lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(Nc1ccnc(CN2CCC(C(=O)NC3CCCCC3)CC2)c1)c1ccc(Cl)cc1
InChIKey: IRYIKFVSZKZKIY-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 455, ALogP 4.65, HBA 4, HBD 2, PSA 74.33, QED 0.67
Compound (SMILES): CC(C)CCNC(=O)CCNC(=O)N1Cc2ccccc2C1
InChIKey: CCVXOLJAYUDMTB-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1: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-1251667.0. 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 = 11200 nM (pChEMBL 4.949999809265137)
Descriptors: MW 303.41, ALogP 2.26, HBA 2, HBD 2, PSA 61.44, QED 0.85
Compound (SMILES): Cc1cc(Cl)ccc1OCC(=O)Nc1cccc(Cc2n[nH]c(=O)c3c2CCCC3)c1
InChIKey: ZLBNXVDLWLVCET-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 56 nM (pChEMBL 7.25)
Descriptors: MW 437.93, ALogP 4.22, HBA 4, HBD 2, PSA 84.08, QED 0.6
Compound (SMILES): COCC(C)N1C(=O)C2(N=C1N)c1cc(C#CC3CC3)ccc1CC21CCC(OC)CC1
InChIKey: RWBSXRBMZAYYSD-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 = 18.1 nM (pChEMBL 7.739999771118164)
Descriptors: MW 435.57, ALogP 2.97, HBA 5, HBD 1, PSA 77.15, QED 0.74
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccccc3)n2)CCN(CC#Cc2ccccc2)CC1
InChIKey: PQHOEDVCVVIECO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 83 nM (pChEMBL 7.079999923706055)
Descriptors: MW 438.54, ALogP 3.48, HBA 6, HBD 2, PSA 99.97, QED 0.57
Compound (SMILES): CN(c1cc(C(=O)Nc2ccc3c(c2)CC(=O)N3)ncn1)C1CCCCC1
InChIKey: NHAFYLPIBJVSIA-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: 35S-GTPgammaS Binding: Measurements of 35S-GTPgammaS Binding: Membranes (1 to 10 ug protein) prepared as described above, were incubated in 96-well Scintiplates (PerkinElmer) with test compounds diluted in DMSO, in 180 ul of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 2 ug/well Saponin, 0.2% fatty acid free BSA (Assay buffer), 140 mM NaCl and 1.7 uM GDP. The assay was initiated with the addition of 20 ul of 1.5 nM [35S]-GTPgammaS (1100 Ci/mmol; GE Healthcare) in assay buffer. After 60 min incubation at 30° C. on a shaker, plates were centrifuged for 10 min at 2000 RPM. Supernatant was discarded and membrane bound radioactivity was measured on a PerkinElmer 1450 MicroBeta counter. Triplicate samples were averaged and expressed as % response relative to S1P activation in absence of compound (n=2).
Measurement: EC50 = 1780 nM (pChEMBL 5.75)
Descriptors: MW 365.44, ALogP 2.99, HBA 5, HBD 2, PSA 87.22, QED 0.87
Compound (SMILES): O=S(=O)(Nc1ccc(C(F)(F)F)c2cccnc12)c1ccc(C(F)(F)F)nc1
InChIKey: VTGJQWUOGSJTNX-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 25000 nM (pChEMBL 4.599999904632568)
Descriptors: MW 421.32, ALogP 4.47, HBA 4, HBD 1, PSA 71.95, QED 0.63
Compound (SMILES): Fc1ccc(C(SCCN2CCN(Cc3ccc(Cl)cc3)CC2)c2ccc(F)cc2)cc1
InChIKey: RZKMAMGURFKTKH-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: P23977
Assay: Displacement of [3H]WIN35,428 from DAT in Sprague-Dawley rat brain membranes incubated for 120 mins by radioligand binding assay
Measurement: Ki = 19.3 nM (pChEMBL 7.710000038146973)
Descriptors: MW 473.03, ALogP 6.26, HBA 3, HBD 0, PSA 6.48, QED 0.38
Compound (SMILES): C[C@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@H](C)F)n1)c1cnc(-c2ccnc(C(F)(F)F)c2)s1
InChIKey: WGHDFKRAKKPLGU-GMXVVIOVSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 67.8 nM (pChEMBL 7.170000076293945)
Descriptors: MW 482.46, ALogP 4.87, HBA 8, HBD 1, PSA 93.13, QED 0.5
Compound (SMILES): Cc1cncc(C(=O)Nc2cccc(CN3CCC(C(=O)N4CCCCC4)CC3)c2)c1
InChIKey: XPDRNYPDWRORIX-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 420.56, ALogP 3.87, HBA 4, HBD 1, PSA 65.54, QED 0.8
Compound (SMILES): CC(C)(C)Nc1c(-c2cccc(Cl)c2)nc2c(Br)cc(Cl)cn12
InChIKey: NVJRTSJVHGFIRF-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Dose Response Assay: Harvested cells with 0.025% trypsin/EDTA. Resuspended cells in growth media without selection antibiotics. Measured cell density and diluted to 2.4×10^5 cells/ml in media containing 1 μg/ml Doxycycline Plate 25 μl/well into 384 well black/clear tissue culture-treated plates. Incubated overnight at 37° C. Growth media was removed from the cell plates (20 μl) and 20 μl of the Replacement Buffer was added followed by addition of 25 μl of diluted dye. All three steps were performed using a Plate Washer BioTek 407. The plates were then incubated for 30' at RT. After incubation, both the cell and compound plates were brought to the FLIPR and 20 μl of the diluted compounds/antagonist/bk were transferred to the cell plates by the FLIPR. Plates were then incubated for 30' at room temperature. After 30' incubation, plates were returned to the FLIPR and 20 ul of 4.5× Cinnamaldehyde was added to the cell plates. During the compound addition as well as agonist addition, fluorescence readings were taken simultaneously from all 384 wells of the cell plate every 1.5 seconds. Five readings were taken to establish a stable baseline, then 20 μl of sample was rapidly (30 μl/sec) and simultaneously added to each well of the cell plate. The fluorescence was continuously monitored before, during and after sample/agonist addition for a total elapsed time of 100 seconds (compound addition) and 120 seconds (agonist addition).
Measurement: IC50 = 1086 nM (pChEMBL 5.960000038146973)
Descriptors: MW 413.15, ALogP 6.28, HBA 3, HBD 1, PSA 29.33, QED 0.54
Compound (SMILES): CN(Cc1cccc(Br)c1)C1CCN(C(=O)Cn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)CC1
InChIKey: YTSUOQJODCDYIB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.27 nM (pChEMBL 9.569999694824219)
Descriptors: MW 728, ALogP 5.99, HBA 9, HBD 1, PSA 109.89, QED 0.19
Compound (SMILES): Cc1nc(NC(=O)c2ccccn2)sc1S(=O)(=O)Cc1ccc(Cl)cc1Cl
InChIKey: GLVUIEJZBBHUHJ-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Receptor Activity Assay: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGluR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured.
Measurement: EC50 = 289 nM (pChEMBL 6.539999961853027)
Descriptors: MW 442.35, ALogP 4.38, HBA 6, HBD 1, PSA 89.02, QED 0.63
Compound (SMILES): Cc1cc2c(Nc3nc(N[C@@H]4CCCC[C@@H]4N)cc4cn[nH]c(=O)c34)cccc2[nH]1
InChIKey: SCZRBEFQLYWCRB-MAUKXSAKSA-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 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 403.49, ALogP 3.53, HBA 6, HBD 5, PSA 124.51, QED 0.35
Compound (SMILES): O=C(O)c1nccc(-c2cccc(Cl)c2F)n1
InChIKey: WHAUHWFSUBXBDQ-UHFFFAOYSA-N
Target: Kynurenine 3-monooxygenase (SINGLE PROTEIN), Homo sapiens
UniProt: O15229
Assay: Inhibition of human KMO
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 252.63, ALogP 2.63, HBA 3, HBD 1, PSA 63.08, QED 0.89
Compound (SMILES): c1ccc(-c2noc(C3CCCCC3c3ccccc3)n2)cc1
InChIKey: UJLGMVSTEHAZJK-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: null: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1x108 cells/pellet) were suspended in buffer containing 20 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM EDTA and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80Â° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA, 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 Owen in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well.
Measurement: IC50 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 304.39, ALogP 5.18, HBA 3, HBD 0, PSA 38.92, QED 0.67
Compound (SMILES): CN(C)CCCC(=O)Nc1ccccc1-c1ccc2c3[nH]ncc3c(=O)n(CC(F)(F)F)c2c1
InChIKey: HZXKEHVVAJCFME-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzymatic Screening Assay: The enzymatic test is a test in two steps.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 32.9 nM (pChEMBL 7.480000019073486)
Descriptors: MW 471.48, ALogP 4.39, HBA 5, HBD 2, PSA 83.02, QED 0.42
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)c(Cl)c1)N1CCC(c2ncc(N3C[C@@H](O)[C@@H](O)C3)cc2Cl)CC1
InChIKey: BQEBPEFKNWQPCA-KDURUIRLSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 uM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 (wash buffer), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 uM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 (assay buffer). Plates are then transferred to a FLIPR for assay.
Measurement: IC50 = 160.1 nM (pChEMBL 6.800000190734863)
Descriptors: MW 519.35, ALogP 4.36, HBA 5, HBD 3, PSA 88.93, QED 0.56
Compound (SMILES): CN(C)CCCCNC(=O)c1ccc(NC(=O)N2Cc3cccnc3C2)cc1
InChIKey: RJBPGDOJZRQTFL-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 = 378 nM (pChEMBL 6.420000076293945)
Descriptors: MW 381.48, ALogP 2.7, HBA 4, HBD 2, PSA 77.57, QED 0.72
Compound (SMILES): Cn1cnc(S(=O)(=O)Nc2ccc(F)c(Nc3ncccc3-c3ncnc4[nH]cnc34)c2F)c1
InChIKey: MGCDELBBUMYCBC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Enzyme Assay: To check the B-Raf kinase inhibitory activity of the compounds of the present invention, the following experiments were conducted.(1) Serial Signaling: 20 ul of diluting solvent (20 mM MOPS, pH 7.2, 25 mM Î²-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol) and 10 ul of Mg/ATP mixed solution (500 uM ATP, 75 mM magnesium chloride) were added into a centrifuge tube, the derivative compound of Formula 1 was added or the compounds of the Examples were not added as a control group, and then 1 ng of activated B-Raf, 0.4 ng of inactivated MEK1, and 1 ug of inactivated MAPK2 were added. The solutions in the tube were collected on the bottom via centrifugation and were reacted at 30° C. for 30 minutes. After taking 4 ul of the mixed solution, the test proceeded to the next step.(2) Phosphorylation of Matrix Protein MBP by MAPK2: 10 ul of diluting solvent, 20 ug of MBP used as a matrix.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 483.46, ALogP 2.97, HBA 9, HBD 3, PSA 143.37, QED 0.33
Compound (SMILES): Nc1ccccc1NC(=O)/C=C/CCCNC(=O)c1cccc(Cl)c1
InChIKey: RQUVXEOPUIOLHF-BIIKFXOESA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 148 nM (pChEMBL 6.829999923706055)
Descriptors: MW 357.84, ALogP 3.63, HBA 3, HBD 3, PSA 84.22, QED 0.4
Compound (SMILES): O=S(=O)(C1CCOCC1)N1CCN(c2ccc(OC[C@@H]3CO[C@@](Cn4ccnc4)(c4ccc(Cl)cc4Cl)O3)cc2)CC1
InChIKey: DVDVFRQEWILNEL-VAVYLYDRSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of recombinant CYP17 (unknown origin) overexpressed in human AD293 cells using [21-3H]17alpha-hydroxyl-pregenolone as substrate preincubated for 60 mins followed by substrate addition measured after 4 hrs by Topcount method
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 637.59, ALogP 4.17, HBA 9, HBD 0, PSA 95.36, QED 0.35
Compound (SMILES): N=C(N)Nc1ccc(C(=O)Oc2ccc(CC(=O)N[C@@H](Cc3ccc(OCC(=O)O)cc3)C(=O)O)c(Cl)c2)cc1
InChIKey: TYHYKNFAOZOJQV-QFIPXVFZSA-N
Target: Serine protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07477
Assay: Inhibition Assay: The compound was dissolved in dimethyl sulfoxide (DMSO), and diluted to an arbitrary concentration (A). A was 100-fold diluted with a buffer (0.1 M Tris (pH 8.0), 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij38) (B). The r-h trypsin was diluted with a buffer to 0.088 ug/mL, and the m-trypsin was diluted with a buffer to 1/50 (C). The dilution ratio of the m-trypsin (1/50) was set to exhibit the same activity as the 0.088 ug/mL r-h trypsin as determined by kinetic analysis. The substrate solution of a substrate for the enzyme reaction, BZiPAR, (Rhodamine Reference Substrate) was diluted with a buffer to 5 umol/L (D). B; 5 uL, C; 5 uL, and D; 10 uL were added to a 384-plate, and incubated at room temperature for 30 minutes. The fluorescent signals were detected with Ex/Em = 497/520 using Tecan Safire Fluorometer. The compound was reviewed from 2500 nM to its 3-fold value, 0.0075 nM, at 12 concentrations, and the inhibitory rate of each compound was calculated.
Measurement: IC50 = 0.88 nM (pChEMBL 9.0600004196167)
Descriptors: MW 568.97, ALogP 2.68, HBA 7, HBD 6, PSA 201.13, QED 0.08
Compound (SMILES): O=C(Nc1ccnc(C(F)(F)F)c1)Nc1ccc(Oc2ccnc(NC(=O)C3CC3)c2)c(F)c1
InChIKey: QIDSOPRPFNFFEC-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition Assay: [Experimental method]: Use Lance@Ultra Ulight™-TK assay kit from PerKinElmer to evaluate the inhibitory activity of compounds at KDR kinase. [Instrument]: PerKinElmer's ENVISION plate reader. [Materials]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 0.25 mM EGTA, 2 mM DTT, 0.01% Tween 20, 10 mM Mg2+, 0.5 mM Mn2+), KDR kinase (790-1356 AA, Crown Bioscience), KDR kinase substrate (PerKinElmer catalogue # TRF0127-M), Lance@Eu-W1024-anti-phosphotyrosine (PT66) (PerKinElmer, catalogue #AD0068), ATP (Invitrogen), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix KDR (final concentration: 20 nM) and compound (final DMSO: 0.5%), pre-incubate for 20 min at 30° C.; then add ATP (final concentration: 90 μM) and the substrate (final concentration 50 μM). React for 2 hrs at 30° C. After the reaction, add antibody and reaction for 60 min at 30° C. Read the plate (615 nm, 665 nm), calculate the ratio of the value at 665 nm vs 615 nm and analyze the data.
Measurement: IC50 = 8.3 nM (pChEMBL 8.079999923706055)
Descriptors: MW 475.4, ALogP 5.42, HBA 5, HBD 3, PSA 105.24, QED 0.41
Compound (SMILES): Cn1cc(-c2nc3ccccc3n2Cc2ccccc2)cc(N)c1=O
InChIKey: CBAYJUFZEDRHCY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (LysS, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
Measurement: IC50 = 1350 nM (pChEMBL 5.869999885559082)
Descriptors: MW 330.39, ALogP 3.03, HBA 5, HBD 1, PSA 65.84, QED 0.63
Compound (SMILES): c1ccc2nc(NCc3nc4c(N5CCOCC5)ccnn4c3-c3ccc(N4CCNCC4)nc3)ccc2c1
InChIKey: HRHQBRNRZWECQX-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 521.63, ALogP 3.2, HBA 10, HBD 2, PSA 95.74, QED 0.35
Compound (SMILES): Cc1cn(-c2cccc(C(=O)Nc3ccc(Oc4ccncc4)cc3)c2)cn1
InChIKey: TXKSFDQJSFSHRB-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 13 (SINGLE PROTEIN), Homo sapiens
UniProt: O15264
Assay: Immobilized Metal Affinity Polarization (IMAP) Assay: The MAPK13 blocking activity of test compounds was determined using an immobilized metal affinity polarization (IMAP) assay containing activated MAPK13, FITC-labeled substrate, and test compound. Full-length 6-His-tagged MAPK13 and constitutively active GST-MKK6 were prepared as described below. Activated MAPK13 was generated in 50 mM Hepes, 10 mM MgCl2 and 1 mM DTT, containing 1 uM MAPK13, 2 uM MKK6 and 50 uM ATP for 1 h at 25° C. MKK6 was removed by incubation with glutathione SEPHAROSE 4B beads (GE Healthcare Biosciences). MAPK13 activation was confirmed by Western blot using anti-phospho-p38-MAPK (T180/Y182) antibody (R& D Systems, Minneapolis, Minn.). IMAP assays were performed in 96-well non-treated half-area black plates or 384-well black plates (Corning Inc., Corning, N.Y.) in a final reaction volume of 20 ul using the linear phase of the rate kinetics. Assay reactions contained 0-100 uM test compound, 5-35 nM (EC80) activated MAPK13, 3 uM.
Measurement: IC50 = 15785 nM (pChEMBL 4.800000190734863)
Descriptors: MW 370.41, ALogP 4.62, HBA 5, HBD 1, PSA 69.04, QED 0.56
Compound (SMILES): COc1ccc(C(=O)C2CCN(C3CCN(Cc4ncc(C)c(=O)[nH]4)C3=O)CC2)cc1
InChIKey: VNXUSDDWRYWPQI-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 uL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 uM, 20 nM of purified enzyme, and 250 uM of p-NAD+ in the 1x Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 uL of 5x quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 uL of the 5x quenching solution per well was added prior to the addition of p-NAD+. The % Inhibition was calculated as: (Control-Sample)/(Control-Background)*100. "Control" is the average value of 8 wells without compound; and "Background" is the average of 8 wells mixed with 5x quenching solution measured prior to initiation of the reaction.
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 424.5, ALogP 1.78, HBA 6, HBD 1, PSA 95.6, QED 0.71
Compound (SMILES): COCCCOc1ccc2[nH]cc(C(=O)OCC34CCN(CC3)CC4)c2c1C
InChIKey: BHHJUQFZEDFVQX-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Alpha-Bungarotoxin Binding Assay: Protocol 2: Studies were carried out using rat brain P2 membrane. All membrane was obtained from male Sprague-Dawley (SD) rats and collected on phosphate buffer. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent. In an α-Bungarotoxin binding assay, membrane was diluted with assay buffer (50 mM potassium phosphate, 1 mM pH 8.0 EDTA, 0.1 mM PMSF, 0.1% BSA). Test compounds were prepared in 100% DMSO solution. The starting concentration of the test compound was 3 μM, 8 points in 3 fold dilution. Assays were performed in a total volume of 200 μL containing [3H] α-Bungarotoxin (final concentration 0.4 nM), membrane suspension (80 μL containing 150 μg of membrane protein) and 20 μL of test compound. Non-specific binding (NSB) was determined with 1 μM α-Bungarotoxin. After 3 hours incubation (37° C.), the assay solution was transferred to a GF/B plate (Millipore). The assay was terminated by adding 100 μL ice-cold PBS dilution and then rapidly filtering over glass fiber filters presoaked in 0.5% polyethylenimine. The filters were washed 4 times with 200 μL/well of ice-cold PBS. They were then were transferred to the reading plate, and 250 μL of microscine 40 was added. The radioactivity trapped by the filters was determined by liquid scintillation counting MicroBeta.
Measurement: Ki = 133.4 nM (pChEMBL 6.880000114440918)
Descriptors: MW 386.49, ALogP 3.53, HBA 5, HBD 1, PSA 63.79, QED 0.56
Compound (SMILES): C=Cc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C(F)(F)F)c3)cn2CCN(C)C)CC1
InChIKey: GBOPQOSLCOHLCP-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 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 503.55, ALogP 4.67, HBA 7, HBD 1, PSA 76.1, QED 0.47
Compound (SMILES): O=C(Nc1nc2ccc(Cl)cc2[nH]1)N1CCC(F)(c2ncc([C@@H](O)CO)cc2F)CC1
InChIKey: PTIKJUYDRZMMER-INIZCTEOSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 1879.8 nM (pChEMBL 5.730000019073486)
Descriptors: MW 451.86, ALogP 3.27, HBA 5, HBD 4, PSA 114.37, QED 0.49
Compound (SMILES): COc1cc(C)cc(OC)c1-c1cccc(C(CC(=O)O)c2nccs2)c1
InChIKey: SVFXWROCSTYJJT-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 383.47, ALogP 4.74, HBA 5, HBD 1, PSA 68.65, QED 0.64
Compound (SMILES): COc1ccc(Oc2nc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)c(Cl)c1
InChIKey: YOFHASCWNYTTSX-MLPAPPSSSA-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 441.84, ALogP 2.33, HBA 9, HBD 3, PSA 131.77, QED 0.39
Compound (SMILES): Cc1ccccc1CCc1c[nH]c(=O)c(O)c1
InChIKey: IQFVXXFWKKFBIZ-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: Enzyme Assay: The functional activity of compounds inhibiting the DAAO enzyme was determined by utilizing the co-product of the catalysis of D-Serine, H2O2 which can be quantitatively measured using the Amplex Red (Invitrogen) detection. Amplex Red reagent is a colorless substrate that reacts with hydrogen peroxide (H2O2) with a 1:1 stoichiometry in the presence of hydrogen peroxide to produce highly fluorescent resorufin (excitation/emission maxima=570/585 nm). The changes in fluorescence were monitored by a fluorescence plate reader, Envision (Perkin Elmer) and increases in DAAO activity were readily detected upon addition of D-Serine and suppression of this response observed with the application of test compounds.Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 229.28, ALogP 2.17, HBA 2, HBD 2, PSA 53.09, QED 0.85
Compound (SMILES): CC[C@H](C)[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@@H](C)NC1=O
InChIKey: CISPALQOSLQHKZ-MIUUGJTFSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 177 nM (pChEMBL 6.75)
Descriptors: MW 508.66, ALogP 2.36, HBA 5, HBD 4, PSA 108.56, QED 0.51
Compound (SMILES): CN(C/C=C/C(=O)N1CCCC(n2nc(-c3ccc(Oc4ccccc4)cc3)c3c(N)ncnc32)C1)CCO
InChIKey: PHFLKOZUXLCTPT-UXBLZVDNSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: HotSpot kinase assay: IC50s were determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 μM ATP.). For enzyme inhibition assays, compounds were tested in range of ten concentrations from 10 uM to 0.0005 uM using purified enzymes and the Hotspot kinase assay. Reaction conditions were 1 uM ATP, one hour incubation with inhibitor, and kinase activity detected using 33-ATP phosphorylation of an appropriately selected peptide substrate.
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 527.63, ALogP 3.51, HBA 9, HBD 2, PSA 122.63, QED 0.32
Compound (SMILES): COc1cc2c(cc1Cc1ccccc1)CC[C@@H]1CNCCCN21
InChIKey: JBGWELFZOGOSPC-LJQANCHMSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Agonist activity at unedited 5-HT2C receptor (unknown origin) expressed in HEK293 cells assessed as [3H]inositol phosphate accumulation after 2 hrs by scintillation counting
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 322.45, ALogP 3.4, HBA 3, HBD 1, PSA 24.5, QED 0.94
Compound (SMILES): Cc1ccc(C2=C(C#N)C(=O)NC(c3ccc(OCCC(F)(F)F)cc3)(C(F)(F)F)C2)cc1
InChIKey: WBACZIGNMMQGOU-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Mus musculus
UniProt: Q921V5
Assay: Inhibition of mouse recombinant MGAT2 expressed in Sf9 cell membrane using 2-oleoylglycerol and oleoyl-CoA as substrates incubated for 10 mins by LCMS assay
Measurement: IC50 = 236 nM (pChEMBL 6.630000114440918)
Descriptors: MW 468.4, ALogP 5.58, HBA 3, HBD 1, PSA 62.12, QED 0.58
Compound (SMILES): Cc1c(N(CCOC(C)(C)C)S(=O)(=O)c2ccc(C(=O)O)cc2)sc2ccccc12
InChIKey: XCSXUOCOXIXTIC-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 447.58, ALogP 4.92, HBA 5, HBD 1, PSA 83.91, QED 0.56