lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(Nc1cc(Cl)ccc1N1CCC(CO)C1)c1cnn2cccnc12
InChIKey: TYDKEVCWEALCFZ-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 = 650 nM (pChEMBL 6.190000057220459)
Descriptors: MW 371.83, ALogP 2.45, HBA 6, HBD 2, PSA 82.76, QED 0.74
Compound (SMILES): COc1cc(CN(CC2CC2)C(CC(=O)O)c2ccc(Cl)cc2Cl)ccc1OCCn1c(=O)ccn(C)c1=O
InChIKey: HBIBUYOXTLVSCE-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Radioligand Binding Assay: The composition of the binding assay buffer is determined in a course of detailed optimization procedure. This resulted in a binding assay buffer constituted by the following components: 25 mM Hepes (pH=7.4), 5 mM MgCl2, 1 mM CaCl2, 100 mM NaCl, supplemented with 0.1% of protease free BSA (as a final concentration). Competition binding assay is performed using 125I-CXCL10 (PerkinElmer, NEX348, specific activity 2200 Ci/mmol) radioligand in a final concentration of 50-70 pM. The nonspecific binding is defined by 150 pM of hr-CXCL10 (R&D Systems, Cat No 266-IP). The total assay volume is equal to 150 ul and contained 1% of DMSO (final concentration). Binding reaction is initiated by adding of membranes (10-20 ug proteins, approximately 5x105 cell equivalents) to the reaction mixture. After 60 minutes of incubation at 25° C. the reaction is terminated by rapid filtration over GF/B glass fibre filters that are pre-soaked with 0.5% polyethyleneimine (Fluka Analytical, P3143) for 1 hour, using a Skatron cell harvester device. Filters then are washed with 8 ml of ice-cold wash buffer (modified binding buffer in which BSA is omitted and the concentration of NaCl is adjusted to 500 mM concentration). The radioactivity retained on the filters is measured by a Wizard 1470 Automatic Gamma counter.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 576.48, ALogP 4.37, HBA 8, HBD 1, PSA 103, QED 0.32
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CCC(N3C[C@@H]4C[C@H]3CO4)CC2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: YTFBLTIFXGAEHJ-PMACEKPBSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.49 nM (pChEMBL 9.3100004196167)
Descriptors: MW 627.05, ALogP 3.56, HBA 10, HBD 1, PSA 119.12, QED 0.32
Compound (SMILES): Cc1c(Cc2ccccc2)nnc(N2CCC(C#N)(c3ccc(C(C)(C)O)cn3)CC2)c1C
InChIKey: MGIMZOSALMVRPI-UHFFFAOYSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 441.58, ALogP 4.37, HBA 6, HBD 1, PSA 85.93, QED 0.63
Compound (SMILES): CCn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCC(O)CC3)n2)cn1
InChIKey: SEWSVZNGUQAMFX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 3.47 nM (pChEMBL 8.460000038146973)
Descriptors: MW 389.46, ALogP 2.66, HBA 8, HBD 1, PSA 84.89, QED 0.58
Compound (SMILES): NC(=O)c1ccc2nc(C3C4Oc5ccc(Oc6ccnc7c6CCC(=O)N7)cc5C43)[nH]c2c1
InChIKey: ANQJLVLLYDBMBE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 453.46, ALogP 3.38, HBA 6, HBD 3, PSA 132.22, QED 0.43
Compound (SMILES): CC(=O)Nc1cccc(O[C@H]2c3ccccc3C[C@@H]2N2CCCC2)c1
InChIKey: UBOGPGCPMSEBJS-SFTDATJTSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Inhibition of CYP2D6 in pooled human hepatic microsomes using dextromethorphan substrate in presence of NADPH
Measurement: IC50 = 640 nM (pChEMBL 6.190000057220459)
Descriptors: MW 336.43, ALogP 3.79, HBA 3, HBD 1, PSA 41.57, QED 0.92
Compound (SMILES): Cc1ccc(-n2nc(-c3ccc(S(=O)(=O)N(C)C)cc3)cc2NC(=O)c2cnn3cccnc23)cc1
InChIKey: NFMZXNNKBUUGPK-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 501.57, ALogP 3.39, HBA 8, HBD 1, PSA 114.49, QED 0.38
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@H]1CSSC[C@@H](C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)C(C)(C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2ccc(O)cc2)NC(=O)CNC1=O)C(C)C)C(C)C
InChIKey: VGNYNIHOZOXROE-DWNZDGQOSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 2433.89
Compound (SMILES): CC(C)(C)c1ccc(CCc2c(C(=O)O)[nH]c(=O)[nH]c2=O)cc1.NCCO
InChIKey: FOTUOOKSBVWMQM-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Inhibition of human dihydroorotate dehydrogenase assessed as reduction in orotate production using L-dihydroorotate as substrate measured after 20 mins by spectrophotometric method
Measurement: Ki = 8400 nM (pChEMBL 5.079999923706055)
Descriptors: MW 316.36, ALogP 1.84, HBA 3, HBD 3, PSA 103.02, QED 0.8
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(S(=O)(=O)C(F)F)cc3)n2)CCC(Nc2ccccc2)CC1
InChIKey: UTAVQTXTWKRVCH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 528.59, ALogP 4.39, HBA 8, HBD 3, PSA 142.9, QED 0.37
Compound (SMILES): Cc1nccc(-c2ccc(C[C@H](NC(=O)[C@@H]3Cc4cc5c(cc4CN3C(=O)c3ccccc3)O[C@@H](c3ccc(OCCC(C)(C)C)cc3)CO5)C(=O)O)cc2)c1C
InChIKey: CTZKSPHCDYFBMK-IWJNXRQDSA-N
Target: Glucagon-like peptide 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43220
Assay: cAMP Functional Assay: The efficacy of GLP-1 receptor agonists was studied in a cAMP functional assay using HEK-293 Cells expressing the cloned human GLP-1 receptor. GLP-1-expressing cells (10,000-20,000 cells per 0.1 mL) were plated in 96-well plates in Dulbecco's modified eagles media (DMEM) containing 10% fetal bovine serum, 2 mg/mL G418 and penicillin-streptomycin. Following overnight incubation of cells, media was removed, and compounds (at concentrations ranging from 0.0001 to 100 μM) were added to monolayer cells in Iscove's modified dulbecco's medium (IMDM), 100 μM RO 20-1724 PDE inhibitor, 0.1% BSA, 2% DMSO in a final volume of 100 μL, and incubated for 30 min at 37° C. 95%02, 5% CO2 in a humidified incubator. Alternatively, cells were harvested and combined with compounds in suspension using the buffer and protocol described above. cAMP was quantified using a homogenous time-resolved fluorescence detection system (cAMP dynamic, CIS bio International).
Measurement: EC50 = 141 nM (pChEMBL 6.849999904632568)
Descriptors: MW 767.92, ALogP 8.07, HBA 7, HBD 2, PSA 127.29, QED 0.14
Compound (SMILES): Cc1ccc(C(=O)N2CC(F)(c3ccc(C#N)cc3)C2)cc1-c1[nH]c(C2CCOCC2)nc1C
InChIKey: HWTYXTGUJWOBHS-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 458.54, ALogP 4.78, HBA 4, HBD 1, PSA 82.01, QED 0.61
Compound (SMILES): CCCC/C=C/c1ccc2c(NCc3ccc(NC(=O)c4ccc(F)cc4)cc3)nc(N(C)C)nc2c1
InChIKey: ZBNFPOPNAPVGTL-BQYQJAHWSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf33.13 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 429 nM (pChEMBL 6.369999885559082)
Descriptors: MW 497.62, ALogP 6.9, HBA 5, HBD 2, PSA 70.15, QED 0.23
Compound (SMILES): CCS(=O)(=O)N(CCOC(C)(C)C)c1sc2ccccc2c1C
InChIKey: UXMYDPWTHOGIBZ-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 = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 355.53, ALogP 4.18, HBA 4, HBD 0, PSA 46.61, QED 0.78
Compound (SMILES): Cn1cc(-c2cc(C(N)=O)nc3cc(Cc4cc(C5CC5)nc(C(F)(F)F)c4)ccc23)cn1
InChIKey: KIHRXNGLVNMIHC-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 451.45, ALogP 4.62, HBA 5, HBD 1, PSA 86.69, QED 0.48
Compound (SMILES): CC(C)c1ccc2c(Nc3cc(C(=O)Nc4ccc(-c5c[nH]c([C@@H]6CCCN6C(=O)Cc6ccccc6)n5)cc4)ccc3Sc3ccc(N)cc3)ncnc2n1
InChIKey: YYGSTGPWNOLXKG-LHEWISCISA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition Assay: The inhibitory effects of the compounds of the invention on HCV replication can be determined by measuring activity of the luciferase reporter gene. For example, replicon-containing cells can be seeded into 96 well plates at a density of 5000 cells per well in 100 μl DMEM containing 5% FBS. The following day compounds can be diluted in dimethyl sulfoxide (DMSO) to generate a 200× stock in a series of eight half-log dilutions. The dilution series can then be further diluted 100-fold in the medium containing 5% FBS. Medium with the inhibitor is added to the overnight cell culture plates already containing 100 μl of DMEM with 5% FBS. In assays measuring inhibitory activity in the presence of human plasma, the medium from the overnight cell culture plates can be replaced with DMEM containing 40% human plasma and 5% FBS. The cells can be incubated for three days in the tissue culture incubators and are then lysed for RNA extraction.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 759.94, ALogP 9.17, HBA 9, HBD 4, PSA 154.81, QED 0.09
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2[C@H](c2cccc(S(=O)(=O)NCCOCCOCCNC(=O)CCC(CCC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)(CCC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)NC(=O)NC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO)c2)C1
InChIKey: MBBHFKPMGSMTNM-JTFILVBRSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 56.23 nM (pChEMBL 7.25)
Descriptors: MW 1907.78
Compound (SMILES): COc1cc(N2CCOCC2)ccc1Nc1nc2c(c(Nc3ccc(F)c(F)c3)n1)C(C)(C)CN2
InChIKey: HCVKKXDEQZMTLX-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 482.54, ALogP 4.79, HBA 8, HBD 3, PSA 83.57, QED 0.46
Compound (SMILES): CC(=O)c1ccc(OCc2ccc(Oc3cccc(-c4nn[nH]n4)c3)cc2)c(C)c1O
InChIKey: QIADWIHDXWIEST-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Positive allosteric modulation of recombinant human mGlu2 receptor expressed in hamster AV12 cells expressing EAAT1 assessed as potentiation of glutamate-induced intracellular calcium level measured after 24 to 48 hrs by FLIPR assay
Measurement: EC50 = 293.5 nM (pChEMBL 6.53000020980835)
Descriptors: MW 416.44, ALogP 4.45, HBA 7, HBD 2, PSA 110.22, QED 0.43
Compound (SMILES): Cc1ccc2nc(NC(=O)Nc3ccccc3N3CC4(CCN(CC(C)(C)C)CC4)c4c(Cl)ccc(O)c43)sc2c1
InChIKey: DYZBVGMXEYNVJC-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 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 uL 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. About 40 uL of the aqueous soluttion was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKERSM (Amersham) Image Reader. Dose-response curves (IC50) were fit by non-linear regression (Toolset an in house data processing program) and binding constants (Ki) calculated using the Cheng-Prusoff relationship (Ki=IC50/(1+L/Kd) in which a Kd for 2MeS-ADP to the P2Y1 receptor was determined to be 14 nM.
Measurement: Ki = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 590.19, ALogP 8.14, HBA 6, HBD 3, PSA 80.73, QED 0.22
Compound (SMILES): Cn1cc(-c2cccc(Nc3nn(C4(CC#N)CCN(C(=O)OCC(F)(F)F)CC4)cc3C(N)=O)c2)cn1
InChIKey: BNKQHKDVXKUESH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 530.51, ALogP 3.53, HBA 9, HBD 2, PSA 144.09, QED 0.48
Compound (SMILES): Cc1ccc(-c2ccc(O[C@H]3C[C@@H](C(=O)N[C@@H](Cc4ccc(OCc5ccc(Br)cc5)cc4)C(=O)NS(=O)(=O)c4ccc(Cl)c([N+](=O)[O-])c4)N(C(=O)OC(C)(C)C)C3)cc2)cc1
InChIKey: HYQSADOTBKLWKO-FOQZPGKDSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Inhibition of 5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG binding to Mcl-1 (unknown origin) preincubated for 30 mins followed by FAM-labeled peptide addition measured after 20 mins by fluorescence polarization assay
Measurement: Ki = 460 nM (pChEMBL 6.340000152587891)
Descriptors: MW 948.29, ALogP 8.55, HBA 10, HBD 2, PSA 183.48, QED 0.08
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CNCCO5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: LGHVXUGTPXYGCJ-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 869.44, ALogP 7.22, HBA 12, HBD 4, PSA 184.06, QED 0.07
Compound (SMILES): CN(c1cccc(CO)c1)c1nc(Nc2cc(N3CCOCC3)cc(S(C)(=O)=O)c2)ncc1F
InChIKey: VWEBEVMRPSIULB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase PAK 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13153
Assay: Inhibition of PAK1 phosphorylation in human MCF10A cells
Measurement: IC50 = 14000 nM (pChEMBL 4.849999904632568)
Descriptors: MW 487.56, ALogP 2.86, HBA 9, HBD 2, PSA 107.89, QED 0.52
Compound (SMILES): CNc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)ccc2[nH]1
InChIKey: WCKJQTNVFAFOSR-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 384.44, ALogP 3.45, HBA 4, HBD 3, PSA 81.25, QED 0.5
Compound (SMILES): COCCNc1ccc2c(c1)-c1cc(Cl)c3cccnc3c1NS2(=O)=O
InChIKey: MBHFZWBLUDGHPK-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 1980 nM (pChEMBL 5.699999809265137)
Descriptors: MW 389.86, ALogP 3.73, HBA 5, HBD 2, PSA 80.32, QED 0.66
Compound (SMILES): Cc1cc(C)c(CNC(=O)c2cc(-c3ccc(CN4CCOCC4)cc3)cc3c2C(C)CN3C2CCOCC2)c(=O)[nH]1
InChIKey: AGFJSOIBLBIMSF-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92800
Assay: Inhibition of human EZH1 complex using histone H3 as substrate after 1 hr in presence of 3H-SAM by filter paper detection analysis
Measurement: IC50 = 3340 nM (pChEMBL 5.480000019073486)
Descriptors: MW 570.73, ALogP 4.52, HBA 6, HBD 2, PSA 86.9, QED 0.43
Compound (SMILES): COc1cc(C(=O)N2CCC(C#N)CC2)ccc1Nc1cc2cc(-c3cnn(CC(C)(C)O)c3)ccc2cn1
InChIKey: DSUKOKLOCZIIRB-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: The enzyme reaction (total volume 10 μl) was carried out in black 384-well low volume plates containing full length MPS1 (12.5 nM or 3 nM), fluorescent labelled peptide [known as H236, which has the sequence: 5FAM-DHTGFLTEYVATR-CONH2](5 μM), ATP (10 μM), either DMSO (1% v/v) or the test compound (in the range 0.25 nM-100 μM in 1% DMSO) and assay buffer (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovandate, 10 μM MgCl2, 1 μM DTT, Roche protease inhibitor). The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μl) containing 20 mM EDTA, 0.05% (v/v) Brij-35, in 0.1M HEPES-buffered saline (Free acid, Sigma, UK). The plate was read on a Caliper EZ reader II (Caliper Life Sciences).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 524.63, ALogP 5, HBA 8, HBD 2, PSA 116.3, QED 0.35
Compound (SMILES): Cn1cc(-c2cnc(N)c(-c3ccc(C(=O)NCc4nccs4)c(F)c3)c2)cn1
InChIKey: QVHBTNANTJRMRE-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 352 nM (pChEMBL 6.449999809265137)
Descriptors: MW 408.46, ALogP 3.26, HBA 7, HBD 2, PSA 98.72, QED 0.53
Compound (SMILES): CC1(O)CCN(c2ccc(C(=O)Nc3ccc(OC(F)(F)F)cc3)cc2-c2cncnc2)C1
InChIKey: AGHYQAWGSYEFCZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radio ABL1 (64-515) Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 uL of the compound pre-diluted with 10 uL of ATP (20 uM ATP with 0.1 uCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 uL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 mM at room temperature, the reaction was stopped by the addition of 50 uL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3x with 0.5% H3PO4, followed by addition of 30 uL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 < 3 nM (pChEMBL 8.52299976348877)
Descriptors: MW 458.44, ALogP 4.26, HBA 6, HBD 2, PSA 87.58, QED 0.59
Compound (SMILES): CC(C)Oc1ccc(C#N)cc1-c1nc(-c2cccc3c2CC[C@H]3N)no1
InChIKey: JBLSPSZGYTUASF-GOSISDBHSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 > 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 360.42, ALogP 4.01, HBA 6, HBD 1, PSA 97.96, QED 0.75
Compound (SMILES): Fc1ncc(C2CC3CCC2N3)cc1-c1cccnc1
InChIKey: YNZDJAQJOUFZIH-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: [125I]Iodo-MLA Binding Assay: Adult male rat cerebral cortices (Pel-Freez Biologicals, Rogers, Ark.) were homogenized (polytron) in 39 volumes of ice-cold 50 mM Tris buffer (assay buffer; pH 7.4 at 4° C.) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2. The homogenate was centrifuged at 35,000 g for 10 min at 4° C. and the supernatant discarded. The pellet was resuspended in the original volume of buffer and the wash procedure repeated twice more. After the last centrifugation step, the pellet was resuspended in one-tenth the original homogenization volume and stored at −80° C. until needed. Triplicate samples were run in 1.4-mL polypropylene tubes (Matrix Technologies Corporation, Hudson, N.H.). Briefly, in a final volume of 0.5 mL, each assay sample contained 3 mg wet weight rat cerebral cortex (added last), 40-50 pM [125I]MLA and 50 nM final concentration of test compound dissolved in buffer containing 10% DMSO, giving a final DMSO concentration of 1%. Total and nonspecific binding were deter
Measurement: Ki = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 269.32, ALogP 2.89, HBA 3, HBD 1, PSA 37.81, QED 0.85
Compound (SMILES): Cc1nn(Cc2ccc(-c3nc4c(C)cccc4[nH]3)cc2)c(C)c1CC(=O)O
InChIKey: ZXMPZSBDTVCPNT-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Binding Assay: The CRTH2 receptor binding assay is performed in a scintillation proximity assay (SPA) format with the radioligand [3H]-PGD2 (Perkin Elmer, NET616000MC). CHO-K1-hCRTH2 cell membranes are again homogenized by passing through a single use needle (Terumo, 23G) and diluted in SPA incubation buffer in suitable concentrations (0.5-10 ug protein/well). The SPA assay is set up in 96 well microtiter plates (Perkin Elmer, CatNo. 6005040) in SPA incubation buffer with a final volume of 200 ul per well and final concentration of 50 mM Tris-HCl, 10 mM MgCl2, 150 mM NaCl, 1 mM EDTA pH 7.4, 0.1% bovine serum albumin). The SPA assay mixture contains 60 ul of the membrane suspension, 80 ul of Wheat Germ Agglutinin coated PVT beads (GE Healthcare, RPNQ-0001, 0.3 mg/well), 40 ul of [3H]-PGD2 diluted in SPA buffer to a final concentration of 1 nM (50 000 dpm) and 20 ul of the test compound (dissolved in dimethylsulfoxid).
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 374.44, ALogP 4.03, HBA 4, HBD 2, PSA 83.8, QED 0.55
Compound (SMILES): COC/C(CN(c1cc(OC)cc(OC)c1)c1ccc2ncc(-c3cnn(C)c3)nc2c1)=N/O
InChIKey: YIDCCLXYFLNYHA-OGLMXYFKSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 389.05 nM (pChEMBL 6.409999847412109)
Descriptors: MW 462.51, ALogP 3.66, HBA 10, HBD 1, PSA 107.12, QED 0.23
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2CN(Cc3ccccc3)CN(CCc3ccccc3)C2=O)cn1
InChIKey: WTSYMPIUHOIKKX-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 = 2640 nM (pChEMBL 5.579999923706055)
Descriptors: MW 470.58, ALogP 4.44, HBA 6, HBD 0, PSA 70.64, QED 0.38
Compound (SMILES): N#CCN1CN(C2CCCCC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: ZKPOLKXRXJQNCH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 309.37, ALogP 2.64, HBA 4, HBD 1, PSA 76.02, QED 0.87
Compound (SMILES): CS(=O)(=O)c1ccc(Nc2nn(C3(CC#N)CCC4(CC3)OCCO4)cc2C(N)=O)cc1
InChIKey: VDKBVSZYIVNJCX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 459.53, ALogP 2.06, HBA 9, HBD 2, PSA 149.33, QED 0.67
Compound (SMILES): Cc1cc2cc(-c3ccc(CO)cc3)c(NC(=O)c3cnn4cccnc34)cc2[nH]1
InChIKey: NBSYWAHBCJDKFY-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 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 397.44, ALogP 3.93, HBA 5, HBD 3, PSA 95.31, QED 0.43
Compound (SMILES): Cn1cc(-c2nc(C(=O)Nc3cnn(C)c3[C@@H]3CC[C@@H](N)[C@@H](F)CO3)cs2)c(C2CC2)n1
InChIKey: DRGYGYVAHXPOPD-VBQJREDUSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO. PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000® (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
Measurement: Ki = 0.253 nM (pChEMBL 9.600000381469727)
Descriptors: MW 459.55, ALogP 2.92, HBA 9, HBD 2, PSA 112.88, QED 0.61
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: SUVAYPGSTAPZTQ-FXWKPZOOSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 98 nM (pChEMBL 7.010000228881836)
Descriptors: MW 2386.79
Compound (SMILES): COc1cc(/C=C/C(=O)CC(=O)/C=C/c2ccc(O)c(CO)c2)ccc1O
InChIKey: UAVJPMNEDOIHEI-YPCIICBESA-N
Target: Nuclear factor NF-kappa-B p105 subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P19838
Assay: Cellular Firefly Luciferase Assay (NFkB): The NF-kB activities of curcumin and analogs were determined by a cellular firefly luciferase assay. This assay utilized a commercially available cell line (Panomics 293T-luc cellular assay) developed for screening inhibitors of NF-kB. This cell line is stably transfected with a luciferase reporter controlled by an NF-kB dependent promoter. The cell is stimulated with tumor necrosis factor alpha (TNFalpha) which activates NF-kB. NF-kB then binds to one of six promoter regions on the cell's DNA leading to the production of a luciferase enzyme. Luciferin is added to the cell lysates and the luciferase enzyme catalyzes a cleavage of luciferin leading to the emission of light.
Measurement: IC50 = 8200 nM (pChEMBL 5.090000152587891)
Descriptors: MW 368.39, ALogP 2.85, HBA 6, HBD 3, PSA 104.06, QED 0.49