lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccccc1/C=C/C(=O)n1nnc2ccccc21
InChIKey: ZSKHPLGLSNVUGA-ZHACJKMWSA-N
Target: Protein-glutamine gamma-glutamyltransferase 2 (SINGLE PROTEIN), Cavia cutleri
UniProt: P08587
Assay: Enzyme Inhibition Assay: For enzymology studies of these compounds, recombinant guinea pig liver TGase was expressed in Escherichia coli and effectively purified (Gillet, S. M. F. G. et al J. N., Prot. Exp. & Purif. 2004, 33, 256). In addition to being easy to obtain in excellent yield and solubility, guinea pig liver TGase was chosen because it shows 80% homology with human tissue TGase (Aeschlimann, D.; Paulsson, M., Throm. Haemost. 1994, 71, 402) and may thus serve as a model for the evaluation of inhibitors of potential therapeutic utility.The IC50 values of synthetic analogues 14a-38a were determined from inhibition of the reaction of 54.4 mM of the chromogenic TGase substrate N-Cbz-Glu(Î³-p-nitrophenyl ester)Gly with 0.010 U of recombinant guinea pig liver TGase as previously reported (Leblanc, A.; Gravel, C.; Labelle, J.; Keillor, J. W. Biochemistry 2001, 40, 8335) and described in detail in the Materials section below. The mode of inhibition was determined for the representative lead compound.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 279.3, ALogP 2.79, HBA 5, HBD 0, PSA 57.01, QED 0.69
Compound (SMILES): CNC(=O)c1c(C(=O)N2CCOCC2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CO4)cc12
InChIKey: QTIGUBZMHDBTBG-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhbition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 232 nM (pChEMBL 6.630000114440918)
Descriptors: MW 633.66, ALogP 3.83, HBA 9, HBD 1, PSA 136.21, QED 0.31
Compound (SMILES): O=C(/C=C/c1ccc(Cl)cc1)NC[C@@H]1CCN(CC(c2ccccc2)c2ccccc2)C(=O)[C@H](CCC(=O)N2CCCCC2)N1
InChIKey: VCPOUCICPQBNHX-YZLBFCITSA-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 < 100 nM (pChEMBL 7)
Descriptors: MW 627.23, ALogP 5.65, HBA 4, HBD 2, PSA 81.75, QED 0.27
Compound (SMILES): COC(=O)CC(=O)Nc1nc2ccc(NC(=O)c3cc(Br)c(Br)[nH]3)cc2s1
InChIKey: AFQMMWARZCTCIV-UHFFFAOYSA-N
Target: DNA gyrase subunit B (SINGLE PROTEIN), Staphylococcus aureus
UniProt: P0A0K8
Assay: Inhibition of Staphylococcus aureus DNA gyrase B ATP binding site assessed as reduction in supercoiling of relaxed pNO1 DNA after 30 mins in presence of biotinylated oligonucleotide by sybrGOLD staining based fluorescence assay
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 516.17, ALogP 3.9, HBA 6, HBD 3, PSA 113.18, QED 0.35
Compound (SMILES): O=C(O)c1ccc(C2(NC(=O)C3CC4(CCN3Cc3cccc(C(F)(F)F)c3)CC4)CC2)cc1
InChIKey: AUYTYEPFKREYLH-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 472.51, ALogP 4.95, HBA 3, HBD 2, PSA 69.64, QED 0.62
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ccccc1Nc1nnc(-c3ccc(C(F)(F)F)cn3)s1)c1c(O)ccc(Cl)c12
InChIKey: AAUMEBRGOQIAOL-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 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 629.15, ALogP 8.25, HBA 8, HBD 2, PSA 77.41, QED 0.23
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CC4CCC(C3)O4)nc(-c3ccc(NC(=O)NCC4CCN(C)CC4)c(F)c3)c2C1=O
InChIKey: KTUPAWYQQVDDEX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The test detects the phosphorylation of the substrate GFP-4EBP1 using a terbium-labelled antibody that is specific for the phospho-Thr46 epitope. Binding of the Tb-antibody to the phosphorylated GFP peptide allows the transfer of energy from Tb, excited at 340 nm, to GFP with an increase in the fluorescence emission of the GFP at 520 nm. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 ul. To 1 ul of inhibitor in 100% DMSO are added (final concentrations) 400 nM of GFP-4EBP1 (Invitrogen PV4759), 8 uM of ATP, 200 ng/ml of enzyme (human recombinant aa mTOR 1360-2549, Invitrogen PV4753) in a buffer of HEPES 50 mM pH 7.5, EGTA 1 mM, MnCl2 10 mM, BSA 0.1 mg/ml, glycerol 0.5%, NV-10 0.1 mg/ml, DTT 2 mM. The mixture is incubated for 30 minutes at room temperature. 10 ul of solution are taken up and the reaction is quenched by adding 10 ul of Tb-antibody solution (Invitrogen PV4757)/EDTA.
Measurement: IC50 = 2120 nM (pChEMBL 5.670000076293945)
Descriptors: MW 595.72, ALogP 3.75, HBA 8, HBD 2, PSA 112.16, QED 0.52
Compound (SMILES): Clc1c(N2CC(c3ccc4ccccc4n3)C2)nc2c(N3CCOCC3)ncc(-c3ccc(N4CCNCC4)nc3)n12
InChIKey: LTRJIIUKZHMZLT-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay rPDE10: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi 3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter and results were expressed as cpm. For blank values the enz
Measurement: IC50 = 871 nM (pChEMBL 6.059999942779541)
Descriptors: MW 582.11, ALogP 3.84, HBA 10, HBD 1, PSA 86.95, QED 0.33
Compound (SMILES): CN(C)c1cc(NC2CC2)n2ncc(/C=C3\SC(=O)NC3=O)c2n1
InChIKey: MYRPEVBCFHHODE-YHYXMXQVSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 = 875.9 nM (pChEMBL 6.059999942779541)
Descriptors: MW 344.4, ALogP 1.69, HBA 8, HBD 2, PSA 91.63, QED 0.82
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCN(c2cncc(C(F)(F)F)c2)CC1
InChIKey: QFAZMYOHCSISHN-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 = 1 nM (pChEMBL 9)
Descriptors: MW 487.46, ALogP 4.19, HBA 7, HBD 2, PSA 112.86, QED 0.5
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCN(C(=O)Cn2ccnc2)CC1
InChIKey: TXZBBDCGMQQTGN-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 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 451.47, ALogP 1.39, HBA 9, HBD 2, PSA 147.75, QED 0.52
Compound (SMILES): Cc1nnc(-c2cc(C(=O)N3CCC(c4ccc(C#N)c(C)c4)CC3)c(C)cc2C2CCC2)[nH]1
InChIKey: SUQRBLFRHSSGCF-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 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 453.59, ALogP 5.56, HBA 4, HBD 1, PSA 85.67, QED 0.56
Compound (SMILES): S=C(NNc1nncc2ccccc12)Nc1ccc(Cl)cc1
InChIKey: PBFFTWJHTSVERV-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR-2 (unknown origin) using poly (Glu, Tyr) 4:1 as substrate after 1 hr by ELISA
Measurement: IC50 = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 329.82, ALogP 3.6, HBA 4, HBD 3, PSA 61.87, QED 0.5
Compound (SMILES): Cc1c(N(CCOC(C)(C)C)S(C)(=O)=O)sc2ccccc12
InChIKey: DPCDWYJUECSXSO-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 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 341.5, ALogP 3.79, HBA 4, HBD 0, PSA 46.61, QED 0.83
Compound (SMILES): CC1(C)CC=C(c2cc(C3(C(=O)O)CCOCC3)ccc2NC(=O)c2cc(C#N)c[nH]2)CC1
InChIKey: CJLKFMALHYRHBJ-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Mus musculus
UniProt: P09581
Assay: Fluorescence Polarization Competition Immunoassay: An autophosphorylation, fluorescence polarization competition immunoassay was used to determine the potency for c-fms inhibition exhibited by selected compounds of Formula I. The assay was performed in black 96-well microplates (LJL BioSystems). The assay buffer used was 100 mM 4-(2-hydroxyethyl)piperazine 1-ethanesulfonic acid (HEPES), pH 7.5, 1 mM 1,4-dithio-DL-threitol (DTT), 0.01% (v/v) Tween-20. Compounds were diluted in assay buffer containing 4% dimethylsulfoxide (DMSO) just prior to the assay. To each well, 5 μL of compound were added followed by the addition of 3 μL of a mix containing 33 nM c-fms (Johnson & Johnson PRD) and 16.7 mM MgCl2 (Sigma) in assay buffer. The kinase reaction was initiated by adding 2 μL of 5 mM ATP (Sigma) in assay buffer. The final concentrations in the assay were 10 nM c-fms, 1 mM ATP, 5 mM MgCl2, 2% DMSO. Control reactions were ran in each plate: in positive and negative control wells, assay buffer (made 4% in DMSO) was substituted for the compound; in addition, positive control wells received 1.2 μL of 50 mM ethylenediaminetetraaceticacid (EDTA). The plates were incubated at room temperature for 45 min. At the end of the incubation, the reaction was quenched with 1.2 μL of 50 mM EDTA (EDTA was not added to the positive control wells at this point; see above). Following a 5-min incubation, each well received 10 μL of a 1:1:3 mixture of anti-phosphotyrosine antibody, 10×, PTK green tracer, 10× (vortexed), FP dilution buffer, respectively (all from PanVera, cat. #P2837). The plate was covered, incubated for 30 min at room temperature and the fluorescence polarization was read on the Analyst. The instrument settings were: 485 nm excitation filter; 530 nm emission filter; Z height: middle of well; G factor: 0.93. Under these conditions, the fluorescence polarization values for positive and negative controls were approximately 300 and 150, respectively, and were used to define the 100% and 0% inhibition of the c-fms reaction.
Measurement: IC50 = 6.4 nM (pChEMBL 8.1899995803833)
Descriptors: MW 447.54, ALogP 4.86, HBA 4, HBD 3, PSA 115.21, QED 0.61
Compound (SMILES): CN(c1ccc(C#N)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: ZZWSVWLKMJYUAE-CYBMUJFWSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z'-LYTE™ Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE™ Tyr 6 peptide or Z-LYTE™ Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100M, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer™) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 = 0.055 nM (pChEMBL 10.260000228881836)
Descriptors: MW 319.37, ALogP 1.94, HBA 6, HBD 1, PSA 84.73, QED 0.79
Compound (SMILES): Cc1cc(NC(=O)c2ccc3cc4n(c3c2)CC(C)CNC4=O)no1
InChIKey: UBMSIZSBVPTYOC-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 338.37, ALogP 2.57, HBA 5, HBD 2, PSA 89.16, QED 0.75
Compound (SMILES): Cc1onc2c1CC1(CC2)Cc2ccc(-c3cccc(C#N)c3)cc2C12N=C(N)N(C)C2=O
InChIKey: KETJJWVTEIRQBP-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.434): Inhibitory activity of compounds was assessed by a fluorescence quench assay of BACE activity using commercially available substrate HiLyte Fluor 488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-(QXLT 520)-OH (AnaSpec, San Jose, Calif.) and truncated human beta-secretase (residues 1-458, His6-tagged at the C-terminus) expressed in insect cells D. melanogaster S2 using a baculovirus expression system (Mallender et al., Characterization of recombinant, soluble beta-secretase from an insect cell expression system., Mol Pharmacol 59:619-26, 2001). The assay was performed at room temperature in 96-well white opaque Optiplates aque Optiplates (PerkinElmer, Waltham, Mass.) in a total volume of 200 ul of the incubation mixture containing 50 mM sodium acetate buffer, pH 4.5, 0.4 uM FRET substrate, 2.4 nM enzyme, 5% DMSO, and 0.05% Brij-35. The tested compounds were serially diluted in DMSO and pre-incubated with the substrate. The reaction was started by addition of enzyme.
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 437.5, ALogP 3.24, HBA 6, HBD 1, PSA 108.51, QED 0.63
Compound (SMILES): CCCCc1nn(C)c(C)c1NS(=O)(=O)c1ccc(CCCCC2CCN(C)CC2)cc1
InChIKey: UEIHUROJPODEIZ-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 460.69, ALogP 4.93, HBA 5, HBD 1, PSA 67.23, QED 0.48
Compound (SMILES): Cc1ccc(Oc2ccnc(NC(=O)C3CC3)c2)cc1NC(=O)Nc1ccc(Cl)c(C(F)(F)F)c1
InChIKey: PGLFODISHUVFOO-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition Assay: [Experimental method]: Use Lance@Ultra Ulight™-TK assay kit from PerKinElmer to evaluate the inhibitory activity of compounds at KDR kinase. [Instrument]: PerKinElmer's ENVISION plate reader. [Materials]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 0.25 mM EGTA, 2 mM DTT, 0.01% Tween 20, 10 mM Mg2+, 0.5 mM Mn2+), KDR kinase (790-1356 AA, Crown Bioscience), KDR kinase substrate (PerKinElmer catalogue # TRF0127-M), Lance@Eu-W1024-anti-phosphotyrosine (PT66) (PerKinElmer, catalogue #AD0068), ATP (Invitrogen), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix KDR (final concentration: 20 nM) and compound (final DMSO: 0.5%), pre-incubate for 20 min at 30° C.; then add ATP (final concentration: 90 μM) and the substrate (final concentration 50 μM). React for 2 hrs at 30° C. After the reaction, add antibody and reaction for 60 min at 30° C. Read the plate (615 nm, 665 nm), calculate the ratio of the value at 665 nm vs 615 nm and analyze the data.
Measurement: IC50 = 12.4 nM (pChEMBL 7.909999847412109)
Descriptors: MW 504.9, ALogP 6.85, HBA 4, HBD 3, PSA 92.35, QED 0.34
Compound (SMILES): Cc1ccoc1C(=O)Nc1cccc(C#Cc2cncc(C(=O)N=S(=O)(CCCO)CCCO)c2)c1
InChIKey: HAXLIGAXLBLFAT-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 ul of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 ul of 0-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 ul of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 509.58, ALogP 3.01, HBA 7, HBD 3, PSA 142.09, QED 0.38
Compound (SMILES): Cc1c(N(CCC(F)(F)F)S(=O)(=O)c2cccc(C#N)c2)sc2ccccc12
InChIKey: XHWULVDWVHCLFT-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 = 18.8 nM (pChEMBL 7.730000019073486)
Descriptors: MW 424.47, ALogP 5.23, HBA 4, HBD 0, PSA 61.17, QED 0.56
Compound (SMILES): CN(C)C(=O)CN1CCC(CCOc2ccc(-c3nc(C#N)nc4c3ccn4CC3CCCO3)cc2C(F)(F)F)CC1
InChIKey: UZGLXBBPPOKCPH-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 = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 584.64, ALogP 4.74, HBA 8, HBD 0, PSA 96.51, QED 0.36
Compound (SMILES): Brc1cnc(Nc2cccc(Oc3ccccc3)c2)nc1Nc1cc(C2CC2)n[nH]1
InChIKey: SPKFIYWRRHDPHP-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 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 463.34, ALogP 6.12, HBA 6, HBD 3, PSA 87.75, QED 0.31
Compound (SMILES): O=C(Nc1ccc(C2=CCN(C(=O)C3CC3(F)F)CC2)cc1)N1Cc2ccncc2C1
InChIKey: PAPZZYCWTHGANS-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 = 2.34 nM (pChEMBL 8.630000114440918)
Descriptors: MW 424.45, ALogP 3.9, HBA 3, HBD 1, PSA 65.54, QED 0.81
Compound (SMILES): CC1(C)C(=O)[C@H](C/C=C\CCCC(=O)NCCO)[C@@H](/C=C/C(O)Cc2cc3ccccc3s2)[C@@H]1O
InChIKey: QVLDQALHAJMTPC-IDIARNOWSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Measurement: IC50 = 4154 nM (pChEMBL 5.380000114440918)
Descriptors: MW 499.67, ALogP 3.79, HBA 6, HBD 4, PSA 106.86, QED 0.26
Compound (SMILES): Cc1cccc(C)c1N1C[C@@](CN2CCC(F)(F)C2)(C(=O)Nc2cc(C3CC3)cc(C(F)(F)F)c2)[C@H](C)C1=O
InChIKey: LLEDNXHJLJYPQC-XHCCPWGMSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 549.58, ALogP 6.15, HBA 3, HBD 1, PSA 52.65, QED 0.44
Compound (SMILES): CCCN1CCC(c2cc(OC)c(Nc3nccc(-c4c(-c5ccc(OCC)c(C(=O)Nc6c(F)cccc6F)c5)nc5ccccn45)n3)cc2C)CC1
InChIKey: YMSGHIRZTKWOJL-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 731.85, ALogP 9.04, HBA 9, HBD 2, PSA 105.91, QED 0.13
Compound (SMILES): Cc1ccc(C(=O)N2Cc3cc4c(cc3C[C@H]2C(=O)N[C@@H](Cc2ccc(-c3ccnc(C)c3C)cc2)C(=O)O)OC[C@H](c2ccc(OCc3ccc(Cl)c(Cl)c3)cc2)O4)s1
InChIKey: ZUELNQLNVWEIFK-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 = 81 nM (pChEMBL 7.090000152587891)
Descriptors: MW 862.83, ALogP 9.51, HBA 8, HBD 2, PSA 127.29, QED 0.13
Compound (SMILES): CC(C)(O)CNC(=O)c1ccc2cc(Oc3ccc(C(F)(F)F)nc3)ccc2n1
InChIKey: ZRPMCOWEVCOVBL-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 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 405.38, ALogP 3.94, HBA 5, HBD 2, PSA 84.34, QED 0.67
Compound (SMILES): COC(=O)CCNC1CCc2c(-c3noc(-c4ccc(OC(C)C)c(C#N)c4)n3)cccc21
InChIKey: UUWSQENZSSGIDH-UHFFFAOYSA-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 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 446.51, ALogP 4.2, HBA 8, HBD 1, PSA 110.27, QED 0.51
Compound (SMILES): COc1cc(Nc2ncnc3ccc4nc(C(N)=O)sc4c23)cc(OC)c1OC
InChIKey: DPSAJOPMKHSBLW-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: HotSpot Technology Assay: The DYRK1A and DYRK1B kinase assays to determine IC50 values were performed by Reaction Biology Corporation using HotSpot technology Worldwide website: reactionbiology.com, Malvern, PA). Kinase reaction with specific kinase/ substrate pair along with required cofactors was carried out in 20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. Purified recombinant kinase was incubated with serial 3-fold dilutions of test compounds starting at a final concentration of 10 μM. Reaction was initiated by addition of a mixture of ATP (Sigma, St. Louis MO) and 33P ATP (Perkin Elmer, Waltham MA) to a final concentration of 10 μM and was carried out at room temperature for 120 min, followed by spotting of the reaction onto P81 ion exchange filter paper (Whatman Inc., Piscataway, NJ). Unbound phosphate was removed by extensive washing of filters in 0.75% Phosphoric acid.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 411.44, ALogP 3.11, HBA 9, HBD 2, PSA 121.48, QED 0.5
Compound (SMILES): Cc1sc(NC(=O)C2=C(C(=O)O)CCC2)c(-c2nc(C3CC3)no2)c1C1CC1
InChIKey: MSBHNVBZWYOJLI-UHFFFAOYSA-N
Target: Fatty acid-binding protein, adipocyte (SINGLE PROTEIN), Homo sapiens
UniProt: P15090
Assay: Time Resolved-Fluorescence Energy Transfer (TR-FRET) Assay: Stock DMSO solutions (1.8 mM) of compounds were serially diluted 3-fold for ten concentrations with 100% DMSO (50 μM to 0.003 μM final compound concentration). 1 μl of these compound dilutions and 1 μl of Bodipy labeled fatty acid 4.5 μM in 100% DMSO (Bodipy FL C11, cat. no. D3862, Invitrogen) were sequentially pipetted in wells of 384-well black polypropylene plates (Thermo Matrix cat. no. 4344). FABP4 or FABP5 protein was then added (28 μl of 64 nM protein in 25 mM Tris pH 7.5, 0.4 mg/ml α-globulin, 1 mM DTT, 0.012% NP40, final protein concentration: 50 nM). Assay blanks contained ligand, but no protein. Neutral controls contained ligand, but no compound. After adding the detection reagent (Tb antiHis6 antibody, Columbia Biosciences, TB-110, 6 μl of a 24 nM Ab solution in 25 mM Tris pH 7.5, 0.4 mg/ml γ-globulin, final Tb antiHis6 Ab concentration: 4 nM), plates were spun one minute at 1000 rpm.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 399.47, ALogP 4.36, HBA 6, HBD 2, PSA 105.32, QED 0.75
Compound (SMILES): COC(=O)N1CCCC(c2cnc(N)c(-c3ccc(C(=O)N[C@H](CO)c4ccccc4)c(F)c3)n2)C1
InChIKey: SCSMELBGNCRFLF-LMNIDFBRSA-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 = 13.4 nM (pChEMBL 7.869999885559082)
Descriptors: MW 493.54, ALogP 3.27, HBA 7, HBD 3, PSA 130.67, QED 0.48
Compound (SMILES): O=C(Nc1ccc(CNc2nc(NCc3ccco3)nc3ccc(C(F)(F)F)cc23)cc1)c1ccc(Cl)cc1
InChIKey: WQDGHNADASGKAA-UHFFFAOYSA-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 = 1284 nM (pChEMBL 5.889999866485596)
Descriptors: MW 551.96, ALogP 7.37, HBA 6, HBD 3, PSA 92.08, QED 0.19