lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc(CN(c2sc3ccccc3c2Br)S(=O)(=O)c2ccccc2)cc1
InChIKey: MSAPUXIEMKAHAC-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 = 31.9 nM (pChEMBL 7.5)
Descriptors: MW 488.43, ALogP 6.07, HBA 4, HBD 0, PSA 46.61, QED 0.33
Compound (SMILES): NC1=N[C@](CF)(c2cc(Oc3cc(Br)cc(Cl)n3)ccc2F)C[C@@H](C(F)(F)F)O1
InChIKey: NNPNVJMRYAFUEL-BLLLJJGKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 1950 nM (pChEMBL 5.710000038146973)
Descriptors: MW 500.65, ALogP 5.26, HBA 5, HBD 1, PSA 69.73, QED 0.46
Compound (SMILES): Cc1nc2ccc(NC(=O)c3ccc4cc5n(c4c3)CCCNC5=O)cc2s1
InChIKey: OXGVHBNTFDUZND-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 = 10 nM (pChEMBL 8)
Descriptors: MW 390.47, ALogP 3.95, HBA 5, HBD 2, PSA 76.02, QED 0.54
Compound (SMILES): CC(C)(NC(=O)c1ccc(Cl)c(-c2cccc(Cl)c2)n1)c1nccs1
InChIKey: JFBCTWDPXWCUEC-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 103.8 nM (pChEMBL 6.980000019073486)
Descriptors: MW 392.31, ALogP 5.18, HBA 4, HBD 1, PSA 54.88, QED 0.67
Compound (SMILES): Cc1ccc(NC(=O)c2ccnc(C(C)(F)F)c2)cc1-c1cc(OCCOP(=O)(O)O)nc(N2CCOC[C@H]2C)c1
InChIKey: HEMZLLXLZPLKRJ-GOSISDBHSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of 1 to 324 residues truncated c-Raf (unknown origin) expressed in baculovirus infected Sf9 insect cells using full length biotinylated MEK1 ATP binding site K97R mutant as substrate preincubated for 60 mins followed by ATP addition measured after 1 hr by alpha screen assay
Measurement: IC50 = 0.1 nM (pChEMBL 10)
Descriptors: MW 592.54, ALogP 4.53, HBA 8, HBD 3, PSA 143.34, QED 0.23
Compound (SMILES): Cc1cccc(OCc2cncc(-c3cc4c(cc3F)N(C)C(=O)CC4)c2)n1
InChIKey: VPYAHOURBINDNQ-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: G402-Based Assay: Herein we identified the use of the G-402 cell line as a host cell to ectopically express (transiently or stably) enzymes of the CYP11 family. Specifically we developed stable G-402 cells expressing ectopically human CYP11B1, human CYP11B2, human CYP11A1, cynmolgus CYP11B1 or cynomolgus CYP11B2 enzyme activity. Importantly the identified cell line G-402 expresses co-factors (adrenodoxin and adrenodoxin reductase) important for the activity of the CYP11 family and no relevant enzyme activity of the CYP11 family (in comparison to H295R cells) was detected in these cells. Therefore the G-402 cell line is uniquely suited as a host cell for the ectopic expression of enzymes from the CYP11 family.Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 377.42, ALogP 4.08, HBA 4, HBD 0, PSA 55.32, QED 0.69
Compound (SMILES): CC1(C)CN(C(=O)Cn2c(=O)c(C(=O)NCc3ccc(Cl)c(Cl)c3)cc3cccnc32)C1
InChIKey: SVSFIRWBHLNJEG-UHFFFAOYSA-N
Target: DNA polymerase catalytic subunit (SINGLE PROTEIN), Human cytomegalovirus (strain AD169) (HHV-5) (Human herpesvirus 5)
UniProt: P08546
Assay: HCMV Polymerase Scintillation Proximity Assay: The assay conditions are the following: 10 mM HEPES pH 7.5, 25 mM KCl, 7.5 mM NaCl, 5 mM MgCl2, 0.2 mg BSA/mL, 1 mM TCEP, 1.5% glycerol, 2 μM dTTP, 90 nM 3H-dTTP (minor variations in concentration are possible due to specific activity of stock), 26 nM Poly(dA)/190 nM BioTEG-dT19; 5% DMSO. The assay volume is 30 μL. Each reagent is added at a 3× conc.: 10 μL a+10 μL b+10 μL c; a: compound diluted in 10 mM Hepes pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM TCEP with 15% DMSO; b: enzyme (GST-UL54) in 10 mM Hepes pH 7.5, 25 mM KCl, 5 mM MgCl2, 22.5 mM NaCl, 4.5% Glycerol, 0.6 mg BSA/mL, 1 mM TCEP w/o DMSO (1 nM GST-UL54 is present in the assay); c: substrate in 10 mM Hepes pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM TCEP, 6 μM dTTP, 270 nM 3H-dTTP, 78 nM Poly(dA)/570 nM BioTEG-dT19 w/o DMSO. To perform the assay, 10 μL enzyme solution is added to columns 2-12 and 14-24. The enzyme is substituted by the blank solution (b solution without enzyme) for columns 1 and 13 (blanks). The plate is centrifuged at 200×g for 30 sec. 10 μL of substrate solution is added to each well. The plate is centrifuged at 200×g for 30 sec. The plates are incubated at 37 °C. for 40 min. To stop the reaction, 25 μL of SPA beads (5 mg/mL in 0.5 M EDTA) are added and mixed by pipetting up and down. The plates are incubated at RT for at least 15 min. 35 μL of 5 M CsCl is added to the bottom of each well. TopSeal is applied to the plate and the plate is incubated at RT for at least 90 min prior to reading. The signal is read on TopCount plate reader (Perkin-Elmer) or equivalent.
Measurement: IC50 = 14000 nM (pChEMBL 4.849999904632568)
Descriptors: MW 473.36, ALogP 3.5, HBA 5, HBD 1, PSA 84.3, QED 0.62
Compound (SMILES): NC(=O)c1cc(-c2ccc(F)cc2)c2ccc(Cn3cc(C(=O)C4CC4)cn3)cc2n1
InChIKey: LTQDWLBJRNEBCU-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 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 414.44, ALogP 3.98, HBA 5, HBD 1, PSA 90.87, QED 0.48
Compound (SMILES): COCCCOc1cc([C@H](C)N(C(=O)[C@H]2CNCCO2)C2CC2)ccc1OC
InChIKey: JLHLQWGXBDSHDU-MGPUTAFESA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: Inhibitory Assay: A substrate of synthetic peptide (Nma-KHPFHLVIHK(Dnp)-NH2) and test compound were mixed, and fluorescence intensity was assayed using a fluorophotometer before staring an enzymatic reaction (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Recombinant human renin was added and the mixture was incubated at 37° C. for 1 hour, and the fluorescence intensity was measured after the reaction using a fluorophotometer (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Renin activity was evaluated on the ground of fluorescence intensity which was obtained by deduction of the intensity before the reaction from the intensity after the reaction, and 50% inhibitory concentration (IC50) was calculated from renin activities under the existence of various concentration of the test compound.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 392.5, ALogP 2.15, HBA 6, HBD 1, PSA 69.26, QED 0.62
Compound (SMILES): Cc1ccncc1S(=O)(=O)c1ccc(NC(=O)/C=C/c2cccnc2)cc1
InChIKey: RFXLTMZJSCZWEJ-RUDMXATFSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 20.4 nM (pChEMBL 7.690000057220459)
Descriptors: MW 379.44, ALogP 3.27, HBA 5, HBD 1, PSA 89.02, QED 0.69
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCOC2Cc2cnn(-c3ccc(F)cn3)c2)c1
InChIKey: NCKZYCBDYGDFRC-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Antagonistic Activity Assay: The antagonistic activities of the test compounds on human orexin-1 receptor (hOX1R) and orexin-2 receptor (hOX2R) were measured by modifying from the method described in literature (Toshikatsu Okumura et al., Biochemical and Biophysical Research Communications 280, 976-981, 2001). Chinese hamster ovary (CHO) cells forcibly expressing the hOX1R and hOX2R were seeded into a 96 well Black clear bottom plate (Nunc) at 20,000 cells per well, which were cultured in Ham's F-12 medium containing 0.1 mM MEM non-essential amino acids, 0.5 mg/ml G418, 10% fetal bovine serum (all by Invitrogen) for 16 hours under the conditions of 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the assay buffer containing fluo-3AM ester, the test compound was dissolved in dimethyl sulfoxide to be 10 mM and diluted with the assay buffer, 150 uL of which was added and incubated for 30 minutes.
Measurement: IC50 = 31.9 nM (pChEMBL 7.5)
Descriptors: MW 433.45, ALogP 2.34, HBA 8, HBD 0, PSA 90.96, QED 0.48
Compound (SMILES): COc1ccc(CN(Cc2ccccc2)S(=O)(=O)c2ccc(-c3nn[nH]n3)cc2)cc1
InChIKey: WMGSBCIPQRZYDN-UHFFFAOYSA-N
Target: Taste receptor type 2 member 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYV8
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 = 3670 nM (pChEMBL 5.429999828338623)
Descriptors: MW 435.51, ALogP 3.27, HBA 6, HBD 1, PSA 101.07, QED 0.46
Compound (SMILES): COc1ncc(-c2c(C)n[nH]c2C)cc1-c1cnc(N2CC(O)C2)nc1CN1C(=O)O[C@H](c2cc(C(F)(F)F)cc(C(F)(F)F)c2)[C@@H]1C
InChIKey: NFGOHUVZTYAGOX-QMTYFTJSSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: In Vitro Radioactive Assays of CETP-Catalyzed CE and TG Transfer (RTA Assay): Radiolabeled donor particles are generated by first combining 100 μl of 200 μM butylated hydroxyl toluene in CHCl3, 216 μL of 21.57 mM DOPC in EtOH, and either 500 μCi [3H]-triolein (Perkin Elmer #NET-431) or 500 μCi [3H]-cholesteryl oleate (GE #TRK886) in a glass tube. Reagents are mixed, dried under nitrogen, and then resuspended in 2 mL of 50 mM Tris, 27 μM EDTA at pH 7.4. After a brief vortex, the solution is sonicated until clear and mixed with 20 mL of fresh human serum. The mixture is incubated overnight at 37° C. The [3H] labeled LDL substrate is separated at 1.063 g/ml density by sequential ultracentrifugal flotation in NaBr according to the method by (Havel, Eder et al. 1955; Chapman, Goldstein et al. 1981). Once isolated the particles are dialyzed 3× in CETP buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA). Human HDL is purchased from Intracel and used as the acceptor particles. Transfer assays are performed in a 96 or 384-well v-bottom polypropylene plate. For the RTA using recombinant CETP (2% RTA), an assay cocktail is prepared with the final concentrations 128 μg/mL HDL, 20 nM rCETP, 2% human serum, and 1×CETP buffer. 1 μL of each test compound diluted in DMSO is added to 47 μL of assay cocktail per well and incubated at 37° C. for 1 hour. To initiate the transfer reaction, 2 μL radiolabeled LDL is added. After an additional 60 min of incubation at 37° C., the transfer action is terminated by precipitation of LDL with an equal volume of 20% W/V PEG 8000. The plates are centrifuged at 2000 rpm for 30 minutes at 4° C. A 40 μL aliquot of the HDL-containing supernatant is transferred to a Packard Optiplate™ with 200 μL of MicroScint™ 20. After mixing, plates are counted by liquid scintillation.
Measurement: IC50 = 332 nM (pChEMBL 6.480000019073486)
Descriptors: MW 677.61, ALogP 5.85, HBA 9, HBD 2, PSA 129.59, QED 0.23
Compound (SMILES): C[C@H]1CCCN1c1nc2cc(-c3nc(=O)o[nH]3)nc(-c3cncc(Cl)c3)c2n1C[C@H]1CC[C@H](C)CC1
InChIKey: MBAQIRLOJAFTEN-ULQDDVLXSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1x Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 508.03, ALogP 5.31, HBA 8, HBD 1, PSA 105.73, QED 0.39
Compound (SMILES): CNC(=O)Nc1nnc(-c2ccc(C(=O)N(C3CC3)C3CCN(C(=O)OC(C)(C)C)CC3)cc2)s1
InChIKey: YLNYLQWBYFLCOR-UHFFFAOYSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] are stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP_848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM β-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37° C. 5% CO2. For the assay, the cells are seeded in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37° C./5% CO2. After the medium is aspirated from the wells completely, 10 ul of the test compound are added, the compounds are diluted using stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes incubation at room temperature (approx. 20° C.), the cAMP concentrations are determined using the AlphaScreen cAMP Assay Kit (Cat. No. #6760625R from PerkinElmer). 10 ul of Biotin-cAMP (final concentration 1 U/well in lysing buffer (5 mM Hepes (pH 7.4), 0.1% BSA, 0.5% Tween) and 10 uL Bead solution (final concentration 1 U/well in lysing buffer) are added. The plates are incubated for another 2 hours at room temperature. The cAMP concentrations are calculated using a cAMP standard curve from the Alpha Screen Counts. The data analysis is carried out by calculating the EC50 value and the maximum value based on a positive control, using suitable software (Graphpad Prism).
Measurement: EC50 = 402 nM (pChEMBL 6.400000095367432)
Descriptors: MW 500.63, ALogP 3.96, HBA 7, HBD 2, PSA 116.76, QED 0.64
Compound (SMILES): COc1cc(Oc2nccc(C)n2)ccc1-c1cc2c(cc1F)ncc1ncn(C3(C)CCN(C(=O)CO)CC3)c12
InChIKey: XQVHUMMXXWIGOY-UHFFFAOYSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: Radioactive Binding Assay: The radioactive filter binding assay is standardized using recombinant human activated BRAF (V599E) kinase (Cat No. 14-557) and kinase dead MEK1 (K97R) (Cat No. 14-737) procured from Upstate. The incorporation of 32P into MEK1 (K97R) by BRAF (V599E) is measured with final assay buffer conditions of 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT, 100 mM sucrose, 100 μM sodium orthovanadate, 5 μM ATP and 2 μCi [γ 32P] ATP and 500 mg MEK1 Kinase dead substrate. The enzymatic reaction is stopped after 120 minutes with 8N HCl (hydrochloric acid) and 1 mM ATP. The solution is spotted on P81 filter paper and washed 4 times with 0.75% orthophosphoric acid and lastly with acetone. The dried P81 filter papers are read in a Micro-beta Trilux scintillation counter. The final concentration of DMSO is 1% in the assay. Compounds are screened at 10 μM concentration with pre-incubation of the enzymes in the presence of test compound for 45 minutes. Compounds of the invention were found to be inactive in this assy, e.g. ex. 33 (13% inhibition at 10 uM), ex. 1A (0% inhibition at 10 uM).
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 556.6, ALogP 4.62, HBA 9, HBD 1, PSA 115.49, QED 0.32
Compound (SMILES): c1ccc(-c2ncc(-c3csc(-c4cccnc4)n3)cn2)cc1
InChIKey: JHVOISNYBCMFRS-UHFFFAOYSA-N
Target: Hematopoietic prostaglandin D synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O60760
Assay: Fluorescence Polarization Binding Assay (FPBA): This assay essentially is available from Cayman Chemical Company as Catalog item #600007. The test data reported for the aforementioned Examples (H-PGDS FPBA IC.sub.50) were generated using a 96-well, instead of the 384-well, format.Detection analyte and H-PGDS-MBP fusion enzyme were incubated in the presence of reduced glutathione (5 mM) for 60-90 minutes at room temperature and FP was measured using a TECAN SAFIRE 2 plate reader equipped with absorbance, fluorescence, fluorescence polarization and FRET capabilities. Assays were performed in 96-well microtiter plates in 100 .mu.L of total sample volume. Excitation and emission wavelengths appropriate for the employed detection analyte were used.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 316.39, ALogP 4.33, HBA 5, HBD 0, PSA 51.56, QED 0.56
Compound (SMILES): CCCn1nc(C)c(-c2cnc(OC)c(-c3cnc(N4CC(F)C4)nc3CN3C(=O)O[C@H](c4cc(C(F)(F)F)cc(C(F)(F)F)c4)[C@@H]3C)c2)c1C
InChIKey: NOJURHHGHNYXHH-SLQAJWMNSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: In Vitro Radioactive Assays of CETP-Catalyzed CE and TG Transfer (RTA Assay): Radiolabeled donor particles are generated by first combining 100 μl of 200 μM butylated hydroxyl toluene in CHCl3, 216 μL of 21.57 mM DOPC in EtOH, and either 500 μCi [3H]-triolein (Perkin Elmer #NET-431) or 500 μCi [3H]-cholesteryl oleate (GE #TRK886) in a glass tube. Reagents are mixed, dried under nitrogen, and then resuspended in 2 mL of 50 mM Tris, 27 μM EDTA at pH 7.4. After a brief vortex, the solution is sonicated until clear and mixed with 20 mL of fresh human serum. The mixture is incubated overnight at 37° C. The [3H] labeled LDL substrate is separated at 1.063 g/ml density by sequential ultracentrifugal flotation in NaBr according to the method by (Havel, Eder et al. 1955; Chapman, Goldstein et al. 1981). Once isolated the particles are dialyzed 3× in CETP buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA). Human HDL is purchased from Intracel and used as the acceptor particles. Transfer assays are performed in a 96 or 384-well v-bottom polypropylene plate. For the RTA using recombinant CETP (2% RTA), an assay cocktail is prepared with the final concentrations 128 μg/mL HDL, 20 nM rCETP, 2% human serum, and 1×CETP buffer. 1 μL of each test compound diluted in DMSO is added to 47 μL of assay cocktail per well and incubated at 37° C. for 1 hour. To initiate the transfer reaction, 2 μL radiolabeled LDL is added. After an additional 60 min of incubation at 37° C., the transfer action is terminated by precipitation of LDL with an equal volume of 20% W/V PEG 8000. The plates are centrifuged at 2000 rpm for 30 minutes at 4° C. A 40 μL aliquot of the HDL-containing supernatant is transferred to a Packard Optiplate™ with 200 μL of MicroScint™ 20. After mixing, plates are counted by liquid scintillation.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 721.68, ALogP 7.72, HBA 9, HBD 0, PSA 98.5, QED 0.16
Compound (SMILES): C[C@H](NC(=O)[C@@H](N)CCCNC(=N)N)C(=O)N[C@H]1CSSC[C@@H](C(N)=O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](CCCNC(=N)N)NC1=O
InChIKey: YBCJXDSBBHYPIM-ODKJCKIQSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Inhibition of PE-conjugated-12G5 anti-CXCR4 antibody binding to CXCR4 in human CEM-CCRF cells preincubated for 30 mins followed by antibody addition by FACS Canto II cytofluorometric analysis
Measurement: IC50 = 5900 nM (pChEMBL 5.230000019073486)
Descriptors: MW 899.12, ALogP -2.87, HBA 12, HBD 14, PSA 367.51, QED 0.03
Compound (SMILES): CNc1nc(Nc2cn(S(=O)(=O)CCOC)nc2C)ncc1C(F)(F)F
InChIKey: VIPHJOOSOVISSJ-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lanthascreen Binding Assay: This assay was used to determine a compound's potency in inhibiting activity of LRRK2 by determining, Kiapp, IC50, or percent inhibition values. In 384 well proxiplates F black, shallow well plates LRRK2, Eu-anti-GST-antibody, Alexa Fluor Kinase tracer 236 and test compound were incubated together.Binding of the Alexa Fluor tracer to a kinase is detected by addition of a Eu-labeled anti-GST antibody. Binding of the tracer and antibody to a kinase results in a high degree of FRET, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET. Assay conditions and materials used were as follows: Final Assay Conditions: GST-LRRK2 G2019S 10 nM Eu-anti-GST-antibody 2 nM Kinase tracer 236 8.5 nM Kinase reaction time: 1 hour Temperature: ambient Total volume: 15 ul DMSO 1% Materials: 384 well proxiplates F black shallow cat# 6008260 well Perkin Elmer Kinase: LRRK2 G2019S, Invitrogen cat # PV4882(LOT 567054A).
Measurement: Ki = 1.85 nM (pChEMBL 8.729999542236328)
Descriptors: MW 394.38, ALogP 1.61, HBA 9, HBD 2, PSA 111.03, QED 0.73
Compound (SMILES): CNC(=O)c1c(-c2cnc(C)s2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CC4)cc12
InChIKey: YECDQFPLMKLHCN-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition 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 = 10.33 nM (pChEMBL 7.989999771118164)
Descriptors: MW 615.71, ALogP 6, HBA 8, HBD 1, PSA 110.33, QED 0.25
Compound (SMILES): CC[C@H](CN1CC[C@H](CNC(=O)c2ccc(Cl)c(Cl)c2)N[C@H](CCN2CCCC(F)(F)C2)C1=O)c1ccccc1
InChIKey: GEVMYKVUQGAFIN-GNMGOMLTSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 595.56, ALogP 5.6, HBA 4, HBD 2, PSA 64.68, QED 0.38
Compound (SMILES): COc1ccc2nc(NC3=NCC4(CN5CCC4CC5)O3)sc2c1
InChIKey: KIDXXFXVPLATJJ-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Agonist activity at rat alpha7 nAChR expressed in HEK293 cells co-expressing human RIC3 measured for 2 mins by FLIPR assay
Measurement: EC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 344.44, ALogP 2.57, HBA 7, HBD 1, PSA 58.98, QED 0.91
Compound (SMILES): CC(C)c1cc(NC(=O)N2CC=C(c3ncccc3Cl)CC2)ccc1OC(F)(F)F
InChIKey: XRVHFCJZIRIMKC-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 > 25000 nM (pChEMBL 4.6020002365112305)
Descriptors: MW 439.87, ALogP 6.08, HBA 3, HBD 1, PSA 54.46, QED 0.63
Compound (SMILES): COc1ccc(/C=N/NC(=O)CCn2cc(C)c3cccnc32)cc1OC
InChIKey: ONUQNRVOIIDUKY-WSDLNYQXSA-N
Target: Transient receptor potential cation channel subfamily M member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ8
Assay: Inhibition of human TRPM5 transfected in HEK293 cells assessed as inhibition of ATP-mediated calcium mobilization by Calcium3 dye-based FLIPR assay
Measurement: IC50 = 680 nM (pChEMBL 6.170000076293945)
Descriptors: MW 366.42, ALogP 2.9, HBA 6, HBD 1, PSA 77.74, QED 0.52
Compound (SMILES): CC(c1nc2c(cnn2C(C)C)c(=O)[nH]1)N1CC(Oc2ccccc2)C1
InChIKey: ACGQEEIBQUHXCU-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of human recombinant PDE9 expressed in insect Sf9 cells using [3H]-cGMP as substrate after 30 mins by scintillation proximity assay
Measurement: IC50 = 81.7 nM (pChEMBL 7.090000152587891)
Descriptors: MW 353.43, ALogP 2.52, HBA 6, HBD 1, PSA 76.04, QED 0.76
Compound (SMILES): Cc1cc(-c2nc3ccncc3n2Cc2ccccc2)cn(C)c1=O
InChIKey: PJOFFRUAZHATMS-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (LysS, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 330.39, ALogP 3.15, HBA 5, HBD 0, PSA 52.71, QED 0.58
Compound (SMILES): CC(C)c1cn(CC(=O)Nc2cc(C(=O)c3cn(C(C)C)c4ccncc34)ccn2)nn1
InChIKey: NLDNOEHDCBJGLE-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 40.6 nM (pChEMBL 7.389999866485596)
Descriptors: MW 431.5, ALogP 3.6, HBA 8, HBD 1, PSA 107.59, QED 0.45
Compound (SMILES): COc1n[nH]c2cc(NC(=O)NC(c3ccc(F)cc3)C(C)O)ncc12
InChIKey: FBFVSUQHZVJYJN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 13.4 nM (pChEMBL 7.869999885559082)
Descriptors: MW 359.36, ALogP 2.35, HBA 5, HBD 4, PSA 112.16, QED 0.56
Compound (SMILES): CN(C)CCN(C)c1ccc(C(=O)Nc2n[nH]c3cc(OCCOCc4ccccc4)ccc23)cc1
InChIKey: JTAKXJMETQCWIZ-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Biochemical Assay: The assay was performed using FLT3 cytoplasmic domain product and purified in house as GST fused protein. The FLT3 protein (1 microM) was pre activated with 800 microM ATP for 1 hour at 28° C in order to obtain a linear kinetic. Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 4 mM MgCl2, 1 mM DTT, 10 microM Na3VO4, and 0.2 mg/mL BSA. The FLT3 kinase assay was run with a final pre activated enzyme concentration of 2 nM, in the presence of 254 microM ATP (residual ATP from KIT pre activation step is negligible), 8 nM 33P-γ-ATP and 55 microM of substrate BioDB n*24 (Aminoacidic sequence: GGKKKVSRSGLYRSPSMPENLNRPR- SEQ ID NO: 1).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 487.6, ALogP 4.41, HBA 6, HBD 2, PSA 82.72, QED 0.29
Compound (SMILES): CCOC(=O)C1c2c(C3=CCNCC3)[nH]c3nccc(c23)-c2cc(F)ccc2N1C
InChIKey: LNZZZORWIXALKJ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay : CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were diluted in 100% DMSO then diluted with 1:10 in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. Equal volume of the compound dilutions was added to a final reaction mixture containing LANCE detection buffer (PerkinElmer CR97-100), 100 nM ULight MBP (PerkinElmer TRF0109M), 1000 μM ATP, and CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 1 hour before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer. The reaction was incubated for 1 hour and the signal was read in Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 406.46, ALogP 3.8, HBA 5, HBD 2, PSA 70.25, QED 0.65
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ccccc1NC(=O)Nc1ccc(C(C)(C)C)cc1)c1c(O)cccc12
InChIKey: PWYNKMDXHGOVLG-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 = 8.3 nM (pChEMBL 8.079999923706055)
Descriptors: MW 540.75, ALogP 7.87, HBA 4, HBD 3, PSA 67.84, QED 0.32
Compound (SMILES): CN(C)CCCNc1ccc(S(=O)(=O)NC(=O)c2ccc(N3CCN(CC4=C(c5ccc(Cl)cc5)CC(C)(C)CC4)CC3)cc2Oc2ccc3[nH]ccc3c2)cc1S(=O)(=O)C(F)(F)F
InChIKey: UOUKYUVJFCOKSR-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 0.126 nM (pChEMBL 9.899999618530273)
Descriptors: MW 941.54, ALogP 9.18, HBA 10, HBD 3, PSA 144.15, QED 0.09
Compound (SMILES): N#Cc1cc(C(F)(F)F)ccc1N1C(=O)COc2cc(S(=O)(=O)Nc3nccs3)ccc21
InChIKey: HYKNTXSQMNNDQG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 1450 nM (pChEMBL 5.840000152587891)
Descriptors: MW 480.45, ALogP 3.89, HBA 7, HBD 1, PSA 112.39, QED 0.61
Compound (SMILES): O=C(Cc1cccc(C(F)(F)F)c1Cl)NC1COc2ncccc21
InChIKey: NOLLGHUYWWIUQZ-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: In Vitro Assay: Cell line generation was performed in general according to established molecular cloning protocols. Specifically, RNA was extracted from human whole blood using the Qiagen RNeasy kit (Qiagen, CH) according to the manufacturer's instructions. Subsequently cDNA was made (Superscript II, Invitrogen AG, CH) and the human P2X7 gene (genbank ref. BC011913) was amplified with the following primers: ATCGCGGCCGCTCAGTAAGGACTCTTGAAGCCACT and CGCCGCTAGCACCACCATGCCGGCCTGCTGCAGCTGCA. The amplified sequence was subsequently ligated into a pcDNA3.1 (+) NotI, NheI digested plasmid. Human embryonic kidney (HEK) cells (ATCC CRL-1573, Manassas, Va., USA) were transfected with the pcDNA3.1 (+). hP2X7 plasmid using lipofectamine 2000 (Invitrogen AG, CH) according to the manufacturer's instructions. Following a 24 h exposure to DNA, cells were trypsinized and re-seeded at low density in the presence of 250 ug Geneticin. Geneticin resistant cells were then selected during two consecutive rounds of cloning by serial limiting dilution with visual inspection.
Measurement: IC50 = 7.4 nM (pChEMBL 8.130000114440918)
Descriptors: MW 356.73, ALogP 3.55, HBA 3, HBD 1, PSA 51.22, QED 0.91
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)N(Cc3cccc(C(F)(F)F)c3)C(C)(C)C2=O)cn1
InChIKey: CGDCALKLKRTNSH-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 = 1540 nM (pChEMBL 5.809999942779541)
Descriptors: MW 461.44, ALogP 4.3, HBA 6, HBD 0, PSA 84.47, QED 0.53