lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(-c2noc(C3(N(C)C)CC3)n2)cc1NC(=O)c1cnc2ccccn12
InChIKey: TTXRVIZQMULATC-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 402.46, ALogP 3.5, HBA 7, HBD 1, PSA 88.56, QED 0.55
Compound (SMILES): O=C(Nc1cc2[nH]nc(OCC(F)F)c2cn1)N[C@@H](c1ccccc1)C1(O)CC1
InChIKey: IPLXFAMLZKIWRP-INIZCTEOSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 403.39, ALogP 2.99, HBA 5, HBD 4, PSA 112.16, QED 0.48
Compound (SMILES): O=C(NCCCCNC(=O)NCCOCCOCCNS(=O)(=O)c1ccc(O[C@H]2c3cc(Cl)cc(F)c3C[C@@H]2N2CC[C@@H](O)C2)cc1)NCCOCCOCCNS(=O)(=O)c1ccc(O[C@H]2c3cc(Cl)cc(F)c3C[C@@H]2N2CC[C@@H](O)C2)cc1
InChIKey: VSLGVHBORZOXHE-CINCTQFYSA-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 > 10000 nM (pChEMBL 5)
Descriptors: MW 1256.29
Compound (SMILES): COCCNc1ccc(CN(CC(C)C)S(=O)(=O)Cc2ccccc2)cc1
InChIKey: SIESSLJIPXCORM-UHFFFAOYSA-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 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 390.55, ALogP 3.73, HBA 4, HBD 1, PSA 58.64, QED 0.59
Compound (SMILES): CCC#CC(=O)Nc1cc(-c2cnc3[nH]cc(C)c3c2)ccc1F
InChIKey: ZWNWMTPLGGPZKE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: In Vitro Inhibition Assay: ITK and JAK3 Kinase assay procedures:Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μL of 3% phosphoric acid. Volume was increased to 100 μL by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μL 0.8% phosphoric acid and dried for an hour at 60° C. 100 μL scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 321.36, ALogP 4.03, HBA 2, HBD 2, PSA 57.78, QED 0.7
Compound (SMILES): O=C(O)CNC(=O)C1=C(O)CCN(Cc2ccc(Oc3ccc(F)cn3)cc2)C1=O
InChIKey: ZRYLATKAQFUATG-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Fluorescence Polarization Assay: The enzyme and the substrate were each diluted with a 50 mM tris-hydrochloric acid buffer (pH 7.5) containing 12.5 mM KCl, 3.75 mM MgCl2, 25 μM iron sulfate, 5 mM ascorbic acid, and 2.5 mM DTT, whereas each test compound was diluted with dimethyl sulfoxide (DMSO).A test compound and the substrate solution were preliminarily added onto 384-well plates and reaction was initiated by adding a human PHD2 enzyme solution (40 ng/well). After 20-min incubation at 30° C., an EDTA-containing quench solution was added and the amount of the proline residues hydroxylated via binding to an added HIF-OH antibody solution was quantified by fluorescence polarization.
Measurement: IC50 = 146 nM (pChEMBL 6.840000152587891)
Descriptors: MW 415.38, ALogP 1.76, HBA 6, HBD 3, PSA 129.06, QED 0.59
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3ccc(CN4CCOCC4)nc3)n2)cnc1N1CCC2(COC2)C1
InChIKey: UAOPNFWYMCGENQ-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 484.56, ALogP 2.61, HBA 10, HBD 1, PSA 112.32, QED 0.56
Compound (SMILES): N[C@]1(C(=O)O)[C@@H]2[C@@H](C(=O)O)[C@@H]2[C@@H](O)[C@H]1OCc1ccc(Cl)c(Cl)c1
InChIKey: WNOZOUHFGZKGNT-AFJGUQMISA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Antagonist activity at recombinant human mGlu2 receptor expressed in hamster AV12 cells co-expressing human EAAT1 assessed as reduction of DCG-4-inhibited forskolin-stimulated cAMP formation after 20 mins by HTRF assay
Measurement: IC50 = 169 nM (pChEMBL 6.769999980926514)
Descriptors: MW 376.19, ALogP 0.98, HBA 5, HBD 4, PSA 130.08, QED 0.6
Compound (SMILES): CCOC(=O)c1c(NC(=O)C23CC4CC(CC(C4)C2)C3)sc2c1CCCC2
InChIKey: KFAHCXHRNCAASD-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Displacement of [3H]-CP-55940 from human CB2 receptor expressed in HEK293 cell membranes after 90 mins by radioligand binding assay
Measurement: Ki = 2.15 nM (pChEMBL 8.670000076293945)
Descriptors: MW 387.55, ALogP 4.96, HBA 4, HBD 1, PSA 55.4, QED 0.74
Compound (SMILES): COc1cc(C(=O)O)ccc1C1CC(C(C)(C)C)=NN1c1ccc(C(=O)O)cc1
InChIKey: PRYOWZYJMVPUMQ-UHFFFAOYSA-N
Target: cGMP-specific 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O76074
Assay: Fluorescence Polarization Assay: PDE activity was measured using the IMAP fluorescence polarization assay (Molecular Devices) in which binding of hydrolyzed cyclic nucleotide substrate to immobilized metal coordination complexes increases fluorescence polarization (FP). Tetramethylrhodamine (TAMRA)-cGMP and fluorescein-cAMP were used as substrates, each at final concentration of 50 nmol/L. The PDE assay was done according to the manufacturer's specifications using either whole cell lysates or recombinant enzymes. FP was measured at excitation, emission wavelengths of either 530,590 nm for TAMRA-cGMP or 485,530 nm for fluorescein-cAMP using a Synergy4 (Biotek) microplate reader.
Measurement: IC50 = 23500 nM (pChEMBL 4.630000114440918)
Descriptors: MW 396.44, ALogP 4.45, HBA 5, HBD 2, PSA 99.43, QED 0.77
Compound (SMILES): O=C(Nc1ccc(-c2noc(CNCC3CCCO3)n2)cc1)N1Cc2ccccc2C1
InChIKey: QOWDLDGHQDNWPQ-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 = 121 nM (pChEMBL 6.920000076293945)
Descriptors: MW 419.49, ALogP 3.55, HBA 6, HBD 2, PSA 92.52, QED 0.63
Compound (SMILES): CCCCCCNC(=O)n1cc(C)c(=O)n(CC2CC2)c1=O
InChIKey: HYJYBQBOCAIURU-UHFFFAOYSA-N
Target: Acid ceramidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q6P7S1
Assay: Enzymatic Assay: r-AC protein samples were pre-incubated with various concentrations of test compounds or vehicle control in 100 mM NaH2PO4/citrate buffer pH 4.5, 0.1% Nonidet P-40, 3 mM DTT for 30 min at 37° C. Samples were incubated with 100 μM N-lauroyl ceramide (Nu-Chek Prep, Elysian, Minn.) at 37° for 30 min. The reaction was stopped by addition of a mixture of chloroform/methanol (2:1 vol/vol) containing 1 nmol of heptadecanoic acid (HDA; NuChek Prep). The organic phases were collected, dried under nitrogen, and analyzed by LC/MS in the negative-ion mode using heptadecanoic acid (HDA) as internal standard (m/z=199 for lauric acid, m/z=269 for HDA). HDA was eluted on an XDB Eclipse C18 column isocratically at 2.2 mL/min for 1 min with a solvent mixture of 95% methanol and 5% water, both containing 0.25% acetic acid, and 5 mM ammonium acetate. The column temperature was 50° C.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 307.39, ALogP 1.87, HBA 5, HBD 1, PSA 73.1, QED 0.78
Compound (SMILES): COc1cc(-c2ccc(Cl)c(C)c2)ccc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ncc(F)cn3)ccc21
InChIKey: JEMJQXXYPDRHPT-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 551, ALogP 5.36, HBA 7, HBD 1, PSA 103.18, QED 0.31
Compound (SMILES): Cc1cc(Nc2nc(Nc3cccc(OCCN4CCOCC4)c3)ncc2F)cc(C)c1O
InChIKey: MOTAYZUQAGJQCJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Biochemical Assay: Several 2,4-pyrimidinediamine compounds were tested for the ability to inhibit Syk kinase catalyzed phosphorylation of a peptide substrate in a biochemical fluorescenced polarization assay with isolated Syk kinase. In this experiment, Compounds were diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin). Compound in 1% DMSO (0.2% DMSO final) was mixed with ATP/substrate solution at room temperature. Syk kinase (Upstate, Lake Placid N.Y.) was added to a final reaction volume of 20 uL, and the reaction was incubated for 30 minutes at room temperature. Final enzyme reaction conditions were 20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin, 0.125 ng Syk, 4 uM ATP, 2.5 uM peptide substrate (biotin-EQEDEPEGDYEEVLE-CONH2, SynPep Corporation). EDTA (10 mM final)/anti-phosphotyrosine antibody (1x final)/fluorescent phosphopeptide tracer (0.5x final) was added in FP Dilution Buffer to stop the reaction for a total volume of 40 uL according to manufacturer's instructions (PanVera Corporation) The plate was incubated for 30 minutes in the dark at room temperature. Plates were read on a Polarion fluorescence polarization plate reader (Tecan). Data were converted to amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit, Green (PanVera Corporation).
Measurement: IC50 = 770.5 nM (pChEMBL 6.110000133514404)
Descriptors: MW 453.52, ALogP 4.14, HBA 8, HBD 3, PSA 91.77, QED 0.44
Compound (SMILES): COCc1cnc(C2=CCN(C(=O)Nc3ccc(C(F)(F)F)cc3)CC2)c(Cl)c1
InChIKey: DVXMITAJSZZILO-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 915.7 nM (pChEMBL 6.039999961853027)
Descriptors: MW 425.84, ALogP 5.22, HBA 3, HBD 1, PSA 54.46, QED 0.73
Compound (SMILES): CCCn1ccc2cc(/N=c3\[nH]c(=O)n(CC(C)(C)C(=O)O)c(=O)n3Cc3ccc(Cl)cc3)ccc21
InChIKey: OWGZMAZZYUSLCJ-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 = 652 nM (pChEMBL 6.190000057220459)
Descriptors: MW 509.99, ALogP 3.75, HBA 7, HBD 2, PSA 114.38, QED 0.38
Compound (SMILES): Cc1ncccc1NC(=O)c1ccc2c(c1)CC[C@@H]1[C@@H]2[C@@H](O)C[C@@]2(C)[C@H]1CC[C@@]2(O)C#C[Si](C)(C)C
InChIKey: FLEOABKAHZYTNM-SXRKIBQPSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Agonist activity at GR in human HepG2 cells assessed as protein mediated-transcriptional activity by MMTV-promoter driven luciferase reporter gene assay
Measurement: EC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 502.73
Compound (SMILES): NC(=O)c1nn(-c2ccc(F)cc2)c2c1CCc1ccc(NC(=O)CNC3CCC3)cc1-2
InChIKey: UNPCLUIVEANAQY-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus using biotinylated IkappaBalpha peptide as substrate assessed as decrease in [gamma-33P]-ATP incorporation in substrate after 30 mins by scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 433.49, ALogP 2.96, HBA 5, HBD 3, PSA 102.04, QED 0.56
Compound (SMILES): Cc1cncc2nc(CCc3nc(N4CCCC4)nn3C)nn12
InChIKey: BZAJFYLFGGRRBB-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Scintillation Proximity Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer Top-Count Scintillation plate reader.
Measurement: IC50 = 251.42 nM (pChEMBL 6.599999904632568)
Descriptors: MW 312.38, ALogP 0.95, HBA 8, HBD 0, PSA 77.03, QED 0.71
Compound (SMILES): CCC(CC)(c1ccc(C(=O)NCCCCCC(=O)NO)cc1)c1ccc(OCC(O)C(C)(C)C)c(C)c1
InChIKey: YNVOUPQJNGMIGD-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Fluorogenic Inhibition Assay: Boc-Lys(Ac)-7-amino-4-methylcoumarin (Boc-Lys(Ac)-AMC) was used as substrate for the HDAC assays. Substrate solution was prepared as follow: Boc(Lys-Ac)-AMC was dissolved in DMSO and diluted with HDAC buffer (15 mM Tris-HCl [pH 8.1], 250 Î¼M EDTA, 250 mM NaCl, 10% glycerol) to give 1 mM solutions containing 1.7% DMSO. Trypsin was used to stop the reaction, releasing free AMC. The trypsin solution was prepared as follow: trypsin was dissolved in HDAC buffer to give a concentration of 10 mg/mL. Release of AMC was monitored by measuring the fluorescence at 460 nm (lex=390 nm) with a microplate reader (SpectraMax Gemini) at 37° C. The AMC signals were recorded against a blank with buffer, substrate and trypsin but without the enzyme. All experiments were carried out at least in triplicate. For HDAC inhibition assays, inhibitor diluted in 50 μL of HDAC buffer was mixed with 10 μL of diluted enzyme solution in HDAC buffer at room temperature. The HDAC reaction was started by adding 40 μL of substrate solution in HDAC buffer followed by 30 min of incubation with stirring at 37° C. The reaction was stopped by adding 100 μL, of trypsin solution. After a 10 min incubation with stirring at 37° C., the release of AMC was monitored by measuring the fluorescence.
Measurement: IC50 = 557 nM (pChEMBL 6.25)
Descriptors: MW 526.72, ALogP 5.68, HBA 5, HBD 4, PSA 107.89, QED 0.14
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(=O)O)NC(=O)CN1CCN(CCNC(=O)CNC(=O)[C@H](CSCNC(C)=O)NC(=O)[C@H](CO)NC(=O)CN(C)C)CC1)C(C)C)C(N)=O
InChIKey: GXCZZATWOZASHE-WIRSSUKHSA-N
Target: Gastrin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30550
Assay: Displacement of [125I]-BBN from GRPR in human PC3 cells after 1 hr by compuGamma counting or microplate scintillation counting method
Measurement: IC50 > 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 1627.92
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ncccn1)c1nc2cccc(Cl)c2c(=O)n1-c1cccnc1
InChIKey: PFPJQUHQSYTNQN-HNNXBMFYSA-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 495.93, ALogP 3.17, HBA 10, HBD 2, PSA 137.39, QED 0.36
Compound (SMILES): O=C(Nc1ccc(Oc2ccnc(NC(=O)C3CC3)c2)c(F)c1)Nc1ccc2[nH]cc(F)c2c1
InChIKey: XYTFGVXDSQVGBZ-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 = 8.2 nM (pChEMBL 8.09000015258789)
Descriptors: MW 463.44, ALogP 5.63, HBA 4, HBD 4, PSA 108.14, QED 0.3
Compound (SMILES): CCCN(/N=C/c1ccccc1C(=O)O)c1nc(-c2ccccc2Cl)cs1
InChIKey: CFIKDBUCHDKKLE-WSDLNYQXSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Method for Testing Activities of Compounds at Enzyme Level: Principles of activity test at enzyme level: firstly, dihydroorotate (DHO) is oxidatively dehydrogenated by DHODH to form orotic acid (Orotate, OA), accompanied by reduction of Flavin mononucleotide (FMN) into reduced flavin mononucleotide (FMNH2) by accepting 2H+ and 2e−, then coenzyme Q (CoQ) as hydrogen acceptor accepting electron and proton from FMNH2 is reduced into reduced coenzyme Q (CoQH2), reduced coenzyme Q transfers electrons to a chromogenic substrate, diclofenac sodium salt (DCIP), and finally DCIP was reduced. DCIP shows maximum absorption at 600 nm, while the reduced DCIP does not show absorption at 600 nm. The degree to which the substrate DHO is oxidized can be judged based on the degree of decrease in absorbance. The degree of oxidation of the substrate DHO per unit time is the initial rate of the enzymatic reaction. Upon addition of a inhibitor, the initial rate of the enzymatic reaction is reduced.The purified DHODH was diluted to 10 nM with a test buffer (50 mM HEPES, pH 8.0, 150 mM KCl, 0.1% Triton X-100), and coenzyme Q and DCIP were added to a final concentration of 100 μM and 120 μM, respectively, mixed well, added into a 96-well plate and incubated for 5 min at room temperature. Substrate DHO was added to start the reaction with a DHO final concentration of 500 μM. The absorbance was read at 600 nm using a BioTek microplate reader and read every 30 s for 6 min. For inhibitor activity test, different concentrations of inhibitors were added into the above reaction system, the initial rate of enzymatic reaction when no inhibitor was added was V0, and the initial rate of enzymatic reaction after an inhibitor was added was Vi, and the inhibition rate of a compound was calculated by the formula (1−Vi/V0)×100%. For calculating IC50 of a compound, Origin 8.0 was used to test the inhibition rate at least 8 concentrations. In the experimental procedure, Brequinar (purchased from Sigma-Aldrich Reagent) was used as a positive control and at least three parallels were set in each experiment.
Measurement: IC50 = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 399.9, ALogP 5.41, HBA 5, HBD 1, PSA 65.79, QED 0.43
Compound (SMILES): NC(=O)c1cc(-c2ccc(F)cc2)c2ccc(Cn3c(-c4ccccn4)nc4ccccc43)cc2n1
InChIKey: AXZSHLZFGWIKBF-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 189 nM (pChEMBL 6.71999979019165)
Descriptors: MW 473.51, ALogP 5.6, HBA 5, HBD 1, PSA 86.69, QED 0.35
Compound (SMILES): CCc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)cc3)cn2CCN2CC3(COC3)C2)CC1
InChIKey: VYDMLEVVUWMMDW-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 = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 491.62, ALogP 3.34, HBA 8, HBD 1, PSA 85.33, QED 0.54
Compound (SMILES): CC(C)n1cc(C(=O)c2cncc(NC(=O)Cc3ccccc3Cl)c2)c2cnccc21
InChIKey: FNAXXBNJEVMVSW-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 = 2370 nM (pChEMBL 5.619999885559082)
Descriptors: MW 432.91, ALogP 5.08, HBA 5, HBD 1, PSA 76.88, QED 0.43
Compound (SMILES): N=C(N)NCCC[C@@H]1N[C@H](CNC(=O)c2cc3ccccc3cn2)CCN(CC(c2ccccc2)c2ccccc2)C1=O
InChIKey: XBQSFLUOEFLRMW-JDXGNMNLSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 577.73, ALogP 3.62, HBA 5, HBD 5, PSA 136.23, QED 0.1
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=N[C@@]2(c2cc(NC(=O)c3ccc(C#N)cn3)cs2)CO1
InChIKey: YIXDWYJNAHJADD-BLWXERESSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG Patch Clamp Assay: All testing was carried out in CHO cells transfected with the hERG gene purchased from Millipore (PrecisION hERG-CHO Recombinant Cell Line CYL3038). The cell line was grown in DMEM/F-12, GlutaMAX with 10% fetal bovine serum, 1% Penicillin-Streptomycin, 1% Geneticin and 1% of 1M HEPES buffer solution, and maintained at approximately 37 °C. in a humidified atmosphere containing 5% carbon dioxide. The cells were passaged every 3-5 days based on confluency. On the day of the experiment, 50%-80% confluent cells were harvested from a 175 cm2 culture flask using Detachin. After 10 minutes of exposure to Detachin at 37 °C., the cells were centrifuged for 1 minute at 1000 RPM. The supernatant was removed and the cell pellet was reconstituted in 5-8 mL of serum free media with 2.5% of 1M HEPES, placed on the Qstirrer, and allowed to recover. After a 30 minute recovery period, experiments were initiated.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 413.53, ALogP 2.95, HBA 8, HBD 2, PSA 113.39, QED 0.8
Compound (SMILES): CC1(C)c2ccc(OCc3ccccc3)cc2C(=O)c2c1[nH]c1cc(Br)ccc21
InChIKey: DYKAUAJLGLVRSU-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 492.73 nM (pChEMBL 6.309999942779541)
Descriptors: MW 446.34, ALogP 6.38, HBA 2, HBD 1, PSA 42.09, QED 0.4
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(CCC3CC3)cc1C21N=C(N)N(Cc2cscn2)C1=O
InChIKey: XLAWOIFIICYOEX-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 = 8.8 nM (pChEMBL 8.0600004196167)
Descriptors: MW 464.64, ALogP 4.17, HBA 6, HBD 1, PSA 80.81, QED 0.7
Compound (SMILES): O=S(=O)(c1ccccc1)N(Cc1ccccc1)c1sc2ccccc2c1Br
InChIKey: XJSJPNRKQKUCBM-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 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 458.4, ALogP 6.06, HBA 3, HBD 0, PSA 37.38, QED 0.36
Compound (SMILES): Cc1cc(N=S(C)(C)=O)cc2ncnc(Nc3ccc(F)cc3OCC3CCO3)c12
InChIKey: LVBYHBWUWBGNCF-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In vitro Kinase Assay: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 430.51, ALogP 4.35, HBA 7, HBD 1, PSA 85.7, QED 0.63
Compound (SMILES): COC(=O)c1cc2occc2n1CC(=O)c1ccc(C)cc1
InChIKey: YKEGUZPBQJMJGX-UHFFFAOYSA-N
Target: N-acetyltransferase Eis (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WFK7
Assay: Inhibition of Mycobacterium tuberculosis H37Rv Eis expressed in Escherichia coli BL21(DE3) using Acetyl-CoA as substrate preincubated for 10 mins followed by substrate addition measured every 30 sec for 10 mins
Measurement: IC50 = 5800 nM (pChEMBL 5.239999771118164)
Descriptors: MW 297.31, ALogP 3.21, HBA 5, HBD 0, PSA 61.44, QED 0.55
Compound (SMILES): CC1CCC(c2cc(C(N)=O)nc3cc(CN4C(=O)CCC4=O)ccc23)CC1
InChIKey: JQDAUOJPMYCOOC-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 379.46, ALogP 3.28, HBA 4, HBD 1, PSA 93.36, QED 0.83
Compound (SMILES): Cc1nc2cnc3cc(F)c(-c4ccc(Oc5cccn(C)c5=O)cc4Cl)cc3c2n1C1CCN(C(=O)[C@H](C)O)CC1
InChIKey: QYBCFSAUZVOGTA-KRWDZBQOSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: TR-FRET Assay: [MEK1 1mM ATP IC50 uM] A BRAF-MEK-ERK cascade assay is used to evaluate the effects of these compounds as inhibitors of the MAP kinase pathway. An enzymatic cascade assay is set up using recombinant human activated BRAF (V599E) kinase (Cat No. 14-557), human full length MEK1 kinase (Cat No. 14-706) and human full length active MAP Kinase 2/ERK2 (Cat No. 14-536) enzymes procured from Upstate. TR-FRET (Time resolved fluorescence resonance energy transfer) detection technology is used for the read out. The assay buffer solution contains 50 mM Tris pH 7.5, 10 mM MgCl2 , 1 mM DTT, 0.01% Tween 20, 0.1 nM activated BRAF, 2 nM inactive MEK1,10 nM inactive ERK2, 1 mM ATP and 500 nM long chain biotin-peptide substrate (LCB- FFKNIVTPRTPPP) in a 384 well format. The kinase reaction is stopped after 90 minutes with 10 mM EDTA and Lance detection mix (2 nM Eu-labeled phospho-serine/threonine antibody (Cat. No. AD0176-Perkin Elmer), 20 nM SA-APC (Cat No. CR130-100-Perkin Elmer) is added. The TR-FRET signal (Excitation at 340 nm, Emission at 615 nm and 665 nm) is read with 50 μs delay time on a Victor3 V fluorimeter.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 590.06, ALogP 5.39, HBA 8, HBD 1, PSA 102.48, QED 0.29
Compound (SMILES): Cc1cc(=O)[nH]c2ccc(OCCCCN3CCN(c4cccc5sccc45)CC3)cc12
InChIKey: XVWAQKVKYOOITD-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P61169
Assay: Dopamine D2 Receptor Binding Assay: The assay was performed according to the method by Kohler et al. (Kohler C, Hall H, Ogren S O and Gawell L, Specific in vitro and in vivo binding of 3H-raclopride. A potent substituted benzamide drug with high affinity for dopamine D-2 receptors in the rat brain. Biochem. Pharmacol., 1985; 34: 2251-2259). The binding assay was performed using 40 ul of the membrane specimen, 20 ul of [3H]-raclopride (final concentration 1 to 2 nM), 20 ul of a test drug and 50 mM Tris-hydrochloric acid buffer (containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) so that the total amount was 200 ul (final dimethylsulfoxide concentration 1%). The reaction was performed at room temperature for 1 hour and terminated by conducting suction filtration with a cell harvester on a glass fiber filter plate. The filter plate made of glass fiber was washed with 50 mM Tris-hydrochloric acid buffer (pH 7.4), and after dried, a microplate liquid scintillation cocktail was added.
Measurement: Ki = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 447.6, ALogP 5.03, HBA 5, HBD 1, PSA 48.57, QED 0.4
Compound (SMILES): CCC(O)c1nc(C)nn1-c1cc(N2CC(c3nc4ccccc4n3C)C2)nc(C)n1
InChIKey: WSLFTXBMJFPNHV-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 nL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/1600 dilution from aliquots; sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP PDE from Molecular Devices (product # R7506), at a final concentration of 50 nM are made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). The enzyme and the substrate are then added to the assay plates in two consecutive additions of 10 μL, and then shaken to mix. The reaction is allowed to proceed at room temperature for 30 minutes. A binding solution is then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction is stopped by addition of 60 μL of the binding solution to each well of the assay plates and the plates are sealed and shaken for 10 seconds. The plate was incubated at room temperature for at least one hour prior to determining the fluorescence polarization (FP). The parallel and perpendicular fluorescence of each well of the plate was measured using a Perkin Elmer EnVision™ plate reader (Waltham, Mass.).
Measurement: Ki = 0.51 nM (pChEMBL 9.289999961853027)
Descriptors: MW 418.51, ALogP 2.61, HBA 9, HBD 1, PSA 97.78, QED 0.53
Compound (SMILES): CCN(c1cc(Br)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1Cl)[C@H]1CC[C@H](N(C)C)CC1
InChIKey: HHWNUBJZWXWWEE-WGSAOQKQSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 3.4 nM (pChEMBL 8.470000267028809)
Descriptors: MW 537.93, ALogP 5.04, HBA 4, HBD 2, PSA 68.44, QED 0.52