lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CC[C@H](F)[C@H](O)C1
InChIKey: MWEQMCAEPGKADY-MAUKXSAKSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 17700 nM (pChEMBL 4.75)
Descriptors: MW 435.38, ALogP 3.99, HBA 5, HBD 1, PSA 75.55, QED 0.63
Compound (SMILES): Cc1nc2ccccc2n1-c1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1
InChIKey: YMEOKQGTHRYIOU-ZSOIEALJSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 415.42, ALogP 1.53, HBA 9, HBD 3, PSA 131.13, QED 0.34
Compound (SMILES): CC1(C)c2cc(OCC(=O)NCCN(CCO)CCO)ccc2C(=O)c2c1oc1ccccc21
InChIKey: QQGKOIAOENIUMD-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 3702.8 nM (pChEMBL 5.429999828338623)
Descriptors: MW 466.53, ALogP 2.08, HBA 7, HBD 3, PSA 112.24, QED 0.42
Compound (SMILES): Cn1cc(S(=O)(=O)NCc2cccc(C(Cc3ccccc3)CN3CCC3)c2)cn1
InChIKey: SPVOBSRIRDTEGQ-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Radioligand Binding Assay: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 424.57, ALogP 2.93, HBA 5, HBD 1, PSA 67.23, QED 0.57
Compound (SMILES): CCCSc1ccc(Nc2nc(=O)n(CCO)c(=O)n2Cc2ccc(Cl)cc2)cc1
InChIKey: WSXYIBMUMGPQNK-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37 C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 446.96, ALogP 3.34, HBA 8, HBD 2, PSA 89.15, QED 0.49
Compound (SMILES): N#CC1(c2ncc(C(=O)Nc3ccc(OC(F)(F)F)cc3)cc2-c2cncnc2)CCOCC1
InChIKey: RAYQKEHJGZBEIM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper 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 a reference compound, plus 16 high and 16 low controls. Liquid handling and incubation steps were done on a Thermo CatX workstation equipped with 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% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 μM ATP, 4 μM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 μL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). 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.
Measurement: IC50 = 6.6 nM (pChEMBL 8.180000305175781)
Descriptors: MW 469.42, ALogP 4.26, HBA 7, HBD 1, PSA 110.02, QED 0.59
Compound (SMILES): O=C(c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1)N1CC2(COC2)C1
InChIKey: VQSLOKHNCGHZMZ-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×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 °C. for 30 min. Compounds were added to a final assay volume of 100 μl 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 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM 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 as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 364.3 nM (pChEMBL 6.440000057220459)
Descriptors: MW 365.38, ALogP 1.8, HBA 5, HBD 0, PSA 54.9, QED 0.8
Compound (SMILES): O=C(Cc1nc(N2CCOCC2)cc(=O)n1-c1ccccc1)N1CCc2ccccc21
InChIKey: IUALSQPOYDCZRS-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Sandwich Immunoassay: This test is based on measurement of the expression of AKT protein phosphorylated on serine 473 (P-AKT-S473) in the PC3 human prostate carcinoma line, by the technique based on a sandwich immunoassay using the Kit MSD Multi-spot Biomarker Detection of Meso Scale Discovery: phospho-Akt kits (Ser473) whole cell lysate (# K151 CAD) or phospho-Akt (Ser473) / Akt whole cell lysate Total (# K15100D). The primary antibody specific for P-AKT-S473 (Kit # K151 CAD) is coated onto an electrode in each well of 96-well plates of the MSD kit: after adding a protein lysate in each well, the revelation of the signal done by the addition of a secondary antibody labeled with an electrochemiluminescent detection compound. The procedure is described in the kit.On day 1, the PC3 cells are inoculated in 96-well plates (TPP # 92096) at a concentration of 35000 cells / well in 200 uL DMEM medium (DMEM Gibco # 1 1960-044) containing 10% fetal calf serum (FBS Gibco, # 10500-056) and 1% glutamine.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 416.48, ALogP 2.2, HBA 6, HBD 0, PSA 67.67, QED 0.65
Compound (SMILES): NC(=O)c1ccc2[nH]nc(-c3cccc(NC(=O)Cc4ccccn4)c3)c2c1
InChIKey: MVGSHUAWAAJEKC-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 <= 1000 nM (pChEMBL 6)
Descriptors: MW 371.4, ALogP 2.9, HBA 4, HBD 3, PSA 113.76, QED 0.5
Compound (SMILES): O=C(N[C@H]1CCOC1)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: BQLKIEAFUFXATC-SFHVURJKSA-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 = 216 nM (pChEMBL 6.670000076293945)
Descriptors: MW 351.41, ALogP 2.75, HBA 3, HBD 2, PSA 70.67, QED 0.89
Compound (SMILES): COc1cc(Nc2ncc3c(n2)CCN(C(=O)c2occc2C)C3)cc(OC)c1OC
InChIKey: SHIGFRIZRYUWMA-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 ul of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 ul of 0-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 ul of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 424.46, ALogP 3.35, HBA 8, HBD 1, PSA 98.95, QED 0.64
Compound (SMILES): CNc1nc(NCc2ccc(NC(=O)c3ccc(Cl)cc3Cl)cc2)c2ccccc2n1
InChIKey: BTRRBAFAFFRSIL-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf33.13 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 1820 nM (pChEMBL 5.739999771118164)
Descriptors: MW 452.35, ALogP 5.84, HBA 5, HBD 3, PSA 78.94, QED 0.34
Compound (SMILES): CN(c1cccc(-c2cc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)c1)S(C)(=O)=O
InChIKey: MXWCUIKTJNSLSS-MFOYZWKCSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 467.51, ALogP 1.55, HBA 8, HBD 3, PSA 137.8, QED 0.37
Compound (SMILES): O=C1CCCC(c2ccnn2-c2ccccc2)N1Cc1cccc(-c2ccccc2)c1
InChIKey: WRXUBKJYXPELAE-UHFFFAOYSA-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 = 654 nM (pChEMBL 6.179999828338623)
Descriptors: MW 407.52, ALogP 5.79, HBA 3, HBD 0, PSA 38.13, QED 0.42
Compound (SMILES): CN(CC1CCOC1)c1ncc(-c2ccnc(Nc3ccc(CN4CCOCC4)nc3)n2)cc1C#N
InChIKey: GXYJFNJQTSRCTJ-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 486.58, ALogP 2.85, HBA 10, HBD 1, PSA 112.32, QED 0.51
Compound (SMILES): CC(C)N(Cc1cccc(-c2ccc(N3CCOCC3)cc2)c1)C(=O)CN.O=C(O)[C@H](O)[C@@H](O)C(=O)O
InChIKey: YUJPVMSKKDOCIS-LREBCSMRSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Inhibition of human VAP-1 expressed in CHO cells using 14C-benzylamine as substrate preincubated for 20 mins followed by substrate addition measured after 1 hr by scintillation spectrometric analysis
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 367.49, ALogP 2.89, HBA 4, HBD 1, PSA 58.8, QED 0.85
Compound (SMILES): Cc1ccc(C(=O)N2CC(c3ccc(C#N)cc3)C2)cc1-c1[nH]ncc1C#N
InChIKey: LJNBGVIIQKMMJC-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 367.41, ALogP 3.37, HBA 4, HBD 1, PSA 96.57, QED 0.77
Compound (SMILES): c1cc2c(c(C3OCCc4c3[nH]c3ccccc43)c1)OCO2
InChIKey: TYIZFFYGZFTNHH-UHFFFAOYSA-N
Target: Sodium/iodide cotransporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q63008
Assay: Inhibition of sodium iodide symporter in rat FRTL5 cells assessed as decrease in [125I]-sodium iodide uptake after 60 mins by scintillation counting method
Measurement: IC50 = 0.41 nM (pChEMBL 9.390000343322754)
Descriptors: MW 293.32, ALogP 3.56, HBA 3, HBD 1, PSA 43.48, QED 0.75
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3cncc(-c4nnc(C5CC5)o4)c3)cc2Cl)CS1(=O)=O
InChIKey: AAXNGXBJBIQTNE-IBGZPJMESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 μl are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 566.2 nM (pChEMBL 6.25)
Descriptors: MW 478.99, ALogP 3.41, HBA 8, HBD 2, PSA 125.07, QED 0.59
Compound (SMILES): CC(C)(C)OC(=O)N1CCC2(CCC(CNC(=O)c3cc4cnccc4[nH]3)OC2)CC1
InChIKey: HIZPCJKBWSTODD-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of human full length C-terminal His6-tagged NAMPT expressed in Escherichia coli Rosetta (DE3) cells using nicotinamide as substrate incubated for 15 mins prior to substrate addition measured after 30 mins in presence of PRPP
Measurement: IC50 = 830 nM (pChEMBL 6.079999923706055)
Descriptors: MW 428.53, ALogP 3.49, HBA 5, HBD 2, PSA 96.55, QED 0.78
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(-c3cncc(C#N)c3)cc1C21N=C(N)N(C(C)C)C1=O
InChIKey: WOIFPJSSHHOWHG-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): 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). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 12.2 nM (pChEMBL 7.909999847412109)
Descriptors: MW 443.55, ALogP 3.51, HBA 6, HBD 1, PSA 104.6, QED 0.78
Compound (SMILES): CC(=O)c1ccc(OCc2cccc(Oc3ccc(-c4nn[nH]n4)cn3)c2)c(F)c1O
InChIKey: ICSUUZAKWUFXEV-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Positive allosteric modulation of recombinant human mGlu2 receptor expressed in hamster AV12 cells expressing EAAT1 assessed as potentiation of glutamate-induced intracellular calcium level measured after 24 to 48 hrs by FLIPR assay
Measurement: EC50 = 1076 nM (pChEMBL 5.96999979019165)
Descriptors: MW 421.39, ALogP 3.68, HBA 8, HBD 2, PSA 123.11, QED 0.43
Compound (SMILES): Cc1n[nH]c(C)c1S(=O)(=O)N1CCC(C#N)(Cc2ccc(Cl)cc2)CC1
InChIKey: DJDHNJIDBFJGHB-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Biological Assay: Prokineticin receptor 1 (PKR1) antagonists may be functionally assessed by measurement of change in intracellular calcium levels induced by Gq mediated increase in inositol triphosphate (IP3) levels. The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro. Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1xHanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 uM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following excitation at 485 nM. Readings from wells that do not contain antagonist enable percentage inhibition curves to be plotted using 4-parameter fit algorithm and IC50 values are calculated for each test compound.
Measurement: IC50 = 890 nM (pChEMBL 6.050000190734863)
Descriptors: MW 392.91, ALogP 3.22, HBA 4, HBD 1, PSA 89.85, QED 0.86
Compound (SMILES): Nc1sc(-c2ccccc2)cc1C(=O)NCCCCc1ccccc1
InChIKey: DMJONCCMYZLQDF-UHFFFAOYSA-N
Target: Polyunsaturated fatty acid lipoxygenase ALOX15 (SINGLE PROTEIN), Homo sapiens
UniProt: P16050
Assay: Inhibition of human 15-LOX-1 assessed as reduction in conversion of linoleic acid to 13(S)-HpODE incubated for 10 mins followed by linoleic acid addition by UV-absorbance analysis
Measurement: IC50 >= 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 350.49, ALogP 4.75, HBA 3, HBD 2, PSA 55.12, QED 0.6
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(CCC3CC3)cc1C21N=C(N)N(Cc2ccc(C(F)(F)F)cn2)C1=O
InChIKey: DYEAVJSNAPJOKL-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): 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). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 24.3 nM (pChEMBL 7.610000133514404)
Descriptors: MW 526.6, ALogP 5.13, HBA 5, HBD 1, PSA 80.81, QED 0.57
Compound (SMILES): CNC(=O)c1cc(Oc2ccc3c(c2)C2C(O3)C2c2nc3ccc(C(F)(F)F)cc3[nH]2)ccn1
InChIKey: JKQCEJLFHYXPNJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 466.42, ALogP 4.77, HBA 5, HBD 2, PSA 89.13, QED 0.45
Compound (SMILES): Cn1nc(C(C)(C)C)c(Cl)c1-c1nc2nc(-c3ccccc3C(F)(F)F)cc(Cl)c2[n-]1.[Na+]
InChIKey: HMGQLJSMNFOJHM-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: HEK293 cells stably expressing canine TRPM8 were routinely grown as monolayers in Dulbecco's minimum essential medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 400 μg/mL G418. Cells were maintained in 5% CO2 at 37° C. At 24 hr prior to assay, cells were seeded in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) at a density of 5,000 cells per well in culture medium 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 incubated 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 treated with compounds of the formula (I) at varying concentrations and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to each well to achieve a final concentration that produces an approximately 80% maximal response.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 468.31, ALogP 6.65, HBA 4, HBD 1, PSA 59.39, QED 0.36
Compound (SMILES): CC(C)C1CN(c2ccc(Nc3ncc4cc5n(c4n3)C3(CCCCC3)CNC5=O)nc2)CCN1
InChIKey: XVRFIUSGDOJQMI-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 695 nM (pChEMBL 6.159999847412109)
Descriptors: MW 474.61, ALogP 3.41, HBA 8, HBD 3, PSA 100, QED 0.53
Compound (SMILES): O=C(CCCc1ccc(F)cc1)NC1COc2ncccc21
InChIKey: ZDUGDUIOWBPIBD-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 = 2027 nM (pChEMBL 5.690000057220459)
Descriptors: MW 300.33, ALogP 2.79, HBA 3, HBD 1, PSA 51.22, QED 0.92
Compound (SMILES): O[C@H]1CC[C@@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: HPMRVSRZBKQEID-DTORHVGOSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 9.1 nM (pChEMBL 8.039999961853027)
Descriptors: MW 257.3, ALogP 1.62, HBA 5, HBD 2, PSA 79.1, QED 0.69
Compound (SMILES): COc1ccc2ccccc2c1[C@@H]1C[C@H](Cl)C(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: QGLHDMYPKCMBCT-OALUTQOASA-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 = 329 nM (pChEMBL 6.480000019073486)
Descriptors: MW 449.86, ALogP 5.83, HBA 3, HBD 0, PSA 38.77, QED 0.46
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1NC1CCN(C(=O)OC(C)(C)C)CC1
InChIKey: WSKIIQILRYHQCK-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 13.2 nM (pChEMBL 7.880000114440918)
Descriptors: MW 476.58, ALogP 4.03, HBA 6, HBD 3, PSA 112.24, QED 0.53
Compound (SMILES): O=C(CN1CCOCC1)N1CC=C(c2ccc(NC(=O)N3Cc4ccccc4C3)cn2)CC1
InChIKey: OZWGZOMPEMDYSC-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 82.1 nM (pChEMBL 7.090000152587891)
Descriptors: MW 447.54, ALogP 2.58, HBA 5, HBD 1, PSA 78.01, QED 0.78
Compound (SMILES): Cc1nn(C)c(C)c1S(=O)(=O)N1CCC(Oc2cccc(Cl)c2)CC1
InChIKey: ZWFKFPIVMQVIJQ-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Biological Assay: Prokineticin receptor 1 (PKR1) antagonists may be functionally assessed by measurement of change in intracellular calcium levels induced by Gq mediated increase in inositol triphosphate (IP3) levels. The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro. Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1xHanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 uM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following excitation at 485 nM. Readings from wells that do not contain antagonist enable percentage inhibition curves to be plotted using 4-parameter fit algorithm and IC50 values are calculated for each test compound.
Measurement: IC50 = 2200 nM (pChEMBL 5.659999847412109)
Descriptors: MW 383.9, ALogP 2.92, HBA 5, HBD 0, PSA 64.43, QED 0.81
Compound (SMILES): COc1cc(NC(=O)OCCN(C)[C@H]2CC[C@H](OC(=O)C(O)(c3cccs3)c3cccs3)CC2)c(C#N)cc1CNC[C@H](O)c1ccc(O)c2[nH]c(=O)ccc12
InChIKey: QREAYRYXWVDMKB-IUEKTVKYSA-N
Target: Beta-2 adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07550
Assay: Adrenergic Receptor Binding Assay: The study of binding to human adrenergic beta1 and beta2 receptors was performed using commercial membranes prepared from Sf9 cells where they are overexpressed (Perkin Elmer). The membrane suspensions (16 μg/well for beta1 and 5 μg/well for beta2) in assay buffer (75 mM Tris/HCl with 12.5 mM MgCl2 and 2 mM EDTA pH=7.4) were incubated with 0.14 or 0.6 nM of 3H-CGP12177 (Amersham) for beta 1 and beta 2 receptors respectively in a final volume of 250 μl, in GFC Multiscreen 96 well plates (Millipore) previously treated with assay buffer containing 0.3% PEI (Sigma). Non specific binding was measured in the presence of 1 μM propanolol. Incubation was maintained for 60 minutes at room temperature and with gentle shaking. The binding reactions were terminated by filtration and washing with 2.5 volumes of Tris/HCl 50 mM pH=7.4.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 801.94, ALogP 5.33, HBA 14, HBD 6, PSA 206.47, QED 0.07
Compound (SMILES): O=C(CCCc1cnn[nH]1)NCC1CCN(C(=O)OCc2cc(Cl)cc(Cl)c2)CC1
InChIKey: XZOSUJGCZYHQHE-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibition Assay: ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 454.36, ALogP 3.6, HBA 5, HBD 2, PSA 100.21, QED 0.64
Compound (SMILES): CCOc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C)c3)cn2CC2CNC2)CC1
InChIKey: GEVIIBINALZINE-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 465.58, ALogP 3.37, HBA 8, HBD 2, PSA 94.12, QED 0.55