lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1CC(C(=O)N2CCOc3c(C#N)cncc3C2)CCN1c1ncc(F)cn1
InChIKey: XDLAWVVLHIXUEY-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding (FP Binding) Assay: Test compounds were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final). In each well of a 384-well Plate were dispensed 0.1 μL of compound solution (or DMSO for controls) followed by 5 μL of GST-hRIPK1 (8-327) at twice the final concentrations in assay buffer (50 mM HEPES pH 7.5, 10 mM NaCl, 50 mM MgCl2, 0.02% CHAPS, 0.5 mM DTT and 0.01% Pluronic F127). For negative control the enzyme addition was replaced by assay buffer only.After addition of 5 μL of fluorescentlabeled ligand at twice the final concentrations in assay buffer, the plate was incubated at RT for 30 mM. At the end, the binding was measured as FP value with the Envision (PerkinElmer) plate reader using filter for an excitation λ=531 nm FP and an emission λ=595 nm FP (S & P-pol). GST-hRIPK1 (8-327) enzyme was produced via Baculovirus expression.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 396.43, ALogP 1.91, HBA 7, HBD 0, PSA 95.24, QED 0.77
Compound (SMILES): CCc1ccc(N(C)c2ccc3c(c2)CCN[C@@H]3CNc2cnccc2C(=O)O)cc1
InChIKey: BCDIBMKCRSWFHV-XMMPIXPASA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: JMJD2C 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 RT for 30 min, 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 hr incubation at RT. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 416.53, ALogP 4.41, HBA 5, HBD 3, PSA 77.49, QED 0.53
Compound (SMILES): N#Cc1ccc(CN2CCC(NC(=O)c3ccc(C(=O)N4CCC(C(=O)c5ccc(N6CCCC6)cc5)CC4)nc3)CC2)cc1F
InChIKey: LQGICZMHJVYZGC-UHFFFAOYSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P54646
Assay: Enzyme-Linked Immunosorbent Assay: Compounds were assayed for their ability to activate AMPK using an enzyme-linked immunosorbent assay. Reagents and procedures for measuring AMPK activation are well known and kits for AMPK activation assays are commercially available.
Measurement: EC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 622.75, ALogP 4.82, HBA 7, HBD 1, PSA 109.64, QED 0.36
Compound (SMILES): CC(C)[C@@H](NC(=O)c1cc(C(F)(F)F)ccc1F)C(=O)N1CCC2(CC1)C(=O)N(C)CN2c1ccc(C(=O)O)c(Cl)c1
InChIKey: VHXSAZIFLREOKP-JOCHJYFZSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Autotaxin LPC Cascade Assay: The efficacy of the Examples described herein, as inhibitors of ATX are demonstrated and confirmed by pharmacological in vitro assays. The following assays and their respective methods are carried out with the compounds according to the present invention. Activity possessed by the compounds may be demonstrated in vivo. Those skilled in the art will appreciate that a variety of assay formats may be used to determine the activity of the compounds of this invention.Autotaxin LPC Cascade Assay:Assay Buffer:100 mM Tris-HCl, pH=9500 mM NaCl5 mM MgCl25 mM CaCl20.05% Triton X 100Reagents:Enzyme Source Stock conc Working conc. Final conc. Autotaxin X-Chem 4.6 μM 20 nM 5 nM LPC Sigma L5254 8 mM in assay buffer 400 μM 100 μM Choline Oxidase Sigma C5896 50 U/mL in water 0.8 U/mL 0.1 U/mL HRP Sigma P8375 1000 U/mL in water 8 U/mL 1 U/mL Ampliflu Red Sigma 90101 100 mM in DMSO 400 μM 50 μM.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 613.01, ALogP 4.25, HBA 5, HBD 2, PSA 110.26, QED 0.47
Compound (SMILES): CC(C)(C)c1ccc(S(=O)(=O)N[C@H]2CC[C@H](CCN3CCN(c4nsc5ccccc45)CC3)CC2)s1
InChIKey: GUBYHCIYOBESBX-MEMLXQNLSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Receptor Binding Test: 1. Experimental Materials:(1) D2 receptor Cell Transfection:This experiment utilizes the plasmid vector containing the gene of D2 receptor protein for transfecting HEK293 cells by calcium phosphate transfection method. By culturing the transfected cells in culture medium containing G418 and selecting cell clones followed by radioligand binding test, a stable cell strain capable of expressing the D2 receptor protein stably is finally obtained.(2) Materials for the Receptor Binding Test:Isotopic ligand [3H] Spiperone (113.0 Ci/mmol) purchased from Sigma Corporation; (+) spiperone purchased from RBI Corporation; GF/B glassfiber filter purchased from Whatman Corporation; imported and subpackaged Tris; PPO and POPOP purchased from Shanghai No. 1 Reagent Factory; and fat-soluble scintillation fluid. Beckman LS-6500 multifunction liquid scintillation counter.2. Experimental Methods:    (1) Cells:HEK-293 cells are infected with the recombinant viruses containing various genes. 48-72 h later, receptor proteins are massively expressed on the membrane. The cells are centrifuged at 1000 rpm for 5 min, then the culture supernatant is discarded and the cell pellets are collected, preserved in refrigerator at −20° C., and resuspended with Tris-HCl reaction buffer (pH=7.5) before use.(2) Competitive Receptor Binding Test:The compound to be tested, the radioligand (20 μL for each) and the receptor protein (160 μL) are added to a reaction tube, wherein the compound to be tested and the positive drug both have the final concentration of 10 μmol/L.After being incubated in water bath at 30° C. for 50 min, the tube is immediately transferred to ice bath to stop the reaction. The mixture is subjected to rapid suction filtration through GF/C glassfiber filter on Millipore cell sample collector, washed with eluent (50 mM Tris-HCl, pH 7.5) (3 mL×3) and dried under microwave for 5-6 min. The filter is transferred to a centrifuge tube (0.5 mL), added with 500 μL fat-soluble scintillation fluid, left in the dark for over min, and counted to determine the radioactivity intensity. The percentage inhibition ratio for each compound to inhibit the isotopic ligand binding is calculated with the following equation:inhibition ratio (1%)=(total binding tube CPM−compound CPM)/(total binding tube CPM−non-specific binding tube CPM)×100%Each compound is tested in duplicate, and the tests are carried out independently twice.Compounds with an inhibition ratio of over 95% are subjected to the receptor binding test at a series of concentrations so as to determine half maximal inhibitory concentration (IC50, the concentration of the compound required for inhibiting 50% binding of [3H]-Spiperone to D2 receptor). Each concentration is tested in duplicate, and tests are carried out independently twice for each compound.
Measurement: Ki = 17.1 nM (pChEMBL 7.769999980926514)
Descriptors: MW 546.83, ALogP 5.7, HBA 7, HBD 1, PSA 65.54, QED 0.41
Compound (SMILES): CCCOCCOc1ccc(-c2cc(=O)[nH]c(-c3c(F)ccc(CNC(=O)C(C)C)c3Cl)n2)cn1
InChIKey: DUJOXEAVSQOHOK-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 502.97, ALogP 4.37, HBA 6, HBD 2, PSA 106.2, QED 0.38
Compound (SMILES): COCCOc1cc2c(cc1OC)nc(NCCCCNC(=O)CC(C)C)c1nnc(C)n12
InChIKey: STDZVZZEFLSUJB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Evaluation of BRD4 binding inhibition ability: The following experiment was performed to evaluate the ability of [1,2,4]triazolo[4,3-a]quinoxaline derivative of the present invention to inhibit the interaction between BRD4 (BD1+BD2) bromodomain, one of BET protein family, and tetraacetylated histone H4 peptide.The compound was serially diluted at the ratio of 1:5 in assay buffer from 10 mM stock in DMSO (initial concentration: 100 μM) on white OptiPlate-384 (PerkinElmer). A mixture comprising 100 nM GST-BRD4 (BD1+BD2) and 100 nM biotinylated acetyl-histone H4 (Lys5,8,12,16) peptide was prepared in assay buffer (50 mM HEPES pH 7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). After adding 6 μl of the mixture to the diluent, 6 μl of the pre-mixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads (PerkinElmer, 10 μg/ml in assay buffer, respectively) was added thereto. The samples were incubated in the dark at room temperature for 30 minutes (shaking at 300 rpm). Then, the signals were measured with PerkinElmer Envision HTS Multilabel Reader using an alpha screen protocol of PerkinElmer. Each plate contained the negative control in which biotinylated acetyl-histone H4 peptide and GST-BRD4 (BD1+BD2) were replaced by assay buffer. In the case of using the software GraphPad Prism for calculation, the negative control point was input as a low standard value. The positive control (probe molecule l-BET762 containing protein/peptide mixture) proceeded to pipetting. IC50 value was determined by using GraphPad Prism 3.03 software (or an updated version thereof).
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 458.56, ALogP 2.97, HBA 9, HBD 2, PSA 111.9, QED 0.38
Compound (SMILES): CCc1cccc(CC)c1-n1nc2c(c1-c1cccc3[nH]cc(F)c13)CN(Cc1ccc(C(F)(F)F)cc1C(F)(F)F)C2(C)C
InChIKey: QQGFKQVMPFXMTF-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: Inhibition Ligand Binding Assay: cAMP treated U937 cells expressing C5aR were centrifuged and resuspended in assay buffer (20 mM HEPES pH 7.1, 140 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, and with 0.1% bovine serum albumin) to a concentration of 3×106 cells/mL. Binding assays were set up as follows. 0.1 mL of cells was added to the assay plates containing 5 μL of the compound, giving a final concentration of 2-10 μM each compound for screening (or part of a dose response for compound IC50 determinations). Then 0.1 mL of 125I labeled C5a (obtained from Perkin Elmer Life Sciences, Boston, Mass.) diluted in assay buffer to a final concentration of 50 pM, yielding 30,000 cpm per well, was added, the plates sealed and incubated for approximately 3 hours at 4° C. on a shaker platform. Reactions were aspirated onto GF/B glass filters pre-soaked in 0.3% polyethyleneimine (PEI) solution, on a vacuum cell harvester (Packard Instruments; Meriden, Conn.). Scintillation fluid (40 μl; Microscint 20, Packard Instruments) was added to each well, the plates were sealed and radioactivity measured in a Topcount scintillation counter (Packard Instruments). Control wells containing either diluent only (for total counts) or excess C5a (1 μg/mL, for non-specific binding) were used to calculate the percent of total inhibition for compound.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 628.64, ALogP 9.57, HBA 3, HBD 1, PSA 36.85, QED 0.19
Compound (SMILES): N[C@]1(COP(=O)(O)O)CC[C@H](c2ccc3c(c2)CC[C@H](Cc2ccccc2)C3)C1
InChIKey: SNMJBCAYROVELN-YFXJRYMSSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1x10^9 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80Â° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 415.47, ALogP 4.11, HBA 3, HBD 3, PSA 92.78, QED 0.62
Compound (SMILES): CN(C)C(=O)C1CCN(c2ccc(CN3CCN(C[C@H](O)CN4CCc5ccccc5C4)C3=O)nc2)CC1
InChIKey: PTODHIYYLRNCOY-HHHXNRCGSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: TR-FRET Assay: Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyze mono- and symmetric demethylation on arginine residues of histone or non-histone proteins in presence of S-adenosylmethionine (AdoMet or SAM) a cofactor responsible for donating the methyl group. PRMT5 is reported to be overexpressed in several human cancers. To identify compounds that inhibit the PRMT5 and decrease its activity, a TR-FRET based assay has been established. Time-resolved fluorescence resonance energy transfer (TR-FRET) HTS assays are homogeneous proximity assays where the interaction of two dye-labeled binding partners is detected by the energy transfer between a donor and an acceptor dye, and the subsequent light emission by the acceptor dye. PRMT5 catalyzes Histone H4 peptide [1-16] which is biotin tagged to the Lysine amino acid at carboxyl end, in presence of S-adenosyl-1-methionine (SAM) to methylate the peptide. The antibody specific to mono methylated H4 peptide (H4R3) with Ig conjugate binds to the methylated peptide, indirectly binding to the Europium lanthanide. SureLight Allophycocyanin-Streptavidin binds to the biotin tag of the peptide, therefore accepting the energy transferred from the Europium lanthanide. This energy transfer between Europium to SureLight Allophycocyanin is a direct measure of the activity/inhibition of the PRMT5 enzyme.
Measurement: IC50 = 119 nM (pChEMBL 6.920000076293945)
Descriptors: MW 520.68, ALogP 2.04, HBA 6, HBD 1, PSA 83.46, QED 0.57
Compound (SMILES): CC(C)NC(=O)c1noc(-c2cc(Cl)c(O)cc2O)c1-c1ccc(CN2CCCCC2)cc1
InChIKey: KZJAWYUJSFALRK-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Fluorescence Polarization Assay: Fluorescence polarization {also known as fluorescence anisotropy} measures the rotation of a fluorescing species in solution, where the larger molecule the more polarized the fluorescence emission.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 469.97, ALogP 5.2, HBA 6, HBD 3, PSA 98.83, QED 0.46
Compound (SMILES): COc1cccc(NC2CCN(c3ccc(-c4cn[nH]c4)c(F)c3F)C2=O)c1
InChIKey: BYMANECJJWRBAR-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assays: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH (SEQ ID NO: 1)). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 384.39, ALogP 3.58, HBA 4, HBD 2, PSA 70.25, QED 0.71
Compound (SMILES): C[C@@H]1CC(n2c(C3CCC(F)(F)C3)nc3cnc4ccc(Cl)cc4c32)CCO1
InChIKey: WELMMGNNNWDWAZ-DNOWBOINSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat #PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: The compound dose response was prepared by diluting compound to a top concentration of 0.3 mM in 100% DMSO and serial diluted by half-log in DMSO to give an 11 point curve, 100× final assay concentration. Using Echo acoustic dispensing, 60 nL of compound was transferred to a low volume Corning 384-well assay plate. 3 μL of a working solution of substrate (200 nM LRRKtide, 2 mM ATP) prepared in assay buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to the 60 nL compound assay plate. The kinase reaction was started with 3 μL of a working solution of LRRK2 enzyme at a concentration of 4 μg/mL. The final reaction concentrations were 100 nM LRRKtide, 1 mM ATP, 2 μg/mL LRRK2 enzyme and a compound dose response with a top dose of 3 μM. The reaction was allowed to progress at room temperature for 30 minutes and then stopped with the addition of 6 μL of detection buffer (20 mM Tris pH 7.6, 0.01% NP-40, 6 mM EDTA with 2 nM terbium labeled anti-phospho-ERM). After an incubation of 1 hour at room temperature, the plate was read on an Envision with an excitation wavelength of 340 nm and a reading emission at both 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data. Inhibition of mutant G2019S LRRK2 (Invitrogen cat #PV4881) was measured in the exact same method. All final concentrations of substrate ATP and enzyme were the same.
Measurement: IC50 = 20.3 nM (pChEMBL 7.690000057220459)
Descriptors: MW 405.88, ALogP 5.88, HBA 4, HBD 0, PSA 39.94, QED 0.53
Compound (SMILES): Cc1ccc2[nH]ncc2c1-c1ccc2nc(NC(=O)OC(C)(C)C)sc2c1
InChIKey: AYMMJMKSSGRCJG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 uL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and SigmaPlot13.0.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 380.47, ALogP 5.5, HBA 5, HBD 2, PSA 79.9, QED 0.48
Compound (SMILES): CC(=O)c1cnc(OCc2cn(-c3ccc(F)c(C(F)(F)F)c3)nn2)nc1
InChIKey: PGHYKQGKLVXRFZ-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 381.29, ALogP 3, HBA 7, HBD 0, PSA 82.79, QED 0.5
Compound (SMILES): O=C(O)c1ccc(-c2noc(-c3cc4c(Cl)ccc(Cl)c4o3)n2)cc1F
InChIKey: PKIOAANQQGQFHH-UHFFFAOYSA-N
Target: Retinoic acid receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P10826
Assay: Transactivation assays: Transcriptional transactivation assays were performed with gal4 fusion receptor constructs, created using each of the RAR ligand binding domains of either mouse or human, co-transfected with the pFR-luc (Stratagene) reporter construct in COS-7 cells. Thus, transfected cells will constitutively express the gal4-RAR fusion protein which in turn may be transactivated by all trans retinoic acid (atRA) to induce the expression of the luciferase that is driven by a gal4UAS.Briefly, on day 1, 96 well plates were seeded with 8000 cells per well then left to recover overnight. On day 2, the cells were co-transfected with 100 ng of reporter plasmid and 10 ng of the appropriate receptor plasmid per well using lipofectamine (Invitrogen). On day 3, the lipofectamine containing media was replaced by a DMEM without phenol red, followed by the addition of test compound dissolved in 1 μL of DMSO to each well's 100 μL total volume. Finally, on day 4, the cells were lysed and their luciferase substrate was provided by the BrightGlo reagent (Promega), the plates were then read on the MicroBeta TriLux (Perkin Elmer).On each plate, an 8 point dose-response curve of atRA was run in duplicate and dose-response curves of test compounds were also generated in duplicate.EC50 data both for test compounds and atRA was generated by fitting dose-response curves using GraphPad Prism. Data for test compounds are quoted as EC50 values. Where replicate data has been generated, the data are quoted as the mean EC50 from the separate experiments.
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 393.16, ALogP 5.29, HBA 5, HBD 1, PSA 89.36, QED 0.51
Compound (SMILES): COc1c(C(C)n2nc(C)c3c(N)ncnc32)cc(Cl)c(C)c1-c1cncc(C(=O)N(C)C)c1
InChIKey: KATAAFRJKJMWFE-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) was purchased from PerkinElmer (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) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from SigmaAldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.).The kinase reaction was conducted in polystyrene 384-well matrix 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 0.5%. The PI3K assays were 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, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI31(6. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (PerkinElmer).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 479.97, ALogP 4.06, HBA 8, HBD 1, PSA 112.05, QED 0.46
Compound (SMILES): C=CC(=O)NC1CN(c2ccncn2)CC1Nc1ncc2c(n1)N(C)C(=O)N(c1c(Cl)c(OC)cc(OC)c1Cl)C2
InChIKey: OIAAQMYTAXVQOD-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme. a) [FGFR4-WT]=10 nM, [ATP]=300 uM, [Y10-Sox]=10 uM (ATP KMapp 300 uM): A 10x stock solution of FGFR4-WT (PR4380C), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 7, was prepared as described below. Alternatively, a 10x stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 7, was prepared as described below. A solution of 1.4xATP (AS001A) and 5x Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1x kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10x stock, KB002A) and 0.2 mM DTT (DS001A). 5 uL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 uL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 ul of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 uL of 1.4xATP. The reactions were monitored every 71 seconds for 240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to ~60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log [Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 600.47, ALogP 3.14, HBA 10, HBD 2, PSA 137.94, QED 0.37
Compound (SMILES): Nc1nccn2c([C@@H]3CCN[C@@H](C(=O)O)C3)nc(-c3ccc(C(=O)Nc4cc(C(F)(F)F)ccn4)cc3)c12
InChIKey: AKEDVPZDTIJMQB-NVXWUHKLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ.ID.NO.: 1), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10)) compound concentrations.
Measurement: IC50 = 4.4 nM (pChEMBL 8.359999656677246)
Descriptors: MW 525.49, ALogP 3.56, HBA 8, HBD 4, PSA 147.53, QED 0.31
Compound (SMILES): CC1(CO)Cc2cc(NC(=O)c3cnn4cc(Cl)cnc34)c(N3CCN(CC(F)F)CC3)cc2O1
InChIKey: LXHBJKLKPARZHK-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 506.94, ALogP 2.71, HBA 8, HBD 2, PSA 95.23, QED 0.53
Compound (SMILES): C=CC(=O)N1CC(NC(=O)Cn2c(C)c(C(=O)N3CCn4c(cnc4C)C3)c3cc(Br)ccc32)C1
InChIKey: OXMRDIPBCDQLSI-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (2 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 9920 nM (pChEMBL 5)
Descriptors: MW 539.43, ALogP 2.39, HBA 6, HBD 1, PSA 92.47, QED 0.5
Compound (SMILES): COC(=O)/C=C/c1c(S(=O)(=O)C(F)(F)F)ccc(Oc2cc(F)cc(C#N)c2)c1C
InChIKey: PQLIJTATPNVDOH-ZZXKWVIFSA-N
Target: Endothelial PAS domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99814
Assay: Scintillation Proximity Assay (SPA): HIF-2a: The total assay volume was about 100 uL in the following configuration: 2 uL compound in 100% DMSO, 88 uL buffer with protein and probe and 10 uL of SPA beads. The compound was diluted in a master plate consisting of a 10-point dose response with a 3-fold compound dilution from 100 nM to 5 nM. Assays were run on a 96-well plate in which one column, designated as the high signal control, contained DMSO with no compound and another column, designated as the low signal control, contained no protein. Prior to plating out of compound, a buffer solution, consisting of 25 mM TRIS pH 7.5 (Sigma), 150 mM NaCl (Sigma), 15% Glycerol (Sigma), 0.15% BSA (Sigma), 0.001% Tween-20 (Sigma), 150 nM Compound 183 and 100 nM HIF-2Î± HIS TAG-PASB Domain, was made and allowed to equilibrate for 30 minutes. Compounds that were to be tested were then plated in to a 96-well white clear bottom Isoplate-96 SPA plate (Perkin Elmer). To the compounds, 88 uL of the buffer solution was then added, the plate was covered with a plastic cover and then aluminum foil, placed onto a shaker and equilibrated for 1 hour. After equilibration, 10 uL of a 2 mg/mL solution of YSi Cu His tagged SPA beads (Perkin Elmer) were then added to each well of the plate, covered and equilibrated for another 2 hours. The plates were then removed from the shaker, placed into a 1450 LSC and luminescence counter MicroBeta Trilux (Perkin Elmer) to measure the extent of probe displacement.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 443.37, ALogP 4.28, HBA 6, HBD 0, PSA 93.46, QED 0.39
Compound (SMILES): CN(C)c1cc(C(=O)N2CCCC(c3nc4ccccc4n3C)C2)ccn1
InChIKey: QMQLHUHVXMANIS-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Biological Assay: Prokineticin receptor 1 (PKR1) antagonists may be functionally assessed by measurement of change in intracellular calcium levels induced by Gq mediated increase in inositol triphosphate (IP3) levels. The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro.Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.
Measurement: IC50 = 1220 nM (pChEMBL 5.909999847412109)
Descriptors: MW 363.46, ALogP 3.05, HBA 5, HBD 0, PSA 54.26, QED 0.72
Compound (SMILES): Cc1[nH]nc2c1C(c1ccccc1)C1=C(CC(C)(C)CC1=O)N2
InChIKey: FFFWFAUFYUNHEL-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Caliper Assay: A solution of 4× inhibitor (5 μL), 4× substrate/ATP-Metal solution (5 μL), and 2× Kinase solution (10 μL) was prepared with assay buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH 7.5) and mixed/incubated in 384 well plates for 1 or 5 hours depending on the kinase being examined, at room temperature. A solution of termination buffer (QuickScout Screening assist MSA; Carna Biosciences) (60 μL) was added to each well. The entire reaction mixture was then applied to a LabChip3000 system (Caliper Life Science) and the product and substrate peptide peaks were separated and quantified. Evaluation of kinase activity was then determined based on ratio of calculated peak heights of product (P) and substrate (S) peptides (P/(P+S)).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 307.4, ALogP 3.92, HBA 3, HBD 2, PSA 57.78, QED 0.84
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cnn(CCN5CCC(OC)CC5)c4)cc3C3(CC3)C2=O)c1F
InChIKey: IWLLEOLQIJXURN-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 553.61, ALogP 3.93, HBA 8, HBD 0, PSA 81.95, QED 0.42
Compound (SMILES): NC(=O)c1cccnc1O[C@H]1CC2(C1)C[C@H](N1C(=O)CN(c3ccc(Cc4ccccc4)cc3)C1=O)C2
InChIKey: GMTHYNAVEYGEPJ-RZWCHVLBSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assays: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH) (SEQ ID No. 1). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 496.57, ALogP 3.93, HBA 5, HBD 1, PSA 105.83, QED 0.5
Compound (SMILES): C[C@@]1(O)CCN(CCc2cccc(-n3cnc(-c4nc(NC5CCN(S(C)(=O)=O)CC5)ncc4C(F)(F)F)c3)c2Cl)C1
InChIKey: KOJFASDTOWQDPX-AREMUKBSSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/mL BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions were stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 628.12, ALogP 3.84, HBA 9, HBD 2, PSA 116.48, QED 0.39
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CCc2ccccc2N(Cc2nccs2)C1=O
InChIKey: MARARTNNFBKKEQ-JSGCOSHPSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA (Peptide A) was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK (SEQ ID NO: 3) was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA as the substrate for the TR-FRET assay.The XIAP protein sequence was obtained from the SWISS-PROT protein sequence database and the BIR2 and BIR3 domains were derived from that. The sequence of the BIR2 domain used for the TR-FRET assay is MRHHHHHHRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNW PDYAHLTPRELASAGLYYTGIGDQVQCFACGGKLKNWEPGDRAWSEHRRHFPNCFFVL GRNLNIRSE (SEQ ID NO: 4).The sequence of the BIR3 domain used for the TR-FRET assay is MRHHHHHHRSDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGF YALGEGDKVKCFHCGGGLTDWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTH SLEECLVRTT (SEQ ID NO: 5).Ten nanomolar of 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/niL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 1597 nM (pChEMBL 5.800000190734863)
Descriptors: MW 358.47, ALogP 1.72, HBA 5, HBD 2, PSA 74.33, QED 0.85