lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2C(c2ccc(S(=O)(=O)NCC(=O)NCC(=O)NCC(=O)NCCCCNC(=O)CNC(=O)CNC(=O)CNS(=O)(=O)c3ccc(C4CN(C)Cc5c(Cl)cc(Cl)cc54)cc3)cc2)C1
InChIKey: IIAFEYNFBCSAQK-UHFFFAOYSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE-3-mediated Na+ dependent H+ antiport expressed in opossum kidney cells in presence of NHE-1 antagonist EIPA by BCECF-AM dye based fluorescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 1138.98
Compound (SMILES): NS(=O)(=O)c1ccc(NC(=O)c2c(-c3c(Cl)cccc3Cl)noc2NC(=O)c2ccccc2)cc1
InChIKey: XCMWGESLCUAXCF-UHFFFAOYSA-N
Target: Carbonic anhydrase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00915
Assay: Inhibition of recombinant human carbonic anhydrase 1 assessed as reduction in CO2 hydration preincubated for 15 mins by stopped flow assay
Measurement: Ki = 3654.6 nM (pChEMBL 5.440000057220459)
Descriptors: MW 531.38, ALogP 4.8, HBA 6, HBD 3, PSA 144.39, QED 0.33
Compound (SMILES): CN(C)c1ccc(C(=O)Nc2n[nH]c3cc(OCc4ccccc4)ccc23)cc1
InChIKey: CQMXHWIVXMYLBS-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Biochemical Assay: The assay was performed using FLT3 cytoplasmic domain product and purified in house as GST fused protein. The FLT3 protein (1 microM) was pre activated with 800 microM ATP for 1 hour at 28° C in order to obtain a linear kinetic. Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 4 mM MgCl2, 1 mM DTT, 10 microM Na3VO4, and 0.2 mg/mL BSA. The FLT3 kinase assay was run with a final pre activated enzyme concentration of 2 nM, in the presence of 254 microM ATP (residual ATP from KIT pre activation step is negligible), 8 nM 33P-γ-ATP and 55 microM of substrate BioDB n*24 (Aminoacidic sequence: GGKKKVSRSGLYRSPSMPENLNRPR- SEQ ID NO: 1).
Measurement: IC50 = 3175 nM (pChEMBL 5.5)
Descriptors: MW 386.46, ALogP 4.46, HBA 4, HBD 2, PSA 70.25, QED 0.51
Compound (SMILES): CN(c1nccc(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)c1C#N)C1CCCCC1
InChIKey: QSIUMYFNRAHVLO-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 643.77, ALogP 6.91, HBA 9, HBD 2, PSA 135.34, QED 0.19
Compound (SMILES): Cc1noc(C2CCCN(C(=O)c3ccc4cc(Oc5ccccn5)ccc4n3)C2)n1
InChIKey: IIGHWIYRODPGEC-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 415.45, ALogP 4.13, HBA 7, HBD 0, PSA 94.24, QED 0.49
Compound (SMILES): Cc1cc(Nc2cc(N3CCC3)nc(O[C@H]3CCN(C(=O)OC(C)(C)C)C3)n2)[nH]n1
InChIKey: SPWVJJKODRLXIM-AWEZNQCLSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Inhibition of human Itk kinase using SAM68 protein (delta 332 to 443 residues) substrate and and [gamma-33P]-ATP pre-incubated for 10 mins before [gamma-33P]-ATP addition and measured after 10 mins by by liquid scintillation counting method
Measurement: Ki < 1000 nM (pChEMBL 6)
Descriptors: MW 415.5, ALogP 2.85, HBA 8, HBD 2, PSA 108.5, QED 0.77
Compound (SMILES): O=C(Nc1ccc(C(=O)N2CCN(c3ncc(C(F)(F)F)cc3Cl)CC2)cc1)N1Cc2ccccc2C1
InChIKey: OORCSWNNSAIVMN-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 = 178 nM (pChEMBL 6.75)
Descriptors: MW 529.95, ALogP 5.26, HBA 4, HBD 1, PSA 68.78, QED 0.5
Compound (SMILES): COC(=O)C[C@@H]1N=C(c2ccc(Nc3ccccc3)cc2)c2c(sc(C)c2C)-n2c(C)nnc21
InChIKey: OOWCQDANWVVOPI-NRFANRHFSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: An expression vector containing cDNA of BRD2, 3 and 4 added with Flag-tag was transfected to CHO cell, and a cell lysate was prepared 24 hr later. Binding of acetylated histone H4 and BRD was confirmed by a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method. To a 384-well white plate (manufactured by Coaster) were added 50 nmol/L biotin-labeled acetylated histone H4 peptide (manufactured by Upstate) and a serially diluted test compound. Furthermore, CHO cell lysate transfected with BRD expression vector, a europium-labeled anti-Flag antibody (manufactured by Cisbio), and XL-665-labeled avidin (manufactured by Cisbio) were added, and the mixture was reacted at room temperature for 30 min to 2 hr. The fluorescence by FRET was measured by EnVision 2103 Multilabel Reader (manufactured by Perkin Elmer).
Measurement: IC50 = 123.5 nM (pChEMBL 6.909999847412109)
Descriptors: MW 471.59, ALogP 5.45, HBA 8, HBD 1, PSA 81.4, QED 0.39
Compound (SMILES): CC1(C)OC(c2ccnc(NCCO)n2)=C(c2cc(Cl)cc(NS(=O)(=O)c3cc(F)ccc3F)c2F)C1=O
InChIKey: RXOXMNNLXKQKEG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: LanthaScreen Kinase Assay: The BRAFV600E enzymatic assay was performed using a LanthaScreen kinase assay kit purchased from Life Technologies (Grand Island, N.Y.). The assay was conducted according to the procedure provided in the assay kit. In brief, the enzyme reaction was carried out in the kinase reaction buffer containing BRAFV600E (20 ng/mL), ATP (2 uM), Fluorescein-MAP2K1 inactive substrate (0.4 uM), HEPES (50 mM, pH 7.5), 0.01% BRIJ-35, MgCl2 (10 mM), and EGTA (1 mM) in the presence or absence of the tested compounds at various concentrations in a 384-well plate at room temperature (22±1° C.) for 60 minutes. The final reaction volume for each reaction was 10 ul. The reaction was stopped by addition of 10 ul of TR-FRET dilution buffer kinase supplemented with kinase quench buffer (10 mM final) and Tb-anti-pMAP2K1 (2 nM final). The plate was further incubated at room temperature for another 60 minutes, and the fluorescent signals were read on Victor 5 (Perkin Elmer) with excitation at 340 nM and emission at 495 and 520 nM. The assay signal was determined as a ratio of FRET-specific signal measured with emission filter at 520 nM to that of the signal measured with Tb-specific emission filter at 495 nM. IC50 value was calculated using appropriate programs in GraphPad Prism by plotting the logarithm of the concentration versus percent inhibition.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 568.96, ALogP 4, HBA 8, HBD 3, PSA 130.51, QED 0.37
Compound (SMILES): Cc1ccc(-n2nc(C3CCN(C4CCC(C(F)(F)F)CC4)CC3)cc2NC(=O)c2cnn3cccnc23)nc1
InChIKey: JTFNKLOSSNBDCO-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 552.61, ALogP 5.17, HBA 8, HBD 1, PSA 93.24, QED 0.37
Compound (SMILES): N#C[C@]1(Cc2cccc(Nc3cc[nH]n3)n2)CC[C@@H](Oc2cccc(Cl)c2F)CC1
InChIKey: DVRASPTYCUVMOS-CIEDQVTBSA-N
Target: Aurora kinase B/Inner centromere protein (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9NQS7
Assay: Inhibition of human full-length N-terminal GST-tagged Aurora B/His-tagged INCENP (803 to 916 residues) using kemptide as substrate and [gamma33P]ATP after 45 mins by liquid scintillation counting method
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 425.89, ALogP 5.41, HBA 5, HBD 2, PSA 86.62, QED 0.55
Compound (SMILES): CC[C@H](CC#N)NC(=O)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: HZSBUBAAHZEUNE-GOSISDBHSA-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 = 109 nM (pChEMBL 6.960000038146973)
Descriptors: MW 362.43, ALogP 3.66, HBA 3, HBD 2, PSA 85.23, QED 0.85
Compound (SMILES): O=C(NCCCCCCNC(=O)N1Cc2ccccc2C1)c1cccc(C(F)(F)F)c1
InChIKey: WNOIAXSXUXJMDE-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 = 8.39 nM (pChEMBL 8.079999923706055)
Descriptors: MW 433.47, ALogP 4.72, HBA 2, HBD 2, PSA 61.44, QED 0.59
Compound (SMILES): NC(=O)c1ccc2[nH]nc(-c3cccc(NC(=O)Cc4ccc(F)cc4)c3)c2c1
InChIKey: IOYIZGRAYJMRSZ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 388.4, ALogP 3.65, HBA 3, HBD 3, PSA 100.87, QED 0.49
Compound (SMILES): C#CCC(C(=O)Nc1ccc(C(=O)O)cc1)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: BVJKTUAUDCSIJC-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 475.89, ALogP 3.95, HBA 6, HBD 2, PSA 121.42, QED 0.5
Compound (SMILES): C/C(=C\c1cc(F)c(Oc2ccc(S(=O)(=O)NCCOCCOCCOCCN)cc2)c(F)c1)C(=O)N=C(N)N
InChIKey: PHPJDFTXBBRTHD-SAPNQHFASA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 125.89 nM (pChEMBL 6.900000095367432)
Descriptors: MW 585.63, ALogP 1.25, HBA 8, HBD 4, PSA 190.58, QED 0.09
Compound (SMILES): CC(C(=O)NC[C@H](C)O)c1ccc(O[C@@H]2CCN(c3ccc(OCC4CC4(F)F)cn3)C2)cc1
InChIKey: QFLBKOYCTHTQES-YJBOJQDKSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction. Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetylCoenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 1883 nM (pChEMBL 5.71999979019165)
Descriptors: MW 475.54, ALogP 3.37, HBA 6, HBD 2, PSA 83.92, QED 0.55
Compound (SMILES): Cc1cccc2nc([C@H](C)Nc3ncnc(N)c3C#Cc3ccc(F)cn3)n(-c3cccc(CN(C)S(C)(=O)=O)c3)c(=O)c12
InChIKey: QJQBVMBUGKZIQB-FQEVSTJZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 612.69, ALogP 3.56, HBA 10, HBD 2, PSA 148.99, QED 0.26
Compound (SMILES): CC(C)CN(Cc1ccc(N[C@H]2CC[C@H](O)CC2)cc1)S(=O)(=O)Cc1ccccc1
InChIKey: PUYZRMSKPCIHIS-RQNOJGIXSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Assay: On day of the assay, 100 uL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20x required final concentration in DMSO (15 uL compound+30 uL DMSO). The 20x compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5x the final test concentration in 25% DMSO (10 uL compound+30 uL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 uL volume. For the assay, 10 uL of 5x compound stock solutions in 25% DMSO were added to the assay plate in duplicate.
Measurement: IC50 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 430.61, ALogP 4.39, HBA 4, HBD 2, PSA 69.64, QED 0.62
Compound (SMILES): C[C@H]1CN(Cc2ccc3c(-c4cnn(C)c4)cc(C(N)=O)nc3c2)C[C@@H](C(F)(F)F)O1
InChIKey: KTYWQIWONODXST-BUXKBTBVSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 433.43, ALogP 2.89, HBA 6, HBD 1, PSA 86.27, QED 0.68
Compound (SMILES): COc1c(Nc2cc3cc(-c4cnn(CC(C)(C)O)c4)ccc3cn2)ccc(-c2cnc(C)n2C)c1F
InChIKey: WMUAOQMVCLAFLU-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: The enzyme reaction (total volume 10 μl) was carried out in black 384-well low volume plates containing full length MPS1 (12.5 nM or 3 nM), fluorescent labelled peptide [known as H236, which has the sequence: 5FAM-DHTGFLTEYVATR-CONH2](5 μM), ATP (10 μM), either DMSO (1% v/v) or the test compound (in the range 0.25 nM-100 μM in 1% DMSO) and assay buffer (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovandate, 10 μM MgCl2, 1 μM DTT, Roche protease inhibitor). The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μl) containing 20 mM EDTA, 0.05% (v/v) Brij-35, in 0.1M HEPES-buffered saline (Free acid, Sigma, UK). The plate was read on a Caliper EZ reader II (Caliper Life Sciences).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 500.58, ALogP 5.47, HBA 8, HBD 2, PSA 90.02, QED 0.31
Compound (SMILES): COc1cc(C(=O)N[C@H](C)c2ccccc2)ccc1-c1cn[nH]c1
InChIKey: ZKQKBOWBNMYREE-CYBMUJFWSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 5.91 nM (pChEMBL 8.229999542236328)
Descriptors: MW 321.38, ALogP 3.58, HBA 3, HBD 2, PSA 67.01, QED 0.75
Compound (SMILES): Cc1nn(C2CCCCC2)c2sc(C(=O)NC3CCC(C(=O)NC4CC4)CC3)cc12
InChIKey: TWGLDLKFUQPZAW-UHFFFAOYSA-N
Target: High affinity 3',5'-cyclic-AMP phosphodiesterase 7A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13946
Assay: Inhibition Assay: The PDE 7 (phosphodiesterase VII) inhibiting effect of the compounds of the present invention was performed by the following method, which was modified assay method described in Biochemical. Pharmacol. 48(6), 1219-1223 (1994).  (1) The active fraction of PDE 7 (phosphodiesterase VII) was obtained. That is, MOLT-4 (obtainable from ATCC as ATCC No. CRL-1582), which was cell line of human acute lymphoblastic lymphoma T cells, was incubated in RPMI1640 culture medium containing 10% fetal bovine serum to obtain 5x108 MOLT-4 cells. The cells were collected by centrifugation and suspended with 10 mL of buffer solution A [25 mM of tris-HCl, 5 mM of 2-mercaptoethnol, 2 mM of benzamidine, 2 mM of EDTA, 0.1 mM of 4-(2-aminoethyl)benzensulfonyl hydrochloride; pH 7.5], then homogenized by Polytron homogenizer. The homogenate were centrifuged under 25,000xG for 10 minutes at 4 C. The supernatant was separated and thus obtained supernatant was further centrifuged under 100,000xG for 60 minutes.
Measurement: IC50 = 7.8 nM (pChEMBL 8.109999656677246)
Descriptors: MW 428.6, ALogP 4.48, HBA 5, HBD 2, PSA 76.02, QED 0.74
Compound (SMILES): CC1(C)CC(=O)C=C(c2ccc3[nH]nc(-c4cc(N5CCC(F)(F)CC5)ncn4)c3c2)C1
InChIKey: BNJMAIOOVQAZCK-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 7.08 nM (pChEMBL 8.149999618530273)
Descriptors: MW 437.49, ALogP 5.03, HBA 5, HBD 1, PSA 74.77, QED 0.62
Compound (SMILES): COc1ccc(C(=O)Nc2cnc(F)c([C@]3(C)C[C@@H](C(F)(F)F)OC(N)=N3)c2)nc1
InChIKey: FLWXUGDYFSVDDG-GUYCJALGSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 119 nM (pChEMBL 6.920000076293945)
Descriptors: MW 427.36, ALogP 2.76, HBA 7, HBD 2, PSA 111.72, QED 0.57
Compound (SMILES): CN(C)CCn1cc(-c2ccc(F)c(C(F)(F)F)c2)nc1C1CCN(c2ncnc(N)c2C(N)=O)CC1
InChIKey: GDBKJDBGCLFSGA-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 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 520.54, ALogP 3.12, HBA 8, HBD 2, PSA 119.19, QED 0.46
Compound (SMILES): CC(O)c1cc(C(=O)NCc2c(F)cccc2Cl)ccc1-c1cn[nH]c1
InChIKey: WQKAWEFODUZYFB-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 373.82, ALogP 3.85, HBA 3, HBD 3, PSA 78.01, QED 0.64
Compound (SMILES): CC(C)Oc1ccc(Nc2ncc(NC(=O)N3CCOCC3)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: BNYLBWZEXYTLPY-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 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 in M MgCl2, 5.0 mM CaCl2, 5.6 D-glucose, 2.5 mM probenecid, 1.0% BSA, and 0.08% Pluronic F-127, pH 7.5) 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 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence inte
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 515.97, ALogP 4.48, HBA 7, HBD 2, PSA 97.72, QED 0.48
Compound (SMILES): Cc1cnn(-c2cc(-c3cc(F)c([C@]4(C)CS(=O)(=O)N(C)C(=N)N4)s3)ccn2)c1
InChIKey: OJGHMQGAJTWXOQ-SFHVURJKSA-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 = 2783 nM (pChEMBL 5.550000190734863)
Descriptors: MW 434.52, ALogP 2.46, HBA 7, HBD 2, PSA 103.97, QED 0.66
Compound (SMILES): CC(C)n1c(=O)nc(Nc2ccc(OC3CCOCC3)c(Cl)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: DDNUNYHYRGZFMC-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 = 116 nM (pChEMBL 6.940000057220459)
Descriptors: MW 505.4, ALogP 4.64, HBA 8, HBD 1, PSA 87.38, QED 0.5
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(NC3CCN(C(=O)c4cccc5c6c([nH]c45)CCCC6)CC3)nc2)c1
InChIKey: LBRSGIXXKZBWJA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 625.7, ALogP 7.69, HBA 6, HBD 3, PSA 98.83, QED 0.18
Compound (SMILES): CCc1cc(CN(CC(C)C)C(CC(=O)O)c2ccc(Cl)cc2)ccc1OCCN1C(=O)CCC1=O
InChIKey: TWFPYTXAUJBOEA-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Radioligand Binding Assay: The composition of the binding assay buffer is determined in a course of detailed optimization procedure. This resulted in a binding assay buffer constituted by the following components: 25 mM Hepes (pH=7.4), 5 mM MgCl2, 1 mM CaCl2, 100 mM NaCl, supplemented with 0.1% of protease free BSA (as a final concentration). Competition binding assay is performed using 125I-CXCL10 (PerkinElmer, NEX348, specific activity 2200 Ci/mmol) radioligand in a final concentration of 50-70 pM. The nonspecific binding is defined by 150 pM of hr-CXCL10 (R&D Systems, Cat No 266-IP). The total assay volume is equal to 150 ul and contained 1% of DMSO (final concentration). Binding reaction is initiated by adding of membranes (10-20 ug proteins, approximately 5x105 cell equivalents) to the reaction mixture. After 60 minutes of incubation at 25° C. the reaction is terminated by rapid filtration over GF/B glass fibre filters that are pre-soaked with 0.5% polyethyleneimine (Fluka Analytical, P3143) for 1 hour, using a Skatron cell harvester device. Filters then are washed with 8 ml of ice-cold wash buffer (modified binding buffer in which BSA is omitted and the concentration of NaCl is adjusted to 500 mM concentration). The radioactivity retained on the filters is measured by a Wizard 1470 Automatic Gamma counter.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 515.05, ALogP 5.1, HBA 5, HBD 1, PSA 87.15, QED 0.37
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3c(F)cncc3F)CC2)C1
InChIKey: LVWFVGNNMGWEOB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 503.52, ALogP 2.72, HBA 8, HBD 1, PSA 119.62, QED 0.44
Compound (SMILES): O=c1c(-c2ccc(Oc3ccccc3)cc2)[n+]([O-])c2ccccc2n1O
InChIKey: ZBEOQOJRQWNLHK-UHFFFAOYSA-N
Target: CDGSH iron-sulfur domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZ45
Assay: Displacement of [3H]rosiglitazone from recombinant human C-terminal His-tagged MitoNEET cytosolic domain (32 to 108 residues) expressed in Escherichia coli BL21 by scintillation proximity assay
Measurement: IC50 = 9657 nM (pChEMBL 5.010000228881836)
Descriptors: MW 346.34, ALogP 3.33, HBA 5, HBD 1, PSA 78.4, QED 0.35
Compound (SMILES): Cc1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)c2ccc(C(=O)NS(C)(=O)=O)cc2)sc2ccccc12
InChIKey: XLSSWWODRCAGEZ-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 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 600.64, ALogP 5.45, HBA 6, HBD 1, PSA 100.62, QED 0.28
Compound (SMILES): CC1CN(C(=O)c2cc(Cc3n[nH]c(=O)c4ccccc34)ccc2F)Cc2nnc(CF)n21
InChIKey: NCVASQJZWCCSRY-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Inhibition of PARP1 (unknown origin) incubated for 1 hr by ELISA analysis
Measurement: IC50 = 11.9 nM (pChEMBL 7.920000076293945)
Descriptors: MW 450.45, ALogP 2.93, HBA 6, HBD 1, PSA 96.77, QED 0.52
Compound (SMILES): CC(C)(CO)n1cc(C(=O)c2cc(NC(=O)Cn3cc(C(F)(F)F)nn3)ccn2)c2cnccc21
InChIKey: IHQWATCZTFERNR-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 79.3 nM (pChEMBL 7.099999904632568)
Descriptors: MW 487.44, ALogP 2.64, HBA 9, HBD 2, PSA 127.82, QED 0.38
Compound (SMILES): CC(C)Oc1ccc2[nH]nc(-c3cc(N4CCC4)ncn3)c2c1
InChIKey: BGDVWAKQHOHIKW-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 7.9 nM (pChEMBL 8.100000381469727)
Descriptors: MW 309.37, ALogP 3.02, HBA 5, HBD 1, PSA 66.93, QED 0.8
Compound (SMILES): COc1ccc2nc(-c3cn4c(C)nnc4c(NC(C)C)n3)n(Cc3ccccn3)c2c1
InChIKey: WKNJQMMPCXOMRN-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4-H4 Tetraacetylated Peptide Inhibition AlphaScreen: This assay is used to determine whether the compounds inhibit the interaction between the first (BRD4-BD1) or the second (BRD4-BD2) bromodomain of BRD4 and the tetraacetylated histone H4 peptide.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 (Lys5, 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.Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (probe molecule JQ1+ with protein/peptide mix) is pipetted. Determination of IC50 values are carried out using GraphPad Prism 3.03 software (or updates thereof).
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 428.5, ALogP 3.72, HBA 9, HBD 1, PSA 95.05, QED 0.44
Compound (SMILES): CCc1cccnc1-n1nnc2c1CCN(C(=O)c1cccc(C(F)(F)F)c1)C2C
InChIKey: OCPWYQMYUFUHMS-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 415.42, ALogP 4, HBA 5, HBD 0, PSA 63.91, QED 0.65