lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNc1ncc(-c2ccc3cnc(NC(=O)C4CCN(C)CC4)cc3c2)s1
InChIKey: MSIFRQGYXWUUBP-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 = 776 nM (pChEMBL 6.110000133514404)
Descriptors: MW 381.51, ALogP 3.68, HBA 6, HBD 2, PSA 70.15, QED 0.72
Compound (SMILES): CNC(=O)c1ccc2c(Cl)c(-c3nn(CC4CCOCC4)c4ncnc(N)c34)[nH]c2c1
InChIKey: ZBOVSHKBSUTFCS-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Enzyme Assay: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer conditions were used for KDR with the addition of 2 mM MnCl2. RETV804M buffer used 1× CisBio enzymatic buffer supplemented with a final concentration of 2 mM MgCl2, 1 mM DTT, 20 nM SEB and 0.01% Triton X100.
Measurement: IC50 = 39.5 nM (pChEMBL 7.400000095367432)
Descriptors: MW 439.91, ALogP 3, HBA 7, HBD 3, PSA 123.74, QED 0.45
Compound (SMILES): C[C@@H]1CCCN1Cc1ccc(-c2cnc(N)c(C(=O)N[C@H]3CC[C@](C)(O)CC3)c2)c(F)c1
InChIKey: XASOXDFSRMCVGS-DNHRDODTSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Caliper Assay: A kinase selectivity panel which measures substrate peptide phosphorylation was set-up for wild-type ALK2 (aa172-499), ALK2 FOP mutant (aa172-499 R206H), ALK1 (aa166-493), ALK5 (aa162-503) and ALK6 (aa168-495). The technology used for the described assay is based on the separation and quantification of substrate and product in an electrical field. In the course of the kinase reaction the peptide substrate is phosphorylated by a kinase. The transfer of a phosphate residue also causes the introduction of two additional negative charges and hence to a change in the net charge of the phospho-peptide compared to the unphosphorylated peptide. Due to this difference in charge the phosphorylated und unphosphorylated peptides migrate with different velocities in an electrical field.In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 m Na3VO4, 10 mM If-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO. Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 5800 nM (pChEMBL 5.239999771118164)
Descriptors: MW 440.56, ALogP 3.88, HBA 5, HBD 3, PSA 91.48, QED 0.66
Compound (SMILES): Cn1cc(-c2cc3c(N4CC(C)(C(=O)NCc5ccc(Cl)cc5F)C4)ncnn3c2)c(N)n1
InChIKey: QJZCCYNUJLXLQY-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 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 468.92, ALogP 2.65, HBA 8, HBD 2, PSA 106.37, QED 0.47
Compound (SMILES): Cc1cccc(-c2nc(-c3ccc(C(=N)NO)cc3)[nH]c2-c2ccncc2)c1
InChIKey: SWEOMWCGPLXIKR-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay : Table 2: The p38 MAPK IC50 values of selected compounds except IM-32 to IM-44 were determined using the HitHunter p38 MAP kinase binding assay from DiscoveRx Corporation (Fremont, Calif., USA). The p38 MAPK IC50 values of IM-32 to IM-44 and the CK1 IC50 values of selected compounds were determined using LanthaScreen Eu kinase binding assay from Invitrogen (Life Technologies, Carlsbad, Calif., USA). The assays were performed following the manufacturers' protocols in white 384-well plates (Cat. No. 3572; Corning Incorporated, Corning, N.Y., USA). SB203580 was used as the control in all assays. For the HitHunter p38 MAP kinase binding assay the compounds were dissolved in DMSO (5 mM stocks) and diluted to a final concentration of 2% (vol/vol) DMSO for all assays. Recombinant GST-tagged active p38α MAP kinase enzyme (Millipore, Billerica, Mass., USA) was used for the HitHunter p38 MAP kinase binding assay. For the LanthaScreen Eu kinase binding assay the compounds were dissolved in DMSO (5 mM stocks) and diluted to a final concentration of 1% (vol/vol) DMSO for all assays. Each data point was done in triplicate. The assay was run using JANUS Automated Workstation according to the protocol developed using WinPREP software (Perkin Elmer Inc., Waltham, Mass., USA). Tecan Infinite® M1000 microplate reader (Tecan Group Ltd., Minnedorf, Switzerland) was used for luminescence measurements (HitHunter p38 MAP kinase binding assay) and fluorescence measurements (LanthaScreen Eu kinase binding assay; ex=340 nm, em=665, 615 nm). All data analysis was performed using GraphPad Prism 5 software (GraphPad Software Inc.). Inhibition curves and IC50 values were generated by nonlinear regression analysis and data represent mean±SEM.
Measurement: IC50 = 295 nM (pChEMBL 6.53000020980835)
Descriptors: MW 369.43, ALogP 4.42, HBA 4, HBD 4, PSA 97.68, QED 0.24
Compound (SMILES): Cc1nc2c(N)cc(N3CCOCC3)nn2c1Cc1cccc2c1CNCC2
InChIKey: FRZDANDRHZSWAX-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: KINOMEscan Enzyme Binding Assays: Kinase enzyme binding affinities of compounds disclosed herein were determined using the KINOMEscan technology performed by DiscoveRx Corporation, San Diego, Calif., USA (www.kinomescan.com).
Measurement: Kd = 2903 nM (pChEMBL 5.539999961853027)
Descriptors: MW 378.48, ALogP 1.69, HBA 7, HBD 2, PSA 80.71, QED 0.72
Compound (SMILES): CCc1c(-c2ccnc(C)c2)[nH]c2ccc(-c3noc(C4CCCNC4)n3)cc12
InChIKey: OXDKJYLOOCWTKQ-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 = 686 nM (pChEMBL 6.159999847412109)
Descriptors: MW 387.49, ALogP 4.62, HBA 5, HBD 2, PSA 79.63, QED 0.53
Compound (SMILES): CN1CCOC(CC(=O)Nc2cc3cc(-c4cnn(C)c4)ccc3cn2)C1
InChIKey: HYXQNGRJHZMKLE-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: The screening assay for DYRK1A kinase activity: Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 μM to 0.00016 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning).The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).The Emission ratio (Em) was calculated as a ratio of the coumarin (C) emission signal (at 445 nm)/Fluorescein (F) emission signal (at 520 nm). The percent phosphorylation was then calculated using the following formula: [1−((Em ratio×F100%)−C100%)/((C0%−C100%)+(Em ratio×(F100%−F0%)))]. Dose-response curves were generated and inhibitory concentration (IC50) values were calculated using non-linear regression curve fit in the Dotmatics' Studies Software (Bishops Stortford, UK).
Measurement: EC50 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 365.44, ALogP 2.29, HBA 6, HBD 1, PSA 72.28, QED 0.77
Compound (SMILES): C[C@@H]1CN(c2ccc(C#N)c(F)c2)CCN1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: KQDLURQMDUCAFL-OAQYLSRUSA-N
Target: Muscarinic acetylcholine receptor M2 (SINGLE PROTEIN), Homo sapiens
UniProt: P08172
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 463.6, ALogP 4.22, HBA 4, HBD 1, PSA 62.61, QED 0.7
Compound (SMILES): FC(F)(F)c1nc(-c2ccc(CNc3cnsc3)nc2)no1
InChIKey: CVROGHVTJUYDQM-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: Enzymatic Assay: HDAC8 reagents: HDAC8 was purchased from Enzo Life Sciences [catalog #BML-SE145]. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 100 mM NaCl [Sigma, catalog #S5150, 5M stock], 20 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock], and 0.1% n-Octyl-β-D-glucopyranoside (N-OG) [Anatrace, catalog #O311, 10% stock]. The HDAC8 enzyme was run at the final concentration of 1.333 nM. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-178], used to evaluate enzyme activity, was added at the final concentration of 200 uM. To enable detection of the signal, Developer II [BioMol Research Laboratories, catalog #KI-176] was added at a 1:200 dilution to the stop solution, which also included 20 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 900 nM (pChEMBL 6.050000190734863)
Descriptors: MW 327.29, ALogP 3.22, HBA 7, HBD 1, PSA 76.73, QED 0.79
Compound (SMILES): CC(NC(=O)c1c(N)nn2cccnc12)c1cc(Cl)c2cnnn2c1-c1ccccc1
InChIKey: JWLQSWCHBZLQLH-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 432.88, ALogP 3.17, HBA 8, HBD 2, PSA 115.5, QED 0.45
Compound (SMILES): COc1ccc(CN(C)C(=O)N2CCCC(CS(=O)(=O)NNC(=O)c3cc(-c4cn(C)cn4)cc(Cl)c3F)C2)cc1
InChIKey: MFRIMWUKTPTVSR-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 607.11, ALogP 3.42, HBA 7, HBD 2, PSA 125.87, QED 0.36
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C(C)(C)C(=O)N[C@@H]4CCCC[C@H]4N)cc3c2C(C)C)ccn1
InChIKey: PYQCHXASKVLTQG-FYYLOGMGSA-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 = 899 nM (pChEMBL 6.050000190734863)
Descriptors: MW 432.61, ALogP 5.33, HBA 3, HBD 3, PSA 83.8, QED 0.51
Compound (SMILES): Cn1cnc2c(ncn2[C@@H]2C[C@@H]3OP(=O)(O)O[C@H]4[C@@H](F)[C@H](n5cnc6c(N)ncnc65)O[C@@H]4COP(=O)(S)O[C@@H]2[C@@H]3O)c1=O
InChIKey: BTCUYIDXTLYAGE-GZQJALTCSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: Radioligand Binding Assay: A radioligand binding assay was developed to determine compound interactions were competitive with a tritium-labeled version of the native STING ligand, 3H-cyclic guanine (2′,5′) monophosphate adenine (3′,5′) monophosphate (3H-cGAMP). The STING constructs (WT and H232R) were comprised of residues 155-341 with both N- and C-terminal truncations; the N-terminal transmembrane domains were removed (1-154), as well as the C-terminal tail (342-379). A highly specific N-terminal biotinylation was achieved enzymatically with the E. coli biotin ligase (BirA) and inclusion of the high-affinity biotinylation peptide AviTag. 100 nM STING protein was immobilized on 20 μg streptavidin polyvinyl toluene (SA-PVT) beads in 150 mM NaCl, 25 mM Hepes (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 0.005% (v/v) Tween-20, 1% (v/v) DMSO. 100 nM 3H-cGAMP and compounds were added and allowed to come to equilibrium at room temperature (20 min). Compounds were tested in three-fold dilution series from a 100 μM starting concentration and normalized to a positive control compound that completely blocked 3H-cGAMP binding and the negative control DMSO.
Measurement: Ki = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 675.49, ALogP 0.42, HBA 18, HBD 4, PSA 243.08, QED 0.17
Compound (SMILES): C=CC(=O)N1CC[C@@]2(CCN([C@H](C(=O)N[C@H]3Cc4cc(O)cc(c4)-c4ccc5c(c4)c(c(-c4ccccc4CCOC)n5CC)CC(C)(C)COC(=O)[C@@H]4CCCN(N4)C3=O)C(C)C)C2=O)C1
InChIKey: LWRSJGNNKIHOCR-JSQBNHNNSA-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 915.14, ALogP 6.2, HBA 10, HBD 3, PSA 162.75, QED 0.12
Compound (SMILES): CC1C[C@@H](NC(=O)Nc2ccc(C(F)(F)F)cc2F)C(=O)N1c1ccc(-c2ccccc2P(C)(C)=O)c(F)c1F
InChIKey: SPJINUIYAUTKQY-YBMSBYLISA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 fluorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 1.21 nM (pChEMBL 8.920000076293945)
Descriptors: MW 583.47, ALogP 6.35, HBA 3, HBD 2, PSA 78.51, QED 0.27
Compound (SMILES): O=C([C@@H]1CC1(F)F)N1[C@@H]2CC[C@H]1CN(c1ccnc(Nc3cnc(CO)c(F)c3)n1)C2
InChIKey: HMZBJXPSSIANDY-MJBXVCDLSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Caliper Enzyme Assay: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 468 nM (pChEMBL 6.329999923706055)
Descriptors: MW 434.42, ALogP 2.08, HBA 7, HBD 2, PSA 94.48, QED 0.74
Compound (SMILES): O=C(Cn1c(=O)ccc2cc(C(F)(F)F)ccc21)Nc1scc(Cl)c1-c1ncn(CCCN2CCCC2)n1
InChIKey: PWODXIIHLFKKKG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: HTRF Assay: (1) Assay buffer #1 with 0.0025% Tween and 1 mM DTT was prepared using JNK buffer stock solution.(2) Assay buffer #2 with 0.025% BSA was prepared using the assay buffer #1.(3) Test compound solution preparation: 5× compound solution was prepared using assay buffer #1 with 5% DMSO. The compound solution (10 μl/well) was added to a 384-well plate (Corning, Cat No. 3654).(4) aJNK1, 2 or 3 preparation: aJNK stock (100 μg/ml) at −80° C. was thawed on ice and an aJNK (1.6 ng/ml) solution was prepared using assay buffer #2. The aJNK solution (20 μl/well) was added to the plate and the plate was shaken. The enzyme was incubated with the compound at RT for 10 min.(5) ATP/substrate solution preparation: ATP and substrate stocks were thawed on ice. 2.5×ATP/substrate (e.g., 25 μM or 2.5 mM ATP/50 nM EPIW-1) was prepared using assay buffer #1. The ATP/substrate (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 15 min. Note: In an exemplary assay, the final ATP concentration was about 1 mM.(6) EDTA preparation: 30 mM EDTA was prepared using 0.5M EDTA stock and assay buffer #1. The EDTA (10 μl/well) was added to the plate to quench the enzyme reaction and the plate was shaken well.(7) Detection reagent preparation: Eu-63 and SA-XL stocks were thawed on ice. 4× Eu-63/SA-XL (2 nM Eu-63/9.4 nM SA-XL) was prepared using JNK detection buffer. The Eu-63/SA-XL solution (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 1 hr before reading the plate on LJL using ratiometric method named HTRF.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 565.02, ALogP 5.12, HBA 8, HBD 1, PSA 85.05, QED 0.32
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@@H]1C[C@@H]2CN(CCC#N)C[C@@H]2C1
InChIKey: XPIZAVXMVAKCMR-BTTYYORXSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: In vitro kinase assay: TBDIn vitro kinase assays described below can be used to determine the activity of a test compound for inhibiting the activity of JAK3 kinase. The test compounds were dissolved in dimethyl sulfoxide and diluted with water to a serial concentration gradient as required by the experiment. JAK3 substrates (Cell Signaling Technology, Catalog Number: 1305s) and ATP (2 mM) solution were diluted with water to obtain a final concentration of 20 M ATP and 1.2 M substrate solution. The appropriate amount of JAK3 kinase (Invitrogen, Catalog Number: pv3 855) was mixed with 4× buffer (prepared by user, and comprising 50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl2, 1.25 mM DTT) to a final concentration of 8 ng/μL. To each well of a microplate [DELFIA® Streptavidin-coated clear plate (Perkin Elmer, Item: AAAND-0005)]17.5 μL of ATP/substrate mixture, 5 μL of aqueous solution of a test compound (L of pure water only were added to the control and blank), and 7.5 μL of the kinase solution prepared above (4× buffer only was added to the control) were added. Each well was mixed sufficiently, then incubated at room temperature (27° C.) for 50 minutes, washed with wash buffer, and dried three times, then HRP conjugated antibody [Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate, Cell signaling Technology, Catalog Number: 5465)] was added, and incubated for 1 hour. The microplate was washed with wash buffer and dried three times, and then TMB (Sigma, Catalog Number: T4444) was added and incubated for 5 to 15 minutes to allow for color change. Stop solution (1 N sulfuric acid solution) was added to stop the reaction. Absorbance was measured on a Novostar microplate reader at a wavelength of 450 nm.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 310.41, ALogP 2.02, HBA 5, HBD 1, PSA 71.84, QED 0.94
Compound (SMILES): COc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2cccc(Oc3ncnc4cc(OC)c(OC)cc34)c2)cc1
InChIKey: IQVJJYJJWIHENX-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 568.63, ALogP 6.58, HBA 9, HBD 2, PSA 121.65, QED 0.22
Compound (SMILES): O=S(=O)(Nc1nccs1)c1cc(Cl)c(N[C@H](CN2CC(F)(F)C2)c2ccccc2)cc1F
InChIKey: JWKATVPFWHKEPJ-QGZVFWFLSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 3030 nM (pChEMBL 5.519999980926514)
Descriptors: MW 502.97, ALogP 4.84, HBA 6, HBD 2, PSA 74.33, QED 0.46
Compound (SMILES): C[C@@H]1CN(c2cnc(C(F)(F)F)cn2)CCN1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: KMDYYADFJDYVEA-LJQANCHMSA-N
Target: Muscarinic acetylcholine receptor M2 (SINGLE PROTEIN), Homo sapiens
UniProt: P08172
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 490.57, ALogP 4.02, HBA 5, HBD 1, PSA 64.6, QED 0.66
Compound (SMILES): CC[C@@H](C(=O)Nc1cccc2c(-c3nc(Nc4cn(C)nc4C)ncc3F)c[nH]c12)N1CCN(C)CC1
InChIKey: RWZJXPSNVCNPSC-QFIPXVFZSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Enzymatic Assay: Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween® 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphorylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition. The results of this assay are shown in Table 16 and indicate that the compounds of Formula (I), (Ia), (Ib) and Table 1 exhibit preferential inhibition of JAK1 over JAK2 (in many cases demonstrating over 100 times selectivity for inhibition of JAK1 vs. JAK2).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 505.6, ALogP 3.51, HBA 8, HBD 3, PSA 107, QED 0.35
Compound (SMILES): CN1C(N)=N[C@](C)(c2cc(/C=C(\F)c3ccc(Br)cn3)ccc2F)CS1(=O)=O
InChIKey: UBXLUDVBTXDSQS-ODZYNRFZSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05 M acetate, pH 4.5, 8% DMSO final, 100 μM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The β-secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2), which was pre-incubated for one hour with the test compound, typically in about 1 uL of DMSO according to a serial dilution, was added thereto. The assay was effectively started by the addition of FRET substrate (50 nM) and the combination was incubated for one hour. The FRET assay was terminated by the addition of tris buffer, which raised the pH to neutrality, and the fluorescence was determined. The FRET substrate was a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. The specific FRET substrate used in this assay was made by Amgen in-house. Commercially available FRET substrates, for example, the FRET substrate offered with the BACE1 FRET Assay Kit sold by ThermoFisher Scientific (Catalog Number P2985), may be used in this assay with the appropriate modifications, which are within the purview of the ability of a person with ordinary skill in the art. Proteolytic cleavage of the FRET substrate released quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 471.33, ALogP 3.26, HBA 5, HBD 1, PSA 88.65, QED 0.74
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCNCC4)nc3c2C(C)C)nn(C)c1=O
InChIKey: DZSQVHDICSOVFD-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
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 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 365.48, ALogP 3.22, HBA 5, HBD 2, PSA 75.6, QED 0.75
Compound (SMILES): C=CC(=O)Nc1cccc(-c2nc(Nc3ccc4c(c3)OCCOCCOCCO4)ncc2F)c1
InChIKey: OALQDJRVWCSCJT-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Inhibitive Activities Against BTK wt, BTK(C481S), BMX, FAK, ITK, EGFR wt Kinases: The inhibiting activities of each of Compound-1 to Compound-24 against BTK wt, BTK(C481S), BMX, FAK, ITK, and EGFR wt kinases were determined using 33P ATP HotSpot kinase assay platform. (See, e.g., Ma, et al, 2008 Expert Opin Drug Discov. 3(6): 607-621.)Each of Compound-1 to Compound-24 were prepared as 10 mM DMSO solution, and then 1:3 serially diluted to a concentration of 1 μM to 0.05 nM. Then, Kinase assays were performed using the HotSpot assay platform.Briefly, specific kinase/substrate pairs along with required cofactors 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 delivered into the reaction, followed about 20 minutes later by addition of a mixture of ATP (Sigma, St. Louis Mo.) and 33P ATP (Perkin Elmer, Waltham Mass.) to a final concentration of 10 μM.Reactions were carried out at room temperature for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman Inc., Piscataway, N.J.). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data was 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 > 1000 nM (pChEMBL 6)
Descriptors: MW 480.5, ALogP 3.96, HBA 8, HBD 2, PSA 103.83, QED 0.53
Compound (SMILES): Cc1nnc(N2CCN(c3ccc(Nc4nccc(-c5ccc(C(=O)NCC#N)cc5)n4)cc3)CC2)o1
InChIKey: MQRFQMNXCPPALU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: In Vitro Assay: NCEs were screened using in vitro JAK (1, 2 and 3) kinase assay on ADP Glo platform (Promega). Fixed amount of recombinant purified human JAK (25 ng of JAK1 and 10 ng of JAK2 and JAK3 per reaction, from Life Technologies Ltd) were incubated with increasing concentration of NCEs in 1× kinase reaction buffer (40 mM Tris-Cl, pH7.5, 20 mM MgCl2, 0.1 mg/ml BSA and 50 μM DTT). Enzymatic reaction was initiated by adding a substrate cocktail containing 50 μM of ATP (final concentration) and 5 μg for JAK1 and 2.5 μg for JAK2 and JAK3 of polyGln4Tyr1 (Signal Chem) in total 25 μl of reaction in 96 well plate. The reaction was incubated at room temperature for 1 hr.After 1 hr of incubation equal volume (25 μl per reaction) of ADP Glo was added and incubated at room temperature for 40 min.This was followed by addition of kinase detection reagent (50 μl per reaction) and incubation at room temperature for 30 min. Finally, plate was read for luminescence at an integration time of 500 millisecond per well.
Measurement: IC50 = 46.9 nM (pChEMBL 7.329999923706055)
Descriptors: MW 495.55, ALogP 3.16, HBA 10, HBD 2, PSA 136.1, QED 0.37
Compound (SMILES): COc1cc2ncnc(Oc3cccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4ccccc4)c3F)c2cc1OC
InChIKey: QKTAGVSPDITYOL-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 556.6, ALogP 6.71, HBA 8, HBD 2, PSA 112.42, QED 0.23