of the plate, and the plate was incubated for 30 minutes at 27° C.,i) the chemiluminescent signal was read with Envision.5. Analysis of the results: calculation of the compound inhibition rate:Inhibition rate (%)=(control measurement without compound−sample measurement)/(control measurement without compound−control measurement without enzyme)*100%The IC50 values of the positive control compound and the test compounds were calculated using the Prism software according to the variable slope of the curve.
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 580.35, ALogP 7.12, HBA 7, HBD 3, PSA 106.63, QED 0.23
Compound (SMILES): Oc1ccc2c3c(cnc2c1)-c1ccc(F)cc1O[C@@H]3c1ccc(OCCN2CC(CF)C2)cc1
InChIKey: OGEAHDJGRDNLQY-MUUNZHRXSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: ERalpha (Wild Type), ERalpha (Y537ERalpha (Wild Type), ERalph (Y537S Mutant) and ERbeta Competition Binding Assay: The purpose of the following ER competition binding assays is to determine the affinity of a test compound against ERα (wild type), ERα (Y537S mutant), and ERβ.Run the competition binding assay in a buffer containing 50 mM HEPES, pH 7.5, 1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mg/mL ovalbumin, and 5 mM DTT, using 0.025 μCi per well 3H-estradiol (118 μCi/mmol, 1 mCi/mL), 7.2 ng/well ERα (wild type), or 7.2 ng/well ERα (Y537S mutant) or 7.7 ng/well ERβ receptor. Add the test compound at 10 different concentrations ranging from 10,000 nM to 0.5 nM, and determine nonspecific binding in the presence of 1 μM of 17-β estradiol. Incubate the binding reaction (140 μL) for 4 hours at room temperature, and then add cold dextran-charcoal buffer (70 μL) (containing per 50 mL of assay buffer, 0.75 g of charcoal and 0.25 g of dextran) to each reaction. Mix the plates for 8 minutes on an orbital shaker at 4° C. and then centrifuge at 3000 rpm at 4° C. for 10 minutes. Transfer an aliquot (120 μL) of the mixture to another 96-well, white flat bottom plate (Costar) and add Perkin Elmer Optiphase Supermix scintillation fluid (175 μL) to each well. Seal the plates and shake vigorously on an orbital shaker. After an incubation of 2.5 hours, read the plates in a Wallac Microbeta counter. Calculate the IC50 using a 4-parameter logistic curve fit and calculate % inhibition at 10 μM. Convert the IC50 values for the compound to Ki using Cheng-Prusoff equation. The results of this assay demonstrate Examples 1, 1A, and 1B (and others) bind to recombinant ERα wild type and ERα mutant (Y537S) as shown in Table 7 below and Example 1B was also determined to bind to ERβ with a Ki (nM) ERβ competition of 0.11±0.07, n=3.
Measurement: Ki = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 474.51, ALogP 5.51, HBA 5, HBD 1, PSA 54.82, QED 0.4
Compound (SMILES): CN(C)C(=O)N[C@@H]1CCCN(c2cnc(C(N)=O)c(Nc3ccc(N4CCS(=O)(=O)CC4)cc3)n2)C1
InChIKey: HUXYMEQAXNVXON-QGZVFWFLSA-N
Target: Tyrosine-protein kinase Lck (SINGLE PROTEIN), Homo sapiens
UniProt: P06239
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 516.63, ALogP 0.79, HBA 9, HBD 3, PSA 153.86, QED 0.51
Compound (SMILES): Nc1ccccc1NC(=O)C1CCCCC1
InChIKey: TZXMXCNAINYVMS-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC3: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. HDAC3-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tween-20 (Zymed 00-3005), -0.05% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC: 0.1 ug/ml HDAC3 (BPS Inc) in assay buffer 3x Substrate+3x Trpsin: 28.5 uM Substrate + 450nM Trpsin in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -802C. BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 3050 nM (pChEMBL 5.519999980926514)
Descriptors: MW 218.3, ALogP 2.79, HBA 2, HBD 2, PSA 55.12, QED 0.75
Compound (SMILES): COCCOCCOCCOc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4cccc(CP(C)(C)=O)c4)c4ccccc34)n2)cc(OC)c1
InChIKey: OKERWTBOFAFQQV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 μL) is incubated with the test compound (either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM. 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM for c-Src, and 60 μM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 851.94, ALogP 9.48, HBA 13, HBD 3, PSA 169.21, QED 0.05
Compound (SMILES): Cc1cnc(C(=O)N2CC3CCC2C(Oc2cnc(C(F)(F)F)cn2)C3)c(-c2ncccn2)c1
InChIKey: JVPQCMCXPWENIH-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 470.46, ALogP 3.73, HBA 7, HBD 0, PSA 93.99, QED 0.57