lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc(CN)cc1Nc1ncc(Cl)c(Nc2ccccc2S(=O)(=O)C(C)C)n1
InChIKey: RJLMIOROTJPHHU-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition of ALK WT Anaplastic Lymphoma Kinase Activity: The following experiment was performed in order to measure the activity of the N2-(2-methoxyphenyl)pyrimidine derivative represented by formula 1 of the present invention to inhibit anaplastic lymphoma kinase (ALK) activity at enzyme level. To measure the activity to inhibit ALK, each of the compounds prepared in examples listed in table 2 (2 μl) was loaded in a 384 well plate. Then, each of the compounds was mixed with anaplastic lymphoma kinase (ALK) enzyme (1 μl) and biotin conjugated peptide substrate (2 μl), followed by culture for 15 minutes. ATP solution (5 μl) was added thereto, followed by kinase reaction at room temperature for 30 minutes. Streptavidin conjugated XL 665 (5 μl) dissolved in ethylenediaminetetraacetic acid solution and europium (Eu3+) conjugated anti-phosphotyrosine antibody (5 μl) were added to the reaction solution to terminate the reaction. Upon completion of the reaction, one hour culture was performed, followed by analysis using homogeneous time-resolved fluorescence (HTRF, Cisbio). OD615/665 was measured with Wallac Envision 2103. IC50 of each compound was determined by using prism software (Version 5.01, Graphpad).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 461.98, ALogP 4.27, HBA 8, HBD 3, PSA 119.23, QED 0.45
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CC5CCC(C4)N5)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: RHTAEFPSHVYAFX-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-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 = 15804 nM (pChEMBL 4.800000190734863)
Descriptors: MW 415.55, ALogP 3.95, HBA 6, HBD 2, PSA 74.14, QED 0.53
Compound (SMILES): COc1ncccc1-c1nc2c(n1C(C)C)[C@@H](c1ccc(Cl)cc1)N(c1cc(C)c3nnn(C)c3n1)C2=O
InChIKey: VQVSQBFKHGXUFK-JOCHJYFZSA-N
Target: Bromodomain-containing protein 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15059
Assay: In-Vitro Binding Assay: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 ul per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5ul per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 ul per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 < 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 529, ALogP 4.92, HBA 9, HBD 0, PSA 103.85, QED 0.32
Compound (SMILES): Cn1cc(-c2cc3c(N4CCN(c5ncc([C@@](C)(N)c6ccc(F)cc6)cn5)CC4)c(F)cnn3c2)cn1
InChIKey: ATJKYAQAXNISKD-MHZLTWQESA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Activity Assay: KIT, KIT mutant, and PDGFRα mutant kinase profiling was performed with recombinant enzymes including wild-type KIT, KIT V560G, KIT V559D/T670I, V559D/V654A, and PDGFRα D842V. Kinase/substrate pairs were prepared in 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). Compounds were dissolved at 10 mM in 100% DMSO, serial diluted and added to the kinase/substrate mix in final concentrations from 10 uM to 0.5 pM. Compounds were incubated with the recombinant enzymes and substrates at room temperature for 20 minutes with their respective Km for ATP. 33P-ATP (10 mCi/mL) was added to initiate the reaction.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 515.57, ALogP 3.35, HBA 9, HBD 1, PSA 93.4, QED 0.38
Compound (SMILES): CC(C)[C@H](C)n1cc(-c2nc(-c3cnn(C[C@@H](O)CO)c3)cn3nccc23)cn1
InChIKey: SSCCJQJLIGWQAF-WMLDXEAASA-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 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 395.47, ALogP 2.03, HBA 9, HBD 2, PSA 106.29, QED 0.5
Compound (SMILES): N#CN1CCCC(n2nc(-c3ccc(Oc4cccc(C(F)(F)F)n4)cc3)c(C(N)=O)c2N)C1
InChIKey: LNMYMSAHQVYVMW-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Human EGFR LanthaScreen Selectivity Assay: TR-FRET LanthaScreen assays were performed by incubating a dilution series of inhibitor concentrations with 20 μM ATP, 100 nM peptide substrate (FITC-C6-KKAEEEEYFELVAKK-NH2 (SEQ ID NO.: 2, American Peptide, #333778) and 600 μM of human EGFR kinase domain (Invitrogen). 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 EGFR 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 > 26800 nM (pChEMBL 4.572000026702881)
Descriptors: MW 471.44, ALogP 3.56, HBA 8, HBD 2, PSA 136.08, QED 0.54
Compound (SMILES): CNC(=O)c1ccc(N2CCN(Cc3ccc4nc(CC(F)(F)F)c(=O)[nH]c4c3)CC2)cn1
InChIKey: GWWKGCCMZJXPIM-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase PARP3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y6F1
Assay: Fluorescence Anisotropy Binding Assay: PARP3: Recombinant full length PARP3 protein was diluted to 100 nM with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, 150 mM NaCl and incubated for four hours with an equivalent volume of 6 nM fluorescent probe diluted with 50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, 150 mM NaCl. The final DMSO concentration of the probe was kept below 1% (v/v).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 460.46, ALogP 2.1, HBA 6, HBD 2, PSA 94.22, QED 0.61
Compound (SMILES): CNC[C@H](c1ccc(Cl)c(Cl)c1)[C@H](O)c1cccc(N(C)C)c1
InChIKey: XKVFRZWDKQJABG-CRAIPNDOSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Uptake Assay Protocol: SERT: This protocol was designed to measure inhibition of uptake by the human serotonin transporter. The reagents were human SERT (HEK293F) cells, fluoxetine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1λ HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human serotonin transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
Measurement: Ki = 178 nM (pChEMBL 6.75)
Descriptors: MW 353.29, ALogP 4.1, HBA 3, HBD 2, PSA 35.5, QED 0.82
Compound (SMILES): Nc1nonc1C(=O)N1CCC2CCC1CN2Cc1c(-c2ccc(Cl)cc2)nc2ccccn12
InChIKey: WTKLDUIAXFXSFO-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: Inhibition of Recombinant TASK-1 and TASK-3 In Vitro: The investigations on the inhibition of the recombinant TASK-1 and TASK-3 channels were conducted using stably transfected CHO cells. The compounds of the invention were tested here with administration of 40 mM of potassium chloride in the presence of a voltage-sensitive dye using the method described in detail in the following references [Whiteaker et al., Validation of FLIPR membrane potential dye for high-throughput screening of potassium channel modulators, J. Biomol. Screen. 6 (5), 305-312 (2001); Molecular Devices FLIPR Application Note: Measuring membrane potential using the FLIPR® membrane potential assay kit on Fluorometric Imaging Plate Reader (FLIPR®) systems, http://www.moleculardevices.com/reagents-supplies/assay-kits/ion-channels/flipr-membrane-potential-assay-kits]. The activity of the test substances was determined as their ability to inhibit a depolarization induced in the recombinant cells by 40 mM potassium chloride. The concentration which can block half of this depolarization is referred to as IC50.
Measurement: IC50 = 3300 nM (pChEMBL 5.480000019073486)
Descriptors: MW 477.96, ALogP 3.5, HBA 8, HBD 1, PSA 105.79, QED 0.48
Compound (SMILES): CN1CCC(N2CCC(C(=O)Nc3cc4cc(-c5cn(C)nn5)ccc4cn3)CC2)CC1
InChIKey: YVWXWIPIQYUOFJ-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: SW480 colon carcinoma cells were transduced with a lentiviral vector expressing luciferase with a human Sp5 promoter consisting of a sequence of eight TCF/LEF binding sites. SW480 cells stably expressing the Sp5-Luc reporter gene and a hygromycin resistance gene were selected by treatment with 150 μg/mL of hygromycin for 7 days. These stably transduced SW480 cells were expanded in cell culture and used for all further screening activities. Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 10-point dose-response curves starting from 10 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well white solid bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. For Sp5-Luc reporter gene assays, the cells were plated at 4,000 cells/well in 384-well plates with a DMEM medium containing 1% fetal bovine serum, and 1% Penicillin-Streptomycin and incubated for 36 to 48 hours at 37° C. and 5% CO2. Following incubation, 15 μl of BriteLite Plus luminescence reagent (Perkin Elmer) was added to each well of the 384-well assay plates. The plates were placed on an orbital shaker for 2 min and then luminescence was quantified using the Envision (Perkin Elmer) plate reader. Readings were normalized to DMSO only treated cells, and normalized activities were utilized for EC50 calculations using the dose-response log (inhibitor) vs. response-variable slope (four parameters) nonlinear regression feature available in GraphPad Prism 5.0 (or Dotmatics).
Measurement: EC50 = 327 nM (pChEMBL 6.489999771118164)
Descriptors: MW 433.56, ALogP 2.78, HBA 7, HBD 1, PSA 79.18, QED 0.68
Compound (SMILES): CNCCN1CCC(c2ccc3[nH]c(-c4cc(C)nc(C)c4)c(CC(F)F)c3c2)CC1
InChIKey: RGNDUCHQWAOHEL-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: 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.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 426.56, ALogP 5.05, HBA 3, HBD 2, PSA 43.95, QED 0.56
Compound (SMILES): CC(C)C1CCN(C(=O)N[C@@H]2CCCN(c3nnc(C(N)=O)c(Nc4ccc(C(=O)N5CCOCC5)cc4)n3)C2)CC1
InChIKey: GNUGYSKRAVVTRC-HSZRJFAPSA-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 579.71, ALogP 2.23, HBA 9, HBD 3, PSA 158.91, QED 0.45
Compound (SMILES): CCc1cc(O)c(F)cc1-c1cc2[nH]nc(-c3ncc[nH]3)c2c(NCc2ccccc2N(C)S(C)(=O)=O)n1
InChIKey: BEEJICRDGFYLFA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.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 = 76.5 nM (pChEMBL 7.119999885559082)
Descriptors: MW 535.61, ALogP 4.43, HBA 7, HBD 4, PSA 139.89, QED 0.23
Compound (SMILES): Cn1cc(-c2cc3c(N4CCN(c5ncc([C@@](C)(N)c6ccc(F)cc6)cn5)CC4)c(Cl)cnn3c2)cn1
InChIKey: YBLTZZSNMXRWQX-MHZLTWQESA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Activity Assay: KIT, KIT mutant, and PDGFRα mutant kinase profiling was performed with recombinant enzymes including wild-type KIT, KIT V560G, KIT V559D/T670I, V559D/V654A, and PDGFRα D842V. Kinase/substrate pairs were prepared in 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). Compounds were dissolved at 10 mM in 100% DMSO, serial diluted and added to the kinase/substrate mix in final concentrations from 10 uM to 0.5 pM. Compounds were incubated with the recombinant enzymes and substrates at room temperature for 20 minutes with their respective Km for ATP. 33P-ATP (10 mCi/mL) was added to initiate the reaction.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 532.03, ALogP 3.87, HBA 9, HBD 1, PSA 93.4, QED 0.37
Compound (SMILES): CNC(=O)c1n[nH]c2nc(-c3cc(F)c(O)cc3CC(F)(F)F)nc(NCc3ccccc3N(C)S(N)(=O)=O)c12
InChIKey: BRQCTFOAORTYKM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.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 = 107 nM (pChEMBL 6.96999979019165)
Descriptors: MW 582.54, ALogP 2.58, HBA 8, HBD 5, PSA 179.22, QED 0.2
Compound (SMILES): C[S+]([O-])c1cc(F)c(-c2nc(C(=O)Nc3cnc4c(c3N3CCC[C@H](N)C3)CC[C@H]4O)ccc2F)c(F)c1
InChIKey: UMFBDHBNGQVFJS-NHODNIPZSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: 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.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 545.59, ALogP 3.46, HBA 7, HBD 3, PSA 127.43, QED 0.42
Compound (SMILES): Cn1cc(-c2cc3c(N4CC(C(=O)NCc5ccc(C(C)(C)C)cc5)C4)ncnn3c2)cn1
InChIKey: GERTXSGVZLHUIV-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Autophosphorylation Assay: 10,000 HMC1.2 cells were incubated in 22 ul culture media (phenol-red free IMDM, no serum) in each well of a 384-well plate and serum starved overnight in a tissue culture incubator (5% CO2, 37° C.). A 10-point dose concentration series of compound (25 uM-95.4 pM) were then added to the cells in a volume of 3.1 ul to each well (0.25% DMSO final concentration). After 90 minutes, 6 ul of 5× AlphaLISA Lysis Buffer (Perkin Elmer) supplemented with a protease and phosphatase inhibitor cocktail (Cell Signaling Technologies) was added to each well and shaken at 450 rpm for 15 minutes at 4° C. 10 ul of phospho-Y719 c-Kit and total c-Kit antibodies (15 nM final concentration, Cell Signaling Technologies) and 50 ug/ml AlphaLISA rabbit acceptor beads (Perkin Elmer) were added to each well and shaken at 300 rpm at room temperature for 2 hours. 10 ul of 100 ug/ml streptavidin donor beads (Perkin Elmer) were added to each well, blocked from light with aluminum adhesive and shaken at 300 rpm at room temperature for 2 hours. Fluorescence signal was obtained on Envision (Perkin Elmer) by AlphaScreen 384 well HTS protocol.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 443.56, ALogP 3.18, HBA 7, HBD 1, PSA 80.35, QED 0.51
Compound (SMILES): C[C@H]1CN(c2ccncc2NC(=O)c2ccc3ccn(C4CCC4)c3n2)C[C@@H](N)[C@@H]1O
InChIKey: XRFGJWXIXBCMST-AVBTWRTFSA-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 μM (Pim-1, Invitrogen PV3503) or 1.25 μM (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 preincubated 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.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 420.52, ALogP 2.55, HBA 7, HBD 3, PSA 109.3, QED 0.6
Compound (SMILES): C=C(C)c1cnc(-c2cc(Cl)ccc2F)cc1Nc1ccncc1C(=O)N[C@@H](CC)CO
InChIKey: CKLULXBYHFYPHR-INIZCTEOSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: In Vitro Kinase Assay Inhibition of ALK1/2/3/4/5/6 Kinases: Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle. IC50 values were generated from activity values performed at multiple concentrations.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 454.93, ALogP 5.21, HBA 5, HBD 3, PSA 87.14, QED 0.43
Compound (SMILES): CC(=O)Nc1nc2ccc(-c3nn(Cc4cc5ccccc5nc4CCN4CCCC4)c4ncnc(N)c34)cc2s1
InChIKey: JYYZNRDQJNYCPX-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00750
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 l 1N HCl followed by 160 μl CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 563.69, ALogP 4.88, HBA 10, HBD 2, PSA 127.74, QED 0.28
Compound (SMILES): O=C(NCc1cccnc1)C1CCN(c2ncnc3[nH]ccc23)CC12CC2
InChIKey: RVRZIGYIYUQOHO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 58.6 nM (pChEMBL 7.230000019073486)
Descriptors: MW 362.44, ALogP 2.28, HBA 5, HBD 2, PSA 86.8, QED 0.74
Compound (SMILES): CN1Cc2cc(Cl)ccc2-n2c(nnc2C2CC3(C2)CN(c2ncccc2F)C3)C1
InChIKey: IJLMXOYQKXELBB-UHFFFAOYSA-N
Target: Vasopressin V2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30518
Assay: Vasopressin V2 Receptor Antagonist Assay: The purpose of the assay was to determine the inhibitory effect of synthesized compounds on the Vasopressin receptor 2. The assay was performed in commercially available 1321N1 cells expressing the human Arginine Vasopressin Receptor V2 (AVPR2) (Perkin Elmer #ES-363-CF). Arginine Vasopressin (AVP) evokes an increase in intracellular cAMP in these cells which is measured in a TR-FRET assay using a Europium cAMP tracer and ULight labelled antibody reagents contained in a LANCE Ultra cAMP kit (Perkin Elmer #TRF0263). Increases in cAMP in the assay result in a reduction in TR-FRET as the cAMP produced by the stimulated cells competes with the Eu-cAMP tracer for binding sites on the ULight labelled antibody. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent decrease in the AVP-mediated reduction in TR-FRET signal.cAMPZen V2 assay ready cells were thawed at 37° C. and resuspended directly from frozen in 9 ml growth medium consisting of DMEM supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 1 mM sodium pyruvate. Cells were spun at 1000 rpm for 3 minutes and the supernatant was carefully removed and discarded. The pellet was resuspended in 5 ml stimulation buffer and the cell density determined using the Vi-Cell automated cell counter. The cell suspension was diluted to a 0.2×106/ml suspension ready for plating. To all wells of a white 384-well Optiplate (Perkin Elmer #6007299) 5 μL of cells in stimulation buffer were dispensed (1,000 cells per well). Stimulation buffer consisted of HBSS (+Ca/+Mg) supplemented with 5 mM HEPES, 0.1% BSA stabiliser and 0.5 mM IBMX.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 424.91, ALogP 3.78, HBA 6, HBD 0, PSA 50.08, QED 0.63
Compound (SMILES): Cn1cc(NC(=O)c2cc(C3CC3NC3CCC(F)(F)CC3)ccc2F)cn1
InChIKey: QUVRSLKZEWZNPT-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 392.43, ALogP 3.83, HBA 4, HBD 2, PSA 58.95, QED 0.81
Compound (SMILES): COc1c(F)cccc1COC[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: UYOYUPNHHAKWJZ-FTGWLVSGSA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: hS1P1 ERK Phosphorylation (S1P1 pERK): hS1P1/CHO cells were plated into BD Amine 384-well plates the day before the assay. On the day of the assay, growth medium was removed and replaced with serum-free medium (Ham's F-12 Invitrogen) and incubated for 2 hours. Test compounds pre-diluted in HBSS (Gibco)/20 mM HEPES (Gibco) were transferred to the cell plates and incubated for 7 minutes at 37° C. Cells were lysed in lysis buffer (Perkin Elmer) and phospho-ERK was measured using the SureFire pERK kit (Perkin Elmer) as described by the manufacturer. Data was plotted as percentage activation of the test compound relative to the efficacy of 10 μM S1P. The EC50 is defined as the concentration of test compound which produces 50% of the maximal response and was quantified using the 4 parameter logistic equation to fit the data.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 493.51, ALogP 4.23, HBA 5, HBD 3, PSA 111.24, QED 0.45
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CCC(N(C)Cc5ccnn5C)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: SKBZABSGHVPSMU-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-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 = 11497 nM (pChEMBL 4.940000057220459)
Descriptors: MW 511.68, ALogP 4.85, HBA 8, HBD 1, PSA 83.17, QED 0.35
Compound (SMILES): C=CC(=O)N1CCC(C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3cc(O)cc(c3)-c3ccc4c(c3)c(c(-c3ccccc3CCOC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)CC1
InChIKey: WWVNOWGMPRWANT-NVVQJRFUSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 917.16, ALogP 6.45, HBA 10, HBD 3, PSA 162.75, QED 0.11
Compound (SMILES): Cc1cccc(C)c1OCCCNCc1ccc(O)cc1
InChIKey: RTXAGNMHOUVFMD-UHFFFAOYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Automated patch clamp (Inactivation State Block): Test Protocol for Determining the Potency of Compounds on Nav1.2 and Nav1.6 Subtypes of Voltage-Gated Sodium Ion Channels Expressed in Mammalian CellsCell culture procedures: Chinese hamster ovary (CHO) cells were stably transfected with the cDNA sequence of either the human SCN2A or SCN8A genes to create hNav1.2 or hNav1.6 cells respectively. The cell lines were cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum and antibiotics in a tissue incubator at 37° C. under a humidified air/CO2 mixture (95/5 v/v). On the day of the experiment, cells were washed twice with Hank's balanced salt solution, treated with trypsin, and resuspended in fresh culture media at a density of 4−6×106 cells in 20 ml. Immediately before placing in the IonWorks Quattro instrument, cells were washed in Hank's balanced salt solution to remove culture medium and re-suspended in 3 ml of Hank's balanced salt solution. Automated patch clamp recordings of cells using the IonWorks Quattro Instrument: The assay used a 384 microwell plate, which enabled 20 compounds to be studied at four different concentrations in quadruplicate as well as allowing a vehicle control group and a positive control group using mexiletine at 8 different concentrations. Cells were added to the wells of the Population Patch Clamp planar electrode using a 48-channel pipettor in a volume of 6 μL, per well. After 5 minutes incubation at ambient temperature with compounds or vehicle, membrane currents were recorded using the patch clamp amplifier. Block of hNav1.2 or hNav1.6 channels was measured using a stimulus voltage pattern as shown in FIG. 1. The pulse pattern was repeated twice, before and after compound addition and peak current amplitudes were measured at TP12 to measure Inactivation State Block. Data analysis: Data acquisition and analyses were carried out using the IonWorks Quattro system operation software which uses an algorithm to correct data for leak currents. Concentration-response curves were fitted by non-linear least squares regression allowing calculation of an IC50 value (μM), being the concentration of the compound producing half-maximal inhibition.
Measurement: IC50 = 2200 nM (pChEMBL 5.659999847412109)
Descriptors: MW 285.39, ALogP 3.57, HBA 3, HBD 2, PSA 41.49, QED 0.76
Compound (SMILES): CCCCOc1nc(N)c2cnn(Cc3ccc(CN4CCCC4)cc3)c2n1
InChIKey: OZXVSIXMIUFERM-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Enzyme Activity Assay: Test Example 4: 100 mM PBS buffer was formulated, which was then used to formulate 2.5 mg/ml microsome solution and 5 mM NADPH solution. The 5× concentration of the compound working solution was diluted with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). The 5× concentration of ketoconazole working solution was diluted with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). Dextromethorphan working solution was diluted with PBS to a concentration of 50 μM.20 μl of 2.5 mg/ml microsome solution, 20 μl of 50 μM testosterone working solution, 20 μl of MgCl2 solution and 20 μl of the compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM, different reaction systems for each concentration) were taken respectively and mixed well. For the positive control group, the compound was replaced with the same concentration of ketoconazole. The mixture together with 5 mM NADPH solution were pre-incubated at 37° C. for 5 minutes. After 5 minutes, 20 μl of NADPH were added to each well, the reaction was started and incubated for 30 minutes. All the incubated samples were present in duplicate. After 30 minutes, 250 μl of acetonitrile containing internal standard were added to all samples, mixed well, shaken at 800 rpm for 10 minutes, and then centrifuged at 3700 rpm for 10 minutes. 80 μl of the supernatant were taken and analyzed by LC-MS/MS.
Measurement: IC50 = 4000 nM (pChEMBL 5.400000095367432)
Descriptors: MW 380.5, ALogP 3.23, HBA 7, HBD 1, PSA 82.09, QED 0.6
Compound (SMILES): FC(F)(F)c1cccc(CSc2nnc(Cc3c[nH]c4ccccc34)n2CCCc2c[nH]cn2)c1
InChIKey: DHMMKEWJQVUGEM-UHFFFAOYSA-N
Target: Somatostatin receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P32745
Assay: Activity Assays: Measurement of forskolin stimulated inhibition of cAMP was performed via time-resolved fluorescence resonance energy transfer (TR-FRET) LANCE assay (AD0262, PerkinElmer Life Science, Inc., Boston Mass.). Recombinant Chinese hamster ovary (CHO-K1) cells expressing human somatostatin SSTR4 cells (ES-524-CF, PerkinElmer Life Science, Inc., Boston Mass.) were thawed (37° C.), resuspended in 10 mL Hanks' balanced salt solution no phenol red (HBSS, Invitrogen, Carlsbad Calif.), and then centrifuged (150×g, 5 minutes). Cellular pellet resuspended in stimulation buffer containing HBSS 1×, HEPES 5 mM, Protease free BSA 0.1% (PerkinElmer), and 3-Isobutyl-1-methylxanthine 0.5 mM (pH 7.4) and seeded in 96-well plates at 4000 cells/well. LANCE cAMP assay was performed per manufacture instruction with assessment of respective compound, somatostatin-28 (Sigma-Aldrich Co., St. Louis, Mo.) as control agonist, against 5 μM forskolin, performed in triplicate across concentration curve. Fluorescence signal was measured at 20 hours (excitation 340 nm and emission 665 nm, 400-μs delay) via TR-FRET (FLUOSTAR Omega-F, BMG Labtech, Inc., Cary, N.C.). Data was calculated via GraphPad PRISM-5 software.
Measurement: EC50 = 38.5 nM (pChEMBL 7.420000076293945)
Descriptors: MW 496.56, ALogP 6.02, HBA 5, HBD 2, PSA 75.18, QED 0.24
Compound (SMILES): COc1nc2c(OCC3CCC(=O)N3)cccc2cc1C(N)=O
InChIKey: IURVALWFNKWINR-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51617
Assay: IRAK1 Enzymatic Assay: IRAK1 is a human purified recombinant enzyme (His-TEV-IRAK1 (194-712)). In this assay, IRAK-1 hydrolyses ATP and autophosphorylates. Measurement of IRAK-1 inhibition was performed in streptavidin coated 384 well FlashPlate (PerkinElmer #SMP410A). His-TEV-IRAK-1 (15 ng/well), ATP (1 μM, [33P]ATP 0.25 μCi/well) and compounds in DMSO (range of concentrations from 20 μM to 1 nM) or controls (2% DMSO) were incubated for 3 hours at 30° C. in assay buffer: Hepes pH7.0 50 mM, Fatty acid-free BSA 0.1%, Dithiothreitol DTT 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Triton-X-100 0.01%. Kinase reaction was stopped by addition of EDTA. Supernatant was discarded, plates were washed three times with 150 mM NaCl and radioactivity was then measured in a Microbeta Trilux reader.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 315.33, ALogP 1, HBA 5, HBD 2, PSA 103.54, QED 0.86
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](CS(=O)(=O)N2CCOCC2)C1
InChIKey: GODNEHJKBMGCML-BETUJISGSA-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 365.46, ALogP 0.83, HBA 6, HBD 1, PSA 91.42, QED 0.84
Compound (SMILES): COc1ccc(-c2[nH]c3ccc(-c4cccc(C(=O)NCC#N)c4)cc3c2CC(F)(F)F)cc1OC
InChIKey: GQXZJSZGCUCSIK-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 = 41083 nM (pChEMBL 4.389999866485596)
Descriptors: MW 493.49, ALogP 5.88, HBA 4, HBD 2, PSA 87.14, QED 0.32