lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(Nc1ccccc1)N1CCN(Cc2cccnc2)CC1
InChIKey: XWYVHQOTIYGBQH-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture was prepared from 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA; Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 200 nM. The reaction mixture was incubated at rt for 1 hour (h). During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 296.37, ALogP 2.43, HBA 3, HBD 1, PSA 48.47, QED 0.95
Compound (SMILES): Cc1cccc(-c2ocnc2C(=O)Nc2cnn(CCn3ccc4ccc(F)cc43)c2)c1
InChIKey: HSVZFGQFEGIBEV-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20'000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 °C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37 °C. in 5% CO2 followed by equilibration at RT for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μl/well, incubated for 10 min and finally 10 μl/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 67 nM (pChEMBL 7.170000076293945)
Descriptors: MW 429.46, ALogP 4.89, HBA 6, HBD 1, PSA 77.88, QED 0.42
Compound (SMILES): O=C(c1cncc(-c2cccc(OCC(O)CN3CCc4ccccc4C3)c2)c1)N1CCCC1
InChIKey: PAFUFJGRVFVNJB-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 457.57, ALogP 3.78, HBA 5, HBD 1, PSA 65.9, QED 0.58
Compound (SMILES): COc1cc(N2CCOCC2)ccc1Nc1nc2c(c(Nc3cccc(C)n3)n1)C1(CC1)CN2
InChIKey: LZMRCDPOTCTCKF-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 459.55, ALogP 3.97, HBA 9, HBD 3, PSA 96.46, QED 0.51
Compound (SMILES): Cn1nc(C(=O)N2CCN(CC(=O)c3ccc(F)cc3)CC2)cc1-c1ccco1
InChIKey: RXLMQYCTADGJGT-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: [125I]DOI Radioligand Binding Assay.: Radioligand binding assays for human 5-HT2A serotonin receptor was conducted using the 5-HT2 agonist [125I]DOI as radioligand. To define nonspecific binding, 10 μM DOI was used for all assays. For competitive binding studies, 0.5 nM [125I]DOI was used and compounds were assayed over a range of 0.01 nM to 10 μM. Assays were conducted in a total volume of 200 μl in 96-well Perkin Elmer GF/C filter plates in assay buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM MgCl2, and 10 μM pargyline). Assay incubations were performed for 60 min at room temperature and were terminated by rapid filtration under vacuum pressure of the reaction mixture over Whatman GF/C glass fiber filters presoaked in 0.5% PEI using a Brandell cell harvester. Filters were then washed several times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4). Plates were then dried at room temperature and counted in a Wallac MicroBeta scintillation counter.
Measurement: IC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 396.42, ALogP 2.46, HBA 6, HBD 0, PSA 71.58, QED 0.62
Compound (SMILES): CCOc1ccc(CN2CCN(C(=O)Nc3ccccc3)CC2)cc1
InChIKey: KQXXSYGOSWXNMM-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture was prepared from 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA; Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 200 nM. The reaction mixture was incubated at rt for 1 hour (h). During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 339.44, ALogP 3.44, HBA 3, HBD 1, PSA 44.81, QED 0.91
Compound (SMILES): O=C(CNC(=O)c1ccc(Cl)cc1)N[C@@H]1CCCc2ccccc21
InChIKey: LVJDAFCIPXALGI-QGZVFWFLSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Inhibition of human recombinant DHODH
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 342.83, ALogP 3.26, HBA 2, HBD 2, PSA 58.2, QED 0.9
Compound (SMILES): [C-]#[N+]/C(=C/c1ccc(Nc2cnc3[nH]cc(C(=O)C(C)(C)C)c3n2)cc1)C(=O)N1CCCCC1
InChIKey: GEWHOOAXEXWIPY-XSFVSMFZSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Phosphorylation Assay: Phosphorylation of the appropriate peptide substrate (fluorescently labeled Srctide) by recombinant JAK3 (catalytic domain, aa 781-1124) was measured in the presence and absence of different concentrations of test compounds. Test compound, enzyme, fluorescently labeled substrate, and cofactors (ATP and Mg2+) were combined in a well of a microtiter plate and incubated for 2 hours at 25° C. The composition of the reaction buffer was 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Enzyme concentration in the final reaction mixture was 0.5 nM while peptide substrate was at 1 uM. ATP was used at 1xKm concentration corresponding to 2 uM. Serial dilutions (3-fold steps) of compounds were prepared in DMSO. At the end of the incubation, the reaction was stopped by the addition of 20 mM EDTA. Substrate and product (i.e. phosphorylated substrate) were separated electrophoretically and both were quantified by fluorescence intensity.
Measurement: IC50 = 1270 nM (pChEMBL 5.900000095367432)
Descriptors: MW 456.55, ALogP 5.2, HBA 5, HBD 2, PSA 95.34, QED 0.31
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2c(OC)ccc3ccccc23)cc(NC(=O)c2ccc(Cl)cn2)n1
InChIKey: GRKIXIAYZKUHAG-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: DELFIA Assay: R3 domains of human XIAP (covering amino acids 241 to 356; XIAP BIR3) and cIAPl (covering amino acids 256 to 363; cIAPl BIR3) were expressed and purified from Ecoli as GST-fusion proteins. Peptide AVPIAQKSE-Lys(Biotin), representing the N-terminus of mature human SMAC (SMAC peptide), was used as interaction partner in the protein- peptide interaction assay.BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonXlOO) for one hour at room temperature in the presence of inhibitiory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-Nl- antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluoresecence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 4967 nM (pChEMBL 5.300000190734863)
Descriptors: MW 489.96, ALogP 4.76, HBA 6, HBD 3, PSA 105.24, QED 0.35
Compound (SMILES): COc1cc2c(Nc3cc(F)c(Br)cc3F)ncnc2cc1OCC1CN(C)CCO1
InChIKey: HKHCOFOLZLSONC-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 495.32, ALogP 4.13, HBA 7, HBD 1, PSA 68.74, QED 0.52
Compound (SMILES): CC[C@H]1c2c(C3=CCN([C@H]4C[C@H](C(=O)O)C4)CC3)[nH]c3nccc(c23)-c2cc(F)ccc2N1C
InChIKey: CAFRPEXZJRPMMG-MDKPJZGXSA-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 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 460.55, ALogP 5.22, HBA 4, HBD 2, PSA 72.46, QED 0.56
Compound (SMILES): CC[C@@H](N[C@@H](C)CC(N)=O)c1ccc(Cl)c(C(=O)c2cncc(N)c2)c1F
InChIKey: OPCXCFQQXLNDGF-ZUZCIYMTSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 840 nM (pChEMBL 6.079999923706055)
Descriptors: MW 392.86, ALogP 2.99, HBA 5, HBD 3, PSA 111.1, QED 0.6
Compound (SMILES): ClC1CC=CC=C1C(c1ccccc1)(c1ccccc1)n1ccnc1
InChIKey: ZJAKIXPPYNHMRA-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: In vitro CYP3A4 Inhibition Assay: Cytochrome P450 is a large and diverse group of enzymes that catalyze the oxidation of organic substances. Some members of the CYP family contribute to the elimination of ATRA by catalyzing its 4-hydroxylation in the mammalian liver and skin, including that of humans as well as swine. Applicant evaluated the potential RAMBA activity of several azoles using pig liver microsomes, a rich source of CYP activity, comprising many different CYP 450 isoforms. Therefore, this approach, while a reasonable way to assess CYP inhibitors with broad activities may or may not be the best way to discover RAMBAs with selectivity for the skin, which has a much more narrow complement of CYP expression. As understanding in this area has progressed, a more specific CYP inhibition assay can be used to provide better predictivity of activity in human skin. Nevertheless, this assay may still be used as a general predictor of overall CYP activity.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 346.86, ALogP 5.17, HBA 2, HBD 0, PSA 17.82, QED 0.6
Compound (SMILES): CS(=O)(=O)N1CC(C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)C1
InChIKey: JPOUEXFNOOIYAK-NRFANRHFSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 634.57, ALogP 3.91, HBA 5, HBD 0, PSA 91.83, QED 0.36
Compound (SMILES): Cc1ccc2c(Nc3nc(N[C@@H]4CCCC[C@@H]4N)cc4cn[nH]c(=O)c34)cccc2n1
InChIKey: HWXNJENYXAISBU-QFBILLFUSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Chip Based Microfluidic Mobility Shift Assay: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, as well as four 8-point serial dilutions of staurosporine as reference compound, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on a Thermo CatX workstation equipped with a Innovadyne Nanodrop Express. Between pipetting steps, tips were cleaned in wash cycles using wash buffer. The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 μl per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 1 mM MgCl2, 3 mM MnCl2, 4 μM ATP, 4 μM peptide (5-Fluo-Ahx-GAPDYENLQELNKK-Amid) and 4.5 μl per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 1 mM MgCl2, 3 mM MnCl2, 4 nM SYK (SYK(2-635), produced in-house from insect cells). Kinase reactions were incubated at 30 °C. for 60 minutes and subsequently terminated by addition of 16 μl per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology. Briefly, samples from terminated kinase reactions were applied to the chip. Analytes are transported through the chip by constant buffer flow and the migration of the substrate peptide is monitored by the fluorescence signal of its label. Phosphorylated peptide (product) and unphosphorylated peptide (substrate) are separated in an electric field by their charge/mass ratio. Kinase activities were calculated from the amounts of formed phospho-peptide.
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 415.5, ALogP 3.6, HBA 7, HBD 4, PSA 121.61, QED 0.4
Compound (SMILES): O=S(=O)(Nc1noc2cc(COc3ccc(Cl)c(Cl)c3)c(Cl)cc12)C1CC1
InChIKey: AGJKCFDQZUWXCU-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay: Sodium currents were measured using the patch clamp technique in the whole-cell configuration using either a PatchXpress automated voltage clamp or manually using an Axopatch 200B (Axon Instruments) or Model 2400 (A-M systems) amplifier. The manual voltage clamp protocol was as follows: Borosilicate glass micropipettes were fire-polished to a tip diameter yielding a resistance of 2-4 Mohms in the working solutions. The pipette was filled with a solution comprised of 5 mM NaCl, 10 mM CsCl, 120 mM CsF, 0.1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA; and adjusted to pH 7.2 with CsOH. The external solution had the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES; and adjusted to pH 7.4 with NaOH. In some studies, the external sodium was reduced by equimolar replacement with choline.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 447.73, ALogP 5.27, HBA 5, HBD 1, PSA 81.43, QED 0.56
Compound (SMILES): CNC(=O)N[C@@H](C)c1ccc(CN2CCc3cc(OCC4CC4(F)F)ccc3C2)cc1
InChIKey: ASZFJTQPOKOMHH-BJQOMGFOSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 429.51, ALogP 4.27, HBA 3, HBD 2, PSA 53.6, QED 0.69
Compound (SMILES): Cn1cnc2cc(-c3ccc4nc(-c5ccco5)cc(C(=O)Nc5cccnc5)c4c3)ccc21
InChIKey: ORNBXXOVMHCTMW-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Biotin Assay: A streptavidin-coated plate (Thermo Scientific, NUNC #436014) was washed three times with 300 μL of PBS-0.05% Tween solution. A biotinylated twenty-six amino acid peptide, corresponding to the BH3 domain of Bim, with the sequence biotin-(β)A-D-M-R-P-E-I-W-I-A-Q-E-L-R-R-I-G-D-E-F-N-A-Y-Y-A-R-R-amide (SEQ ID No. 14), hereafter referred to as biotin-Bim, was obtained (Tufts). The biotin-Bim peptide was diluted to 0.018 μg/mL (5 nM) in SuperBlock blocking buffer in PBS (Thermo Scientific, #37515), and 100 μL of this solution was incubated in the streptavidin-coated plate for 2.5 hours while shaking. Separately, a compound of Formula I or Formula II, which had been prepared in a 10 mM stock DMSO solution, was then incubated with 20 nM GST-Mcl-1 fusion protein in PBS. Compounds were tested in six three-fold dilutions ranging from 20 μM to 0.6 μM, 10 μM to 0.3 or 5 μM to 0.15 μM. In addition, Bim peptide (New England Peptide) was utilized as a control and tested in six-fold dilutions ranging from 100 nM to 3.3 nM. The streptavidin-coated plate which had previously been treated with biotin-Bim peptide was then washed three times with 300 μL of PBS-0.05% Tween solution. A solution of GST-MCL-1 and a compound of Formula I or Formula II (100 μL) was then added to the streptavidin-coated plate, with two wells utilized as controls (PBS buffer only, no GST-MCL-1 or biotin-Bim) and two wells as an alternative control (containing GST-MCL-1 but no biotin-Bim), and four three-fold dilutions of DMSO (0.2%, 0.06%, 0.02%, and 0.008%). All wells containing compounds of Formula I or Formula II were loaded in duplicate. The plate was incubated for 2 hours at RT, then washed three times with 300 μL of PBS-0.05% Tween solution. Anti-GST HRP (GE Healthcare, #RPN1236) is diluted 1:20,000 in freshly prepared PBS, 0.1% Tween20, and 0.5% BSA was then added to the plate and the plate was incubated for 30 minutes. The plate was then washed five times with 300 μL of PBS-0.05% Tween solution. A solution of color reagents A (stabilized peroxide solution) and B (stabilized chrommogen solution) (R&D Systems, #DY999) is mixed in a 1:1 ratio and added to each well in a quantity of 80 μL. The wells were allowed to develop until control wells containing DMSO are blue (approximately 5-10 minutes). A solution of ELISA stop solution (1M sulfuric acid, 20 μL per well) was then added. Absorbance at 450 nM was read on a Tecan GeniosPro plate reader.
Measurement: IC50 = 2200 nM (pChEMBL 5.659999847412109)
Descriptors: MW 445.48, ALogP 5.7, HBA 6, HBD 1, PSA 85.84, QED 0.38
Compound (SMILES): COc1cc(CN(Cc2ccc(C(=O)O)cc2)[C@H](C)c2ccc(Cl)cc2)ccc1OCCN1C(=O)CCC1=O
InChIKey: DEYHMHZUEVDHBR-HXUWFJFHSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 345 nM (pChEMBL 6.460000038146973)
Descriptors: MW 551.04, ALogP 5.34, HBA 6, HBD 1, PSA 96.38, QED 0.3
Compound (SMILES): COc1cccc(C)c1[C@@H]1C[C@H](Cl)C(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: DWMCXQLHQGWMOG-HOTGVXAUSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 1517 nM (pChEMBL 5.820000171661377)
Descriptors: MW 413.82, ALogP 4.98, HBA 3, HBD 0, PSA 38.77, QED 0.65
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2c(F)ccc(C#N)c2c(=O)n1-c1cncc(F)c1
InChIKey: PPFDZFSCLCVTJT-VIFPVBQESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 460.41, ALogP 1.93, HBA 11, HBD 3, PSA 185.21, QED 0.41
Compound (SMILES): COc1cc(C(=O)N2CC(OC)C2)ccc1Nc1cc2cc(-c3cnn(C4CCCC4O)c3)ccc2cn1
InChIKey: OEJJTGBPFAXDHG-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: The enzyme reaction (total volume 10 μl) was carried out in black 384-well low volume plates containing full length MPS1 (12.5 nM or 3 nM), fluorescent labelled peptide [known as H236, which has the sequence: 5FAM-DHTGFLTEYVATR-CONH2](5 μM), ATP (10 μM), either DMSO (1% v/v) or the test compound (in the range 0.25 nM-100 μM in 1% DMSO) and assay buffer (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovandate, 10 μM MgCl2, 1 μM DTT, Roche protease inhibitor). The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μl) containing 20 mM EDTA, 0.05% (v/v) Brij-35, in 0.1M HEPES-buffered saline (Free acid, Sigma, UK). The plate was read on a Caliper EZ reader II (Caliper Life Sciences).
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 513.6, ALogP 4.41, HBA 8, HBD 2, PSA 101.74, QED 0.38
Compound (SMILES): Cc1ccc(CN2CCN(C(=O)Nc3ccccc3)CC2)cc1C
InChIKey: MFWPLJBROWDMQO-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture was prepared from 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA; Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 200 nM. The reaction mixture was incubated at rt for 1 hour (h). During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 630 nM (pChEMBL 6.199999809265137)
Descriptors: MW 323.44, ALogP 3.65, HBA 2, HBD 1, PSA 35.58, QED 0.94
Compound (SMILES): COc1cc(N(CC2CC2)c2ccc(Cl)cc2)cnc1C(=O)C1CC1C(=O)NS(=O)(=O)C1CC1
InChIKey: WRQJEUJCLVYDJW-UHFFFAOYSA-N
Target: Leukotriene C4 synthase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16873
Assay: In Vitro hLTC4 Synthase Enzyme Assay (Test A): In the assay, LTC4 synthase catalyses the reaction where the substrate LTA4 is converted to LTC4. Recombinant human LTC4 synthase is expressed in Piccia pastoralis and the purified enzyme is dissolved in 25 mM tris-buffer pH 7.8 supplemented with 0.1 mM glutathione (GSH) and stored at −80 °C. In order to obtain IC50-values for the compounds, the following procedure was used: A volume (24 μL) of 0.25 g/mL LTC4 synthase in 75 mM tris (pH ~8.5), 0.5 mM MgCl2 and 3 mM GSH was preincubated for 10 min with 0.5 μL of compound of interest in DMSO at ten different concentrations, typically in the range 10^−9,5-10^−5 M as well as with DMSO only. The buffer is used as background. Runs are performed in duplicates. The enzymatic reaction is initiated by addition of 0.5 μL LTA4 in diglyme (final assay concentration 8 μM). The reaction is stopped after 1 min by addition of double the reaction volume of a stop solution (MeOH:H2O:acetic acid 70:30:1).
Measurement: IC50 = 356 nM (pChEMBL 6.449999809265137)
Descriptors: MW 504.01, ALogP 3.72, HBA 7, HBD 1, PSA 105.67, QED 0.49
Compound (SMILES): O=C(O)c1cc(-c2nc(-c3ccc(Cl)c(Cl)c3)cs2)ccc1-c1ccccc1
InChIKey: YJBVHNLHUDBXPO-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E (SINGLE PROTEIN), Homo sapiens
UniProt: P06730
Assay: Binding Assay: Human eIF4E (aa 28-217) with a C-terminal His tag (HH-eIF4E) was expressed in E. coli in inclusion bodies. The protein was solubilized with 8 M urea and purified under denaturing conditions using nickel-charged HisTrap HP columns (GE Healthcare). The protein was refolded by diluting to approximately 0.25 mg/mL with 6 M urea, 20 mM Hepes pH 7.0, 500 mM NaCl, 1 mM DTT, 1 mM EDTA, and 0.5 M arginineÂ»HCl, and then dialyzing overnight into the same buffer without the urea. The protein was further dialyzed into 20 mM Hepes pH 6.5, 50 mM NaCl, 1 mM EDTA, and 1 mM DTT, filtered, and then concentrated using Hitrap SP sepharose FF columns (GE Healthcare). The protein was dialyzed into 20 mM Hepes pH 7.0, 500 mM NaCl, 5 mM DTT, and 10% glycerol and stored at -80 Â°C until use. Test compounds (1.6 mM stock in DMSO) were diluted 3 fold in series in DMSO. Compound solutions were diluted 4 fold in Assay Buffer (50 mM sodium phosphate, pH 6.5, 50 mM KC1, 1 mM DTT and 0.5 mg/mL gammaglobulin).
Measurement: IC50 = 5900 nM (pChEMBL 5.230000019073486)
Descriptors: MW 426.32, ALogP 7.15, HBA 3, HBD 1, PSA 50.19, QED 0.38
Compound (SMILES): CCn1c(=O)c2cnc3c(OC)cccc3c2n(C2CCCC2)c1=O
InChIKey: SHXAYINEIAIQRK-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: ELISA Assay: Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature. Repeat the aspiration/wash as in step 2 of Plate Preparation. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature. Repeat the aspiration/wash as in step 2 of Plate Preparation. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light. Repeat the aspiration/wash as in step 2. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 339.4, ALogP 2.86, HBA 6, HBD 0, PSA 66.12, QED 0.69
Compound (SMILES): O=C1C(N2CCC(c3ccc(O)cc3)C(F)(F)C2)CCN1Cc1ccc(Cl)c(Cl)c1
InChIKey: CLNCTPCQWVHYIL-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 455.33, ALogP 4.92, HBA 3, HBD 1, PSA 43.78, QED 0.71
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2cncn2C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: WXCQNRFWIHJMLJ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Inhibition of human FGFR1 using Btn-Flt3 as substrate after 60 mins by TR-FRET assay
Measurement: IC50 = 17.38 nM (pChEMBL 7.760000228881836)
Descriptors: MW 479.54, ALogP 3.97, HBA 9, HBD 0, PSA 83.12, QED 0.34
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(OCC5CN(C6CC6)CCO5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: LCOMZWJSJOFOEF-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.01 nM (pChEMBL 11)
Descriptors: MW 910.49, ALogP 8.06, HBA 12, HBD 2, PSA 172.47, QED 0.08
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(OCC(F)(F)F)cc1C21N=C(N)N(CCF)C1=O
InChIKey: GCNZQNXCFKMJOJ-WUFFQQIKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham).
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 443.44, ALogP 3.08, HBA 5, HBD 1, PSA 77.15, QED 0.71
Compound (SMILES): CC(C)N1CCC2(CC1)CN(c1ccccc1NC(=O)Nc1ccc(OC(F)(F)F)cc1)c1c(O)ccc(-c3nc4cc(Cl)ccc4s3)c12
InChIKey: XQWVKDZZPXGAFS-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 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 708.21, ALogP 9.76, HBA 7, HBD 3, PSA 89.96, QED 0.16
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1cc(F)cc2c1OCOC2
InChIKey: AYHZKTTUBQETRW-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 502.33, ALogP 6.27, HBA 7, HBD 1, PSA 74.73, QED 0.33
Compound (SMILES): CN(C)C(=O)CN1CC=C(c2[nH]c3nccc4c3c2CN(C)c2ccc(F)cc2-4)C(C)(C)C1
InChIKey: VYBYOLOBVGPCHH-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 = 870 nM (pChEMBL 6.059999942779541)
Descriptors: MW 447.56, ALogP 4.13, HBA 4, HBD 1, PSA 55.47, QED 0.66
Compound (SMILES): O=C(Nc1cccc(C(=O)c2ccc3c(/C=C/c4ccccn4)n[nH]c3c2)c1)c1ccccc1
InChIKey: UMNQEFXKDFGFNN-FYWRMAATSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of unphosphorylated human 50 residues truncated cytoplasmic domain of VEGFR2 E990V mutant expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using poly(E4Y1) as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 0.046 nM (pChEMBL 10.34000015258789)
Descriptors: MW 444.49, ALogP 5.61, HBA 4, HBD 2, PSA 87.74, QED 0.33
Compound (SMILES): C[C@@H]1CN(Cc2ccccc2)c2ccncc2[C@H](C)N1C(=O)NCc1ccccc1
InChIKey: FXQIXJJVPMVJQZ-UXHICEINSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Channel blocking activity at recombinant human CaV 3.1 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 6280 nM (pChEMBL 5.199999809265137)
Descriptors: MW 400.53, ALogP 4.76, HBA 3, HBD 1, PSA 48.47, QED 0.69
Compound (SMILES): CC[C@H](C)CNC(=O)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: ZLOZQNWQYQWSHJ-HNNXBMFYSA-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 = 20.2 nM (pChEMBL 7.699999809265137)
Descriptors: MW 351.45, ALogP 4.01, HBA 2, HBD 2, PSA 61.44, QED 0.85
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CCc4c(cnn4-c4ccccc4)C3)n2)c1
InChIKey: RAJVHPQXVCWTAA-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 20950 nM (pChEMBL 4.679999828338623)
Descriptors: MW 426.48, ALogP 2.72, HBA 8, HBD 2, PSA 100.86, QED 0.51
Compound (SMILES): c1coc(-c2n[nH]c3ccc(-c4nnn[nH]4)cc23)c1
InChIKey: IOWFMMGSUZKPFB-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 252.24, ALogP 2, HBA 5, HBD 2, PSA 96.28, QED 0.57
Compound (SMILES): O=C(Cn1nnc(-c2ccncc2)c1-c1ccccc1)Nc1ccc(-c2ccco2)cn1
InChIKey: JNCNARGJGZOOEE-UHFFFAOYSA-N
Target: Protein-serine O-palmitoleoyltransferase porcupine (SINGLE PROTEIN), Mus musculus
UniProt: Q9JJJ7
Assay: Inhibition of porcupine-mediated Wnt signaling in mouse L-cells after 24 hrs by springerImage-Topflash reporter assay
Measurement: EC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 422.45, ALogP 4.3, HBA 7, HBD 1, PSA 98.73, QED 0.44