lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCCCN1CCN(c2ccc3[nH]c(-c4cc(OC)c5ncnn5c4)c(C(C)C)c3n2)CC1
InChIKey: KPRLDXQGXNCZOJ-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 447.59, ALogP 4.33, HBA 7, HBD 1, PSA 74.58, QED 0.45
Compound (SMILES): C=CCOC(=O)N1[C@@H]2CC[C@H]1C[C@H](Nc1nc(Nc3cc(C)[nH]n3)cc3ncccc13)C2
InChIKey: NCMWHBXJSDSGLY-OSYLJGHBSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 433.52, ALogP 4.13, HBA 7, HBD 3, PSA 108.06, QED 0.5
Compound (SMILES): Cc1nc(-c2ccc(F)cc2)c(-c2ccc3nc(NC(=O)c4cccc(CN5CCCC5)c4)cn3n2)n1C
InChIKey: CBWYPZFLYVWTGO-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 6.81 nM (pChEMBL 8.170000076293945)
Descriptors: MW 509.59, ALogP 5.09, HBA 7, HBD 1, PSA 80.35, QED 0.34
Compound (SMILES): O=C(c1ccccn1)N1CCN(Cc2c(-c3ccc(Cl)cc3)nc3ccccn23)CC1
InChIKey: DZPYYCKJQUWSRK-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: Inhibition In Vito Assay: The investigations on the inhibition of the recombinant TASK-1 and TASK-3 channels were carried out using stably transfected CHO cells. Here, the compounds according to the invention were tested with administration of 40 mM of potassium chloride in the presence of a voltage-sensitive dye for high-throughput screening of potassium channel from Molecular Devices FLIPR Application Note: Measuring membrane potential using the FLIPR membrane potential assay kit on Fluorometric Imaging Plate Reader (FLIPR) systems. 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.
Measurement: IC50 = 590 nM (pChEMBL 6.230000019073486)
Descriptors: MW 431.93, ALogP 4.01, HBA 5, HBD 0, PSA 53.74, QED 0.49
Compound (SMILES): C=CC(=O)N1CCN(CC2(Cn3c(=O)c(-c4c(Cl)c(OC)cc(OC)c4Cl)cc4cnc(NC)nc43)CC2)CC1
InChIKey: QMXGFRWKHLJTJA-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Activity Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of FGFR family (FGFR1, FGFR2, FGFR3, FGFR4) kinase activity of a compound of Formula (III). Serial dilutions of test compounds were incubated with either human recombinant FGFR1 (0.5 nM), FGFR2 (0.1 nM, FGFR3 (0.9 nM), or FGFR4 (2 nM), ATP (FGFR1: 100 μM; FGFR2: 75 μM; FGFR3: 120 μM; FGFR4: 250 μM) and a phosphoacceptor peptide substrate FAM-KKKKEEIYFFF-CONH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper LabChip 3000. Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 587.51, ALogP 3.93, HBA 9, HBD 1, PSA 101.82, QED 0.38
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CC3CCC2C(Nc2cnc(C(F)(F)F)cn2)C3)n1
InChIKey: SVHKTRKRXMFRIK-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 458.45, ALogP 2.88, HBA 8, HBD 1, PSA 101.72, QED 0.64
Compound (SMILES): CO[C@H]1CN(C(=O)c2cc(OC(F)(F)F)ccc2-n2nccn2)C(C)(c2nc3c(C)c(Cl)ccc3[nH]2)C1
InChIKey: JKAQEFIPJYJNQX-NKTHEXPSSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 534.93, ALogP 4.78, HBA 7, HBD 1, PSA 98.16, QED 0.4
Compound (SMILES): COc1ccc2c(c1)[C@]1(C[C@H]1c1ccc3c(-c4ccc(OCCN5CCCCC5)cc4)n[nH]c3c1)C(=O)N2
InChIKey: VRRDCJGHECQBRM-HVNZXBJASA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Inhibition Assay: Aurora B inhibition was determined using the Z-Lyte assay kit from Invitrogen. The assay was performed using the recommended manufacturer's instructions with 128 μM ATP and 28 nM Aurora B (Invitrogen cat #PV3970). The % inhibition values were determined according to the manufacturer's directions and IC50 values were obtained using a non-linear 4 point logistic curve fit (XLfit4, IDBS)
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 508.62, ALogP 5.48, HBA 5, HBD 2, PSA 79.48, QED 0.35
Compound (SMILES): Cn1cc(-c2ccc(C3CC3NCC3CCN(Cc4ccc(C(=O)NO)cc4)CC3)cc2)ccc1=O
InChIKey: QCWJSKPFTMLIKX-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Histone Deacetylase Assay : Histone deacetylase assay was done as per manufacturer's instructions. Briefly, assay buffer, 200 uM HDAC substrate (fluorogenic HDAC acetylated peptide substrate for class I HDACs (HDACs 1, 2, and 3) and class 2b HDACs (HDACs 6 and 10) and 1% BSA are taken as a master mix and aliquoted as 40 ul per well. Compounds (10×) were diluted in assay buffer and were added to respective wells of a black 96 well plate. HDAC6 human recombinant enzyme was thawed on ice and 5 μl (7 ng/ul) enzyme was added per well. The plate was incubated at 37° C. for 1 hour. Developer solution was then added (50 μl per well) and incubated at room temperature for 10 minutes. Fluorescence was measured at an excitation wave length of 350-380 nm and emission wavelength of 440-480 nm.
Measurement: IC50 = 199 nM (pChEMBL 6.699999809265137)
Descriptors: MW 486.62, ALogP 3.53, HBA 6, HBD 3, PSA 86.6, QED 0.34
Compound (SMILES): N[C@]1(COP(=O)(O)O)CC[C@H](c2ccc3c(c2)CC[C@@H](c2ccccc2)C3)C1
InChIKey: DEKOIOTXPOCTSP-VOQZNFBZSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: [35S] GTPgammaS Binding Assays: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 ul/well, final protein 3 ug/well) with MULTIDROP. [35S] GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11 Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 ul) was added to each well for counting on the Packard TOPCOUNT. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 401.44, ALogP 4.03, HBA 3, HBD 3, PSA 92.78, QED 0.66
Compound (SMILES): CCC1CC(Cc2nc(Nc3cc(C)[nH]n3)ccc2F)(C(=O)O)CCN1Cc1cccc(Cl)c1F
InChIKey: LWIKRSTVFVSLEM-UHFFFAOYSA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Evaluation of Aurora a and Aurora B Inhibitory EffectKinase Assay: Experimental Methods:(1) Prepare a 1× kinase buffer [50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), PH 7.5, 0.01% Brij-35, 10 mM MgCl2, 2 mM dithiothreitol (DTT)].(2) Prepare compound:The testing compound was dissolved with DMSO. The testing compound was diluted up to 100× final concentration in a 384-well plate. Transfer 250 nL of the compound dilution to a 384-well assay plate using Echo 550. 100% DMSO of 250 nL was added to the negative control well and the positive control well.(3) Prepare a 2.5× enzyme solution using the above 1× kinase buffer.(4) Add 10 μL of the 2.5× enzyme solution to the compound well and the positive control well of the 384-well assay plate. Add 10 μL of the 1× kinase buffer to the negative control well.(5) The 384-well plate was centrifuged at 1000 rpm for 30 seconds and incubated at room temperature for 10 min.(6) A mixture of ATP and Kinase substrate 21 with a final concentration of 25/15× was prepared by the 1× kinase buffer.(7) Add 15 μL of the mixture of 25/15×ATP and kinase substrate solution 21 to start reaction.(8) The 384-well plate was centrifuged at 1000 rpm for 30 seconds and incubated at room temperature.(9) 30 μL of the stop and detection buffer was added to stop the kinase reaction and centrifuged at 1000 rpm for 30 seconds.(10) Collect data on Caliper EZ ReaderII.Data Analysis:
Measurement: IC50 = 3584 nM (pChEMBL 5.449999809265137)
Descriptors: MW 503.98, ALogP 5.48, HBA 5, HBD 3, PSA 94.14, QED 0.38
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)c3cccc(N)n3)c2)c1
InChIKey: IADYJBHZAHVWMD-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. 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-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 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 424.85, ALogP 1.48, HBA 7, HBD 3, PSA 132, QED 0.53
Compound (SMILES): Clc1ccc(Sc2cnn[nH]2)cc1
InChIKey: RGIJNMPSUADKFJ-UHFFFAOYSA-N
Target: 2-Hydroxyacid oxidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UJM8
Assay: HepaRG-CAR Cell-Based Assay for Quantitation of Glycolate Oxidase Inhibition: A HepaRG human hepatic cell line was transfected for stable overexpression of the constitutive androstane receptor (i.e., HepaRG-CAR cells), as reported by van der Mark et al. (Drug Metab. Dispos., 2017, 45:56-67. Overexpression of CAR in these cells resulted in higher levels of glycolate oxidase (GOX) expression compared to the parental HepaRG cells. HepaRG-CAR cells were plated in a 12-wells plate and incubated for 4 weeks until fully differentiated.To measure cellular glycolate flux, the HepaRG-CAR cells were incubated in Williams medium supplemented with 10% fetal bovine serum (FBS), 5 μg/mL insulin, 50 μM hydrocortisone hemisuccinate, 2 mM glutamine, 5000 U/mL penicillin and 5 mg/mL streptomycin. Test compounds were added to the medium at 0, 0.3, 1, 3 or 10 μM and incubated for 30 minutes, after which 500 μM glycolate was also added. After incubation for 48 hours, 400 μL medium was taken from the culture plate and added to 60 μL 37% HCl.Internal standards (2,2-d2 glycolate, 1,2-13C2 oxalate and 13C2-glyoxylate) and hydroxylamine were added followed by another 30 minute incubation at 80° C. The acids were extracted using ethyl acetate with NaCl. The organic phase was dried under nitrogen and derivatized with N-tert-butyldimethylsilyl-N-methyl trifluoroacetamide (MTBSTFA) for 30 minutes at 80° C. The amounts of glycolate, glyoxylate and oxalate were determined by gas chromatography-mass spectrometry (GC-MS) analysis, using a 25 meter CP-Sil 5 CB low bleed column. A standard curve was used to calculate the concentrations of each acid in the culture medium.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 211.68, ALogP 2.61, HBA 3, HBD 1, PSA 41.57, QED 0.83
Compound (SMILES): CC(=O)N1CCC(c2nc(-c3ccc4c(c3)c(-c3cnn(C)c3)nn4C)c3n2CCN(C(C)=O)C3)CC1
InChIKey: JKOMGWIGYRCKBQ-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaScreen Assay: The binding of 2.5 nM of BRD4(49-170) to 10 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) was assessed following the same procedure described for the CBP assay. The standard dose response curves were fitted by Genedata Screener software using the variable-slope model:Signal=Signalnegative control+(SignalDMSO control−Signalnegative control)/(1+(IC50/Dose){circumflex over ( )}Hill slope).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 500.61, ALogP 2.93, HBA 8, HBD 0, PSA 94.08, QED 0.43
Compound (SMILES): Cc1cn2c(-c3cc(S(=O)(=O)N[C@H]4CC[C@](C)(O)CC4)ccc3C)cnc2c(N)n1
InChIKey: YFXRFMXJZJVBAN-WEHONNDLSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: PI3Kδ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) Recombinant Human Protein was purchased from Eurofins (St Charles, Mo.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kδ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 3.4 nM PI3Kδ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 429.55, ALogP 2.57, HBA 7, HBD 3, PSA 122.61, QED 0.59
Compound (SMILES): Nc1ncc(-c2cc(C(=O)C3CCOCC3)cc(N3CCOCC3)n2)cn1
InChIKey: URYLXGQUXXMCGL-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: pAKT Protocol: Inhibition of the PI3K-AKT-mTOR pathway was measured by quantifying the loss of (Ser-473) pAKT using AlphaScreen (Perkin Elmer). B103 (Rat Neuroblastoma) cells were seeded in serum containing medium (High Glucose DMEM (-Phenol Red)+10% FBS+2× Glutamax+1 mM Sodium Pyruvate+10 mM HEPES+1× Non-Essential Amino Acids+1× Pen/Strep) on a 96-well tissue culture treated plate and grown for 20 hours. Cells were then serum starved in serum free medium (High Glucose DMEM (-Phenol Red)+1× Glutamax+1 mM Sodium Pyruvate+1× Pen/Strep) for 6 hours prior to a 2-hour pretreatment with inhibitors of the pathway, including reference inhibitor LY294002. These inhibitors were prepared at a 200× final concentration as a 6-point, 1:3 serial dilution in DMSO series, with DMSO as the 7th point. The inhibitors were then diluted in experimental medium (High Glucose DMEM (-Phenol Red)+1× Glutamax+1 mM Sodium Pyruvate+1× Pen/Strep+25 mM HEPES+0.1% BSA) and combined with the cells at 1× final concentration in 0.5% DMSO. The cells were then stimulated for 20 minutes with (2.5 μg/mL) insulin, an activator of the PI3K-AKT-mTOR pathway and a demonstrated (Ser-473) pAKT agonist. Cells were promptly lysed using Perkin Elmer proprietary lysis buffer and the (Ser-473) pAKT and total AKT contained in the lysate was measured by AlphaScreen. In AlphaScreen, donor beads were coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads were coated with Protein A to immobilize the other antibody. In the presence of target protein, the two antibodies bring the donor and acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of target protein present in the sample. For each inhibitor tested: the ratio of measured (Ser-473) pAKT/totalAKT was plotted in GraphPad Prism as a 7-point, non-linear regression, 4-parameter curve with bottom constrained to reference control bottom and unconstrained top anchored to DMSO.
Measurement: IC50 = 560 nM (pChEMBL 6.25)
Descriptors: MW 369.43, ALogP 1.57, HBA 8, HBD 1, PSA 103.46, QED 0.81
Compound (SMILES): CCS(=O)(=O)N1[C@@H]2CCC[C@H]1C[C@H](N(C)c1nc(Cl)cc(O[C@@H]3CCOC3)n1)C2
InChIKey: QCSWDWJUAVLPMF-QKPAOTATSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Biochemical JAK and Tyk2 Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 444.99, ALogP 2.47, HBA 7, HBD 0, PSA 84.86, QED 0.62
Compound (SMILES): CC(C)c1cccc(N(C)c2ccc(Oc3nc(O)c4ccncc4n3)cc2)c1
InChIKey: BHGRKCHJTFKAPL-UHFFFAOYSA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: In Vitro Enzyme Inhibition Assay: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 386.45, ALogP 5.41, HBA 6, HBD 1, PSA 71.37, QED 0.49
Compound (SMILES): Cc1nc(C(=O)N2CC[C@H](C)[C@H]2c2nc3cc(C(F)(F)F)ccc3[nH]2)c(-c2ccc(Br)cc2)s1
InChIKey: JAQGOGOCUAMXQY-YUNKPMOVSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 2160 nM (pChEMBL 5.670000076293945)
Descriptors: MW 549.42, ALogP 7, HBA 4, HBD 1, PSA 61.88, QED 0.3
Compound (SMILES): CCS(=O)(=O)c1ccc2c(c1)CN(C(=O)CC1CCC1)C2C(=O)Nc1ccc(C(O)(C(F)(F)F)C(F)(F)F)cc1
InChIKey: DEOPPCNBGSZENX-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: FRET Assay: The assay was run in black 384 well plates (Greiner cat no: 784900). Various concentrations of test ligands in 0.1 microlitres DMSO were dispensed to assay plates using an Echo acoustic dispenser. Two pre-mixes were prepared and incubated for 1 h at room temp in the dark. Pre-mix 1 comprised 100 nM Protein (Biotinylated HN-Avi-MBP-TCS-hRORÎ³ (258-518)) and 60 nM Streptavidin APC in assay buffer, 50 mM MOPS pH7.4, 50 mM KF, 0.003% (w/v) CHAPS, 10 mM DTT and 0.01% (w/v) BSA and pre-mix 2 comprised 160 nM biotinylated SRC-1 peptide (NCOA1-677-700) and 20 nM Europium-W8044 labelled Streptavidin in assay buffer. Five microlitres of pre-mix 2 was dispensed to assay plates containing 0.1 microlitres of test compound and was incubated for 15 minutes prior to adding five microlitres of pre-mix 1. Plates were incubated at room temperature for 1 hour in the dark, prior to reading in a Pherastar multi-mode plate reader using HTRF filter set (ex 320, em 612 and 665). The FRET signal at 665 nm was divided by the signal at 612 nm and multiplied by 10,000 to generate a signal ratio value for each well. The raw data was transformed to % effect using the equation:Compound % effect=100*[(Xâmin)/(maxâmin)],where X represents the normalized value for the compound based on the Min (vehicle) and Max (reference compound) inhibition control.
Measurement: IC50 = 63.1 nM (pChEMBL 7.199999809265137)
Descriptors: MW 592.56, ALogP 5, HBA 5, HBD 2, PSA 103.78, QED 0.44
Compound (SMILES): COc1cc2nccc(Oc3ccc(NC(=O)C4(C(=O)Nc5ccc(F)cc5)CC4)cc3)c2cc1-c1cccc(C#N)c1F
InChIKey: CRZSZRSFXGWQFK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Mer Kinase Assay: Human Mer (residues R557-E882 with H628Q and R794A, 0.7 nM) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 mM NaCl, 250 μM GGMEDIYFEFMGGKKK (SEO ID NO. 44). 10 mM magnesium acetate, and 10 μM [γ-33P-ATP]. The reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of phosphoric acid to a concentration of 0.5%. A reaction aliquot of 10 μL was then spotted onto a P30 filtermat and washed four times for 4 minutes in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting. Incorporated 33P was measured using the Wallac Microbeta scintillation counter (Perkin Elmer).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 590.59, ALogP 7.21, HBA 6, HBD 2, PSA 113.34, QED 0.18
Compound (SMILES): Cc1cnc(-c2cc(OC3CCOCC3)cc(C(=O)N[C@@H](C)c3nnc(C(F)(F)F)s3)c2)s1
InChIKey: NWTVSPCFEGLAFO-LBPRGKRZSA-N
Target: P2X purinoceptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBL9
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity at Human P2X3 and Human P2X2/3 Receptors: A fluorescent imaging plate reader (FLEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Fluo-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of Loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C. The final assay DMSO concentration is 1%. The agonist, α,β-Me-ATP, was added in a volume of 20 μl at a concentration representing the EC80 value. The fluorescence was measured for an interval of 90 sec at 2 sec intervals and analyzed based on the increase in peak relative fluorescence units (RFU) compared to the basal fluorescence. Peak fluorescence was used to determine the response to agonist obtained at each concentration of test compound by the following equation: % Response=100*(RFU(test compound)−RFU(control)/RFU(DMSO)−RFU(control)).
Measurement: IC50 = 293 nM (pChEMBL 6.53000020980835)
Descriptors: MW 498.55, ALogP 5.04, HBA 8, HBD 1, PSA 86.23, QED 0.51
Compound (SMILES): CC(C)(C)c1nc(N2CC[C@H](O)C2)c2nn(Cc3ccccc3CSC#N)nc2n1
InChIKey: UNFFXTGEFRBOPX-INIZCTEOSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: The affinity of the compounds of the invention for cannabinoid receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 mL for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 423.55, ALogP 2.85, HBA 9, HBD 1, PSA 103.75, QED 0.62
Compound (SMILES): COc1ccc(COc2ccc(Nc3ccc4ncc(N5CCOCC5)nc4c3C#N)cc2OC)cc1
InChIKey: SVGRIJAWIHWIDS-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kinase Assay: c-KIT: Inhibition of c-KIT kinase activity by a test compound disclosed herein was estimated by radiometric assay. Standard assay conditions were 20 ng of recombinant c-Kit (SignalChem) with 2 μg of substrate Poly-(Glu 4:Tyr 1) (sigma) in the assay buffer in kinase reaction buffer (10 mM MOPS pH 7.0, 0.21 mM EDTA, 0.5% glycerol, 0.001% Brij-35, 0.01 2-mercaptoehtanol, 1 mg/ml BSA, 10 mM MgCl2, 10 mM MnCl2, 10 μM ATP, 0.1 μCi per well [33P]-ATP, in the presence of test compound (diluted in final concentration of 4% DMSO) or DMSO control, with a final volume of 25 μl for 60 minutes at 30° C. The reaction was stopped by adding 5 μl of 3% phosphoric acid solution. Total reaction solution was then harvested onto a filter plate (UniFilter-96 GF/B, PerkinElmer), and washed 20 times for 5 min with dH2O. 30 μl of MicroScint-20 Cocktail (PerkinElmer) was added to dried plate. The plate was sealed and counted using a TopCount scintillation detector (PerkinElmer). The readout from control reaction (complete reaction mixture) was designated as 0% inhibition and the readout for the reaction without enzyme as 100% inhibition. The IC50 values of quinoxaline compounds of this invention against c-KIT kinase were determined using the GraphPad Prism 5 software.
Measurement: Ki = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 497.56, ALogP 4.68, HBA 9, HBD 1, PSA 101.76, QED 0.37
Compound (SMILES): CCC(Sc1cc(N)nc2[nH]nnc12)c1ccccc1
InChIKey: DDWXGJFDKMYFNK-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: MPO Peroxidation (Amplex Red) Assay: MPO peroxidation activity was measured in 100 mM KPi (pH 7.4) by utilizing the non-fluorescent reagent Amplex Red (Invitrogen catalog #A12222) which can be oxidized to the highly fluorescent resorufin. Amplex Red is oxidized by the peroxidase action of MPO to resorufin. Reactions were carried out in 50 μL total volume by adding a 25 μL mixture of 200 pM myeloperoxidase and 40 nM H2O2 (Sigma #349887) to 100 nL inhibitor in 100% DMSO in a 384 well Perkin Elmer Optiplate. Enzyme and compound were preincubated for ten minutes at rt.After the ten minute preincubation, 25 μL of an Amplex Red mixture containing 200 μM Amplex Red and 10 mM H2O2 was added to the plate. Kinetic determinations were carried out immediately on a Perkin Elmer Envision (15 minute kinetic read, Ex: 535 nm, Em: 590 nm).IC50 values were calculated by determining the slope of the linear portion of the kinetic trace (180-540 secs), and using that calculated slope to determine % inhibition occurring at each concentration of inhibitor using the following equation:Y = A + B - A 1 + ( C / x ) Dwhere A=minimal Y value (activity level of inhibited sample), B=maximal Y value (activity level of uninhibited sample), C=Log IC50, D=Hill Slope, x=concentration of inhibitor.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 285.38, ALogP 3.18, HBA 5, HBD 2, PSA 80.48, QED 0.72
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(-c5cn[nH]c5)n4c3)ccc2o1
InChIKey: CWZXHQQFVURMSS-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 316.32, ALogP 3.11, HBA 6, HBD 2, PSA 98.03, QED 0.52
Compound (SMILES): Cc1nc(Oc2ccccc2)ccc1N1C(=O)Nc2c(C(=O)N[C@@H]3CCCC[C@H]3N)sc3nccc1c23
InChIKey: VRGUWATVDVFSOQ-QZTJIDSGSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan). Data were analyzed using Xlfit version 5.3 from ID Business Solutions (Guildford), Microsoft Excel add-in. pIC50 refers to the negative log of the IC50 in molar
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 514.61, ALogP 5.48, HBA 7, HBD 3, PSA 122.47, QED 0.32
Compound (SMILES): C[C@@H]1CN(Cc2cccc(OC(F)(F)F)c2)CCN1C(=O)Oc1cncc(F)c1
InChIKey: AOQXQEXFKWRHGD-CYBMUJFWSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Assay: Proteomes (mouse brain membrane fraction or cell lysates for mouse assays; human prefrontal cortex or cell membrane fractions for human assays) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FPRh or HT-01 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software.
Measurement: IC50 > 51000 nM (pChEMBL 4.291999816894531)
Descriptors: MW 413.37, ALogP 3.82, HBA 5, HBD 0, PSA 54.9, QED 0.71
Compound (SMILES): COc1ccc(CNC(=O)C2CN(c3ncnn4cc(-c5cnn(C)c5)cc34)C2)cc1
InChIKey: VVRHWKCWIJXHBG-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit D816V Assay: In each well of a 384-well plate, 0.04 ng/ul (0.5 nM) of D816V Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 15 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 417.47, ALogP 1.89, HBA 8, HBD 1, PSA 89.58, QED 0.52
Compound (SMILES): Nc1nc2cc(-c3cccc4[nH]nc(N)c34)ccc2o1
InChIKey: XWUSCTPIPARKEV-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 265.28, ALogP 2.54, HBA 5, HBD 3, PSA 106.75, QED 0.49
Compound (SMILES): Cc1cc(Cn2cc(-c3ccc(F)c(C(F)F)c3)cn2)[nH]n1
InChIKey: MACPRACNPYALGP-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of Specific Binding to the hERG-Receptor (HERGBD): After thawing on the day of the assay, the membrane suspension was diluted further with 20 mM HEPES/0.1 mM KCl/pH 7.2.The incubation mixture of 200 μl contained 1.5 nmol/l 3H-Dofetilide, optimized amount of membrane preparation, 20 mM HEPES/0.1 mM KCl/(pH 7.2) and inhibitor in 1% DMSO. Nonspecific binding was estimated in the presence of 10 M Dofetilide. The samples were incubated for 90 min. at RT.Binding was terminated by filtration of the incubated membrane preparations using Filtermat B (Pharmacia, Uppsala Sweden) and a Micro Cell Harvester (Skatron, Lier, Norway). The Filtermat B had been presoaked with 1% polyethylen imine and carefully washed with 0.05 M Tris/HCl-buffer pH=7.7 after the filtration to separate free and bound radioactivity. The filters were counted in a scintillation counter (Betaplate 1205, Berthold, Wildbad, Germany) in order to determine the specific binding of [3H]-Dofetilide.
Measurement: IC50 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 306.29, ALogP 3.71, HBA 3, HBD 1, PSA 46.5, QED 0.8
Compound (SMILES): CNC[C@H](c1ccc2ccccc2c1)[C@H](O)c1cccs1
InChIKey: PKZICZJQJAOMKU-AEFFLSMTSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Uptake Assay Protocol: DAT: This protocol was designed to measure inhibition of uptake by the human dopamine transporter. The reagents were human DAT (HEK293F) cells, GBR 12909 (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 pt polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human dopamine 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.12 μL of test compound (10 mM) in DMSO was added to the wells, and nomifensine was used as a control. GBR 12909 (final assay concentration of 10 μM) was used for background signal. The neurotransmitter transporter dye was prepared in AB prior to use.
Measurement: Ki = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 297.42, ALogP 3.94, HBA 3, HBD 2, PSA 32.26, QED 0.75