lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): FC(F)(F)Oc1cccc(-c2nnc3ccc(N[C@H]4CC[C@@H](Br)CC4)cn23)c1
InChIKey: OMLIXNDNKDQSNH-OKILXGFUSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase-Glo Assay: These assays are set up in duplicate 50 ul volumes in white, flat bottom 96 well plates. Inhibitors are added to the solution of 1× kinase buffer, 10 uM ATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, and water in serial dilutions ranging from micromolar to nanomolar concentrations. This solution is incubated at 30 degrees Celsius at 360 rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagent is added to each well, including all positive and negative control wells, and incubated at room temperature for 15 minutes. The plate is then read by the Luminoskan Ascent instrument and the results displayed with the Ascent Software version 2.6.
Measurement: IC50 = 2490 nM (pChEMBL 5.599999904632568)
Descriptors: MW 455.28, ALogP 5.41, HBA 5, HBD 1, PSA 51.45, QED 0.53
Compound (SMILES): CC1CN(C(=O)c2cc(Cc3n[nH]c(=O)c4ccccc34)ccc2F)Cc2nnc(C(F)F)n21
InChIKey: KLEMFAOWDQXMRO-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: ELISA Assay: The HTb-PARP1 positive clones were obtained using the full-length PARP1 plasmid, through PCR amplification, enzyme digestion, ligation, and transformation into DH5a. The plasmids were extracted and determined by enzyme cleavage, and then transformed into DH10Bac. Bacmid/PARP is determined by PCR and sequencing. TNI was transfected, the viruses were collected, and cells were lysed. PARP1 protein was purified by affinity chromatography and determined by Western blotting. A plate was coated by substrate histone, NAD+ and DNA, as well as expressed PARP1 enzyme, was placed into 96-well plate reaction system. Various reaction conditions were optimized and ultimately determined.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 468.44, ALogP 3.4, HBA 6, HBD 1, PSA 96.77, QED 0.5
Compound (SMILES): CNC(=O)Cn1cc(-c2ccc(N3CCC(n4ccc5ccc(F)cc54)CC3)nn2)cn1
InChIKey: OAZDCTMDSUWDDQ-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Enzymatic Assay: The SCD1 enzymatic assay was done in a volume of 50 μL using 10 μg of RLM (prepared as described above) in a 96-well polypropylene plate (enzyme reaction buffer contains 0.1 M K-Phosphate Buffer, 10 mM ATP, 6 mM MgCl2. 1 mM CoA, 1 mM β-NADH, 1.6 mM L-glutathione, 20 μM Stearoyl-CoA). Stearoyl-[9,10-3H]-CoA (ARC-0390, 1 mCi/mL, 60 Ci/mmol,) was added at a final concentration of 2 μCi/mL. Test compound was then added to the reaction mixture at the selected concentration. After incubation at room temperature for 2 hours, 5 μL 1 N HCl was added to stop the reaction, followed by addition of 25 μL of 10% charcoal. The reaction mixture was then transferred to 96-well Multiscreen plate (Millipore, Cat#MSFCN6B50). [3H2O] was collected into Opti-plate (PE, Cat#6005290) by centrifuge, at 1,000 rpm for 2 minutes. 150 μL Microscint 40 (PE, cat #6013641) was then added to each well and counted on Topcount for [3H] counts per minute (cpm).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 433.49, ALogP 3.02, HBA 7, HBD 1, PSA 80.87, QED 0.52
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)N(Cc3cccc(C#N)c3)C(C)(C)C2=O)cn1
InChIKey: LXPBHKQPWOTJHJ-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 418.46, ALogP 3.16, HBA 7, HBD 0, PSA 108.26, QED 0.59
Compound (SMILES): COc1cccc(-c2ccc(CC[C@](C)(C(=O)NO)S(C)(=O)=O)cc2)c1
InChIKey: BDVUXWNJIVZJIT-LJQANCHMSA-N
Target: UDP-3-O-acyl-N-acetylglucosamine deacetylase (SINGLE PROTEIN), Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG12228)
UniProt: P47205
Assay: Enzyme Assay: IC50 determination in the LpxC enzyme assay was carried out in a similar manner to that described by Malikzay et al in the 2006 Poster, Screening LpxC (UDP-3-O(R-3-hydroxymyristoyl)-GlcNAc deacetylase) using BioTrove Rapid Fire HTS Mass Spectrometry (aNew Lead Discovery and bInflammation and Infectious Disease, cStructural Chemistry, Schering-Plough Research Institute, Kenilworth, N.J. 07033, (BioTrove, Inc. 12 Gill St., Suite 4000, Woburn, Mass. 01801). Briefly, Pseudomonas aeruginosa LpxC enzyme (0.1 nM) purified from E. coli-overexpressing bacteria was incubated at 25° C. in a final volume of 50 ul containing 0.5 uM UDP-3-O(R-3-hydroxydecanoyl)-N-acetylglucosamine, 1 mg/mL BSA, and 50 mM sodium phosphate buffer, pH 8.0 in the presence and absence of inhibitor compound. At the end of 1 hour, 5 ul of 1N HCl was added to stop the enzyme reaction; the plates were centrifuged, and then processed with the BioTrove Rapidfire HTMS Mass Spectrometry System.
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 377.46, ALogP 2.6, HBA 5, HBD 2, PSA 92.7, QED 0.57
Compound (SMILES): Cc1cc(Nc2cc(C)c(C#N)c(NCCOc3ccccc3F)n2)n[nH]1
InChIKey: AWLBOIUGNGQJSU-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of recombinant Aurora A kinase derived from human Hela cells using kemptide as substrate in presence of [c-32P]ATP
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 366.4, ALogP 3.67, HBA 6, HBD 3, PSA 98.65, QED 0.55
Compound (SMILES): Cc1ncc(C)n2nc(CCc3ccc4ccc(F)cc4n3)nc12
InChIKey: VOASYVQFJORMOW-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: A PDE10A assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 321.36, ALogP 3.21, HBA 5, HBD 0, PSA 55.97, QED 0.58
Compound (SMILES): C[C@@H](CC(=O)O)NC(=O)c1nc(C#N)c2cc(Oc3ccccc3)ccc2c1O
InChIKey: CTADYWAQKGRMMW-LBPRGKRZSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 3270 nM (pChEMBL 5.489999771118164)
Descriptors: MW 391.38, ALogP 3.2, HBA 6, HBD 3, PSA 132.54, QED 0.59
Compound (SMILES): O=C(Nc1ccncc1)N1CCN(Cc2ccc3c(c2)OC(F)(F)O3)CC1
InChIKey: NOLNVTCPLFOTPX-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: FAAH Assay: T84 frozen cell pellets or transfected SK-N-MC cells (contents of 1x15 cm culture dishes) were homogenized in 50 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% Glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture consisted of 50 uL of the cell homogenate, 10 uL of the test compound, and 40 pt of anandamide [1-3H-ethanolamine] (3H-AEA, Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 80 nM. The reaction mixture was incubated at rt for 1 h. During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 376.36, ALogP 2.75, HBA 5, HBD 1, PSA 66.93, QED 0.89
Compound (SMILES): CC(c1nc2c(cnn2C2CCOCC2)c(=O)[nH]1)N1CC(Oc2ccc(F)c(C#N)c2Cl)C1.O=C(O)C(F)(F)F
InChIKey: YAUGNKGSHVKGME-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of human recombinant PDE9 expressed in insect Sf9 cells using [3H]-cGMP as substrate after 30 mins by scintillation proximity assay
Measurement: IC50 = 36.2 nM (pChEMBL 7.440000057220459)
Descriptors: MW 472.91, ALogP 2.96, HBA 8, HBD 1, PSA 109.06, QED 0.61
Compound (SMILES): CCCC[C@H](c1ccc(C(=O)NCCC(=O)O)cc1)C(c1ccc(Cl)cc1)c1csc2c(C#N)cc(Cl)cc12
InChIKey: WUDPSHWLKSHJTQ-MIJJZIGMSA-N
Target: Glucagon receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P47871
Assay: Binding Assay: A stable CHO (Chinese hamster ovary) cell line expressing cloned human glucagon receptor was maintained as described (Chicchi, et. al. J Biol Chem 272, 7765-9 (1997); Cascieri, et. al. J Biol Chem 274, 8694-7 (1999)). To determine antagonistic binding affinity of compounds, 0.001-0.003 mg of cell membranes from these cells were pre-incubated with 0.100 mg WGA-coated PVT SPA beads (Amersham) for 20 minutes at room temperature in 25 μL of a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM EDTA, 0.1% BSA and 3% glycerol in Costar 384 well plates with clear bottoms (#3706). Next, 25 μL of 125I-Glucagon (New England Nuclear, MA) (1×10−14 mol per well) and either 1 μL solutions of test compounds or 0.001 mM unlabeled glucagon or DMSO were added and mixed. After 4-12 hours incubation at room temperature, the radioactivity bound to the cell membranes was determined in a radioactive emission detection counter (Wallac-Microbeta).
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 579.55, ALogP 8.39, HBA 4, HBD 2, PSA 90.19, QED 0.19
Compound (SMILES): CN1CCC(C(=O)N2CCC(CCOc3ccc(-c4nc(C#N)nc5c4ncn5C)cc3C(F)(F)F)CC2)C1
InChIKey: YSWHOZAXJCXOPA-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Inhibition Assay: 5 μL of a human cathepsin S enzyme (R&D1183-CY-010) was added to a 96-well plate so as to be 20 ng/well. Next, 10-fold dilution of the test compound (a 10 mM DMSO solution) was serially performed with a buffer solution for assay (a solution of 50 mM sodium methoxide, 250 mM sodium chloride and 5 mM dithiothreitol (DTT), adjusted to pH=4.5 with 1 N hydrochloric acid) six times such that the final concentration becomes 0.1 nM to 10 μM then, 20 μL of each of the solutions was added to the wells (the final DMSO concentration was 0.1%), and 25 μL of synthetic substrate VVR-AMC (3211-V manufactured by PEPTIDE INSTITUTE, INC.) was added thereto such that the final concentration becomes 40 μM, thereby starting a enzyme reaction. A fluorescence intensity (an excitation wavelength: 380 nm and a fluorescent wavelength: 460 nm) was measured at 37° C. for 10 minutes immediately after the start of the reaction using a fluorescence spectrophotometer (SPECTRA MAX GEMINI, manu
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 541.58, ALogP 3.88, HBA 8, HBD 0, PSA 100.17, QED 0.47
Compound (SMILES): COC[C@H](C)Oc1ccc2[nH]nc(-c3cc(N4CCC5(CCCO5)CC4)ncn3)c2c1
InChIKey: YJUNXTITQVBNKL-INIZCTEOSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 2.02 nM (pChEMBL 8.699999809265137)
Descriptors: MW 423.52, ALogP 3.58, HBA 7, HBD 1, PSA 85.39, QED 0.65
Compound (SMILES): CN1CCC(CNC(=O)c2ccc(NC(=O)N3Cc4ccc(C#N)cc4C3)cc2)CC1
InChIKey: SRAGHGLPVFLVJA-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 3.67 nM (pChEMBL 8.4399995803833)
Descriptors: MW 417.51, ALogP 3.18, HBA 4, HBD 2, PSA 88.47, QED 0.8
Compound (SMILES): NCCC[C@H]1N[C@@H](CNC(=O)c2ccc3ccccc3c2)CCN(Cc2ccc(Cl)cc2Cl)C1=O
InChIKey: XTKNLEWVDDEEIW-ILBGXUMGSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 513.47, ALogP 4.37, HBA 4, HBD 3, PSA 87.46, QED 0.42
Compound (SMILES): COC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(NS(=O)(=O)O)cc1)c1nc2c(s1)CCC2
InChIKey: FBHDOKDDKKSQTM-SFTDATJTSA-N
Target: Receptor-type tyrosine-protein phosphatase beta (SINGLE PROTEIN), Homo sapiens
UniProt: P23467
Assay: Inhibition of recombinant HPTPbeta (unknown origin) preincubated for 10 mins followed by substrate addition measured after 15 mins by DiFMUP probe-based fluorescence assay
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 544.66, ALogP 3.21, HBA 7, HBD 4, PSA 146.72, QED 0.29
Compound (SMILES): CCN(CC)C(=O)c1ccc(-c2ccc3[nH]nc(-c4cc(N5CCOCC5)ncn4)c3c2)cc1
InChIKey: JYYXRQFXEDBQKY-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 607.5 nM (pChEMBL 6.21999979019165)
Descriptors: MW 456.55, ALogP 4.01, HBA 6, HBD 1, PSA 87.24, QED 0.47
Compound (SMILES): CNC(=O)c1ccc2c(c1)[nH]c(=O)n2-c1nc2ccccc2[nH]1
InChIKey: RLHHTNGKNOTFMX-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of His-tagged human Aurora A kinase (122 to 40 residues) expressed in Escherichia coli BL21 (DE3) Rosetta cells using biotinylated STK2 substrate incubated for 30 mins by HTRF assay
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 307.31, ALogP 1.55, HBA 4, HBD 3, PSA 95.57, QED 0.52
Compound (SMILES): COc1cccc(-c2cccc(C(CC(=O)O)c3nccs3)c2)c1OC
InChIKey: YUNVHQSVLSQZCF-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 950 nM (pChEMBL 6.019999980926514)
Descriptors: MW 369.44, ALogP 4.43, HBA 5, HBD 1, PSA 68.65, QED 0.67
Compound (SMILES): O=S(=O)(c1ccc(F)cc1)[C@@]1(c2ccc(I)c(F)c2)CCN(Cc2ccccc2)C1
InChIKey: HSPFELKFCXSWMU-QHCPKHFHSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 1137 nM (pChEMBL 5.940000057220459)
Descriptors: MW 539.39, ALogP 5.14, HBA 3, HBD 0, PSA 37.38, QED 0.33
Compound (SMILES): O=C(NCC(O)C(F)(F)F)c1ccc2cc(Oc3ccc(C(F)(F)F)nc3)ccc2n1
InChIKey: OVHAKRKWBWZJAC-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 2770 nM (pChEMBL 5.559999942779541)
Descriptors: MW 445.32, ALogP 4.09, HBA 5, HBD 2, PSA 84.34, QED 0.58
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3ccc(CN4CCOCC4)nc3)n2)cnc1N1CCC(OC(F)F)C1
InChIKey: ZSJKDURWVGXFJO-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 508.53, ALogP 3.2, HBA 10, HBD 1, PSA 112.32, QED 0.49
Compound (SMILES): CCn1c(CNc2ccc(C(=N)N)cc2)nc2cc(C(=O)N(CCC(=O)O)c3ccccc3C)ccc21
InChIKey: PCJRLTPRBXAYJZ-UHFFFAOYSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Inhibition of human thrombin pre-incubated for 10 mins before Ac-FVR-AMC substrate addition and measured after 10 mins by fluorescence based assay
Measurement: IC50 = 3.11 nM (pChEMBL 8.510000228881836)
Descriptors: MW 498.59, ALogP 4.38, HBA 6, HBD 4, PSA 137.33, QED 0.19
Compound (SMILES): COc1cc2ncccc2cc1NC(=O)c1csc2cnc(NC3CC3)nc12
InChIKey: JFKRMJRQGVELFG-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: In vitro IRAK-1 and IRAK-4 Assay: Purified recombinant IRAK-4 protein was incubated with 250 uM synthetic peptide(KKARFSRFAGSSPSQSSMVAR) in 30 ul of kinase buffer including (20 mM MOPS pH7.2, 25 mM beta glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 50 uM ATP, 20 mM MgCl2, 10 uCi gamma-33P, 0.1% BSA) for the indicated time. For purified recombinant IRAK-1 protein kinase assay, 50 uM ATP was used. A 25 ul aliquot of the reaction mixture was transferred on to p81 phosphocellulose squares (Upstate Biotechnology, Lake Placid, N.Y.). The assay squares were washed three times with 0.75% phosphoric acid and once with acetone. Enzyme activity was measured by determining the bound radioactivity by liquid scintillation counting.
Measurement: IC50 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 391.46, ALogP 4.07, HBA 7, HBD 2, PSA 89.03, QED 0.53
Compound (SMILES): c1ccc(-c2cc(CNc3nccc(Nc4cc(C5CCCC5)n[nH]4)n3)on2)cc1
InChIKey: CMAQGLFHNAGBSI-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 401.47, ALogP 4.87, HBA 7, HBD 3, PSA 104.55, QED 0.41
Compound (SMILES): CC(=O)N[C@H]1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: VAMYQOUPUFFEEZ-JYJNAYRXSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 381.48, ALogP 2.09, HBA 6, HBD 2, PSA 91.21, QED 0.72
Compound (SMILES): Cc1ccc2c(c1)N(Cc1ccccc1)C[C@@H](C)N(C(=O)Nc1cccnc1)[C@H]2C
InChIKey: SHBCBFDPLIFTBC-UXHICEINSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Channel blocking activity at recombinant human CaV 3.1 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 227 nM (pChEMBL 6.639999866485596)
Descriptors: MW 400.53, ALogP 5.39, HBA 3, HBD 1, PSA 48.47, QED 0.64
Compound (SMILES): CCc1cc(CNc2nc(Nc3cc(C4CC4)n[nH]3)cc(OCCN(C)C)n2)on1
InChIKey: JAMDHEBMJGXSJP-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 412.5, ALogP 2.92, HBA 9, HBD 3, PSA 117.02, QED 0.44
Compound (SMILES): Cn1cc(CNC(=O)c2cc(NC(=O)c3cc(-c4ccccn4)c(Cl)cc3Cl)n(-c3ccccc3)n2)nn1
InChIKey: LSFHVEHSZCNNDG-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 4.92 nM (pChEMBL 8.3100004196167)
Descriptors: MW 547.41, ALogP 4.55, HBA 8, HBD 2, PSA 119.62, QED 0.31
Compound (SMILES): C[C@@H]1CN(c2scnc2-c2nc3cc(C(C)(C)C)ccc3[nH]2)CCN1C(=O)Cn1cnc2cccnc21
InChIKey: MTVAHZZIVWOUEP-QGZVFWFLSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 514.66, ALogP 4.47, HBA 8, HBD 1, PSA 95.83, QED 0.38
Compound (SMILES): Cc1ncc(C(CNC(=O)c2cccc(F)c2Cl)N2CCCC(F)(F)C2)cn1
InChIKey: FVXYBJRQYPAMFI-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 412.84, ALogP 3.78, HBA 4, HBD 1, PSA 58.12, QED 0.81
Compound (SMILES): Cc1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)CC(F)(F)F)sc2ccccc12
InChIKey: TYBIDOANARREDX-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 51.2 nM (pChEMBL 7.289999961853027)
Descriptors: MW 485.45, ALogP 6.27, HBA 3, HBD 0, PSA 37.38, QED 0.4
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ccccc1Nc1nnc(-c3ccc(Cl)cc3)s1)c1c(O)ccc(F)c12
InChIKey: RGKWYLIWKHCGJG-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA) Assay: SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Yi receptors (The P2Yi receptor membranes were provided by Biology and the cloning of the receptor and P2Yi receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384- well OptiPlates (PerkinElmer Life Sciences, Cat # 6007299) in a volume of 50 ui containing -15 fmol of P2Y1 receptor (1.7 ug of total protein), 0.3 nM [33P] 2MeS-ADP(PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 uM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium Chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 ug of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 578.16, ALogP 7.98, HBA 7, HBD 2, PSA 64.52, QED 0.25
Compound (SMILES): COc1ccc([C@H](NC(=O)c2cc(F)c3cnc(Nc4cn(C)nc4C)nc3c2)c2cnn(C)c2)cc1F
InChIKey: QYIZTCVUYUDJAX-DEOSSOPVSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an N-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 1 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 6.8 nM (pChEMBL 8.170000076293945)
Descriptors: MW 518.53, ALogP 3.96, HBA 9, HBD 2, PSA 111.78, QED 0.34
Compound (SMILES): Cc1cnc(Nc2ccc(N3CCOCC3)cc2)nc1-c1cc2cc(COCC(F)(F)F)ccc2o1
InChIKey: CTCWNBMNJSPICY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of GST-tagged recombinant human JAK2 expressed in baculovirus using TK-substrate-biotin as substrate preincubated for 5 mins followed by substrate addition after 30 mins by HTRF assay
Measurement: IC50 = 25.7 nM (pChEMBL 7.590000152587891)
Descriptors: MW 498.51, ALogP 5.86, HBA 7, HBD 1, PSA 72.65, QED 0.34
Compound (SMILES): CC(C)NCC1(C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CCS(=O)(=O)CC1
InChIKey: KWBPKGOWXKIHBQ-SANMLTNESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 818 nM (pChEMBL 6.090000152587891)
Descriptors: MW 704.7, ALogP 5.21, HBA 6, HBD 1, PSA 100.62, QED 0.3
Compound (SMILES): C[C@H]1CCN(c2ncc3nc(C4(n5cccn5)CC4)[nH]c3n2)C[C@@H]1C(=O)N1CCCC1
InChIKey: HEAIEEPDCXJUPZ-HOTGVXAUSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: In Vitro DGAT2 Assay: For determination of IC50 values, the reactions were carried out in 384-well white Polyplates (Perkin Elmer) in a total volume of 20 uL. To 1 uL of compounds dissolved in 100% DMSO and spotted at the bottom of each well, 5 uL of 0.04% bovine serum albumin (BSA) (fatty acid free, Sigma Aldrich) was added and the mixture was incubated at room temperature for 20 minutes. To this mixture, 10 uL of hDGAT2 membrane fraction (0.01 mg/mL) diluted in 100 mM Hepes-NaOH, pH 7.4, 20 mM MgCl2 containing 200 nM methyl arachidonyl fluorophosphonate (Cayman Chemical; dried from ethyl acetate stock solution under argon gas and dissolved in DMSO as 5 mM stock) was added. After this mixture was preincubated at room temperature for 2 hours, DGAT2 reactions were initiated by the addition of 4 uL of substrates containing 30 uM [1-14C]decanoyl-CoA (custom-synthesized by Perkin Elmer, 50 mCi/mmol) and 125 uM 1,2-didecanoyl-sn-glycerol (Avanti Polar Lipids) dissolved in 12.5% acetone. The reaction mixtures were incubated at room temperature for 40 min and the reactions were stopped by addition of 5 uL of 1% H3PO4. After the addition of 45 uL MicroScint-E (Perkin-Elmer), plates were sealed with Top Seal-A covers (Perkin-Elmer) and phase partitioning of substrates and products was achieved using a HT-91100 microplate orbital shaker (Big Bear Automation, Santa Clara, Calif.). Plates were centrifuged at 2,000×g for 1 min in an Allegra 6R Centrifuge (Beckman Coulter) and then were sealed again with fresh covers before reading in a 1450 Microbeta Wallac Trilux Scintillation Counter (Perkin Elmer). DGAT2 activity was measured by quantifying the generated product [14C]tridecanoylglycerol in the upper organic phase.
Measurement: IC50 = 37400 nM (pChEMBL 4.429999828338623)
Descriptors: MW 420.52, ALogP 2.17, HBA 7, HBD 1, PSA 95.83, QED 0.7
Compound (SMILES): CC(=O)Nc1ccc2nc(-c3cccc(NS(=O)(=O)c4ccc5ccccc5c4)c3)nc(N3CCOCC3)c2c1
InChIKey: FFNCFGWKXFOXPA-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00750
Assay: Inhibition of GST-tagged human recombinant PI3KC2beta expressed in Sf9 cells
Measurement: IC50 = 530 nM (pChEMBL 6.28000020980835)
Descriptors: MW 553.64, ALogP 5.05, HBA 7, HBD 2, PSA 113.52, QED 0.3