lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc([C@H]2C[C@@H]2COc2cc(NCc3cnc(C)s3)c(=O)n(C)n2)nc1
InChIKey: CYPMGOZGXXKRNR-ZBFHGGJFSA-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).
Measurement: Ki = 0.011 nM (pChEMBL 10.960000038146973)
Descriptors: MW 397.5, ALogP 3.04, HBA 8, HBD 1, PSA 81.93, QED 0.66
Compound (SMILES): CC(CO)NC(=O)C(C)c1ccc(O[C@@H]2CCN(c3ncnc(OCC(C)(F)F)c3F)C2)cc1
InChIKey: GRWMAMGXIZKDRR-JTTJXQCZSA-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 = 838 nM (pChEMBL 6.079999923706055)
Descriptors: MW 482.5, ALogP 2.91, HBA 7, HBD 2, PSA 96.81, QED 0.54
Compound (SMILES): Cc1cc(N=S(C)(C)=O)cc2ncnc(Nc3ccc(F)cc3O[C@H](C)C(=O)NC3COC3)c12
InChIKey: QLMITVVFQXTVID-CQSZACIVSA-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 487.56, ALogP 3.46, HBA 8, HBD 2, PSA 114.8, QED 0.52
Compound (SMILES): CC[C@@H](C)NC(=O)C(C)c1ccc(O[C@@H]2CCN(c3ncnc(OCC(C)(C)O)c3Cl)C2)cc1
InChIKey: PNXPTVIWQHCENN-RMRANGLJSA-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 = 472 nM (pChEMBL 6.329999923706055)
Descriptors: MW 491.03, ALogP 3.96, HBA 7, HBD 2, PSA 96.81, QED 0.52
Compound (SMILES): NC(=O)C1CCCCNC(=O)[C@@H](Cc2ccc(C(=O)c3ccccc3)cc2)NC(=O)[C@H](CC2CCCCC2)NC(=O)C(CO)NC(=O)/C=C/C(=O)N1
InChIKey: FYHDGQGGLUWGBZ-BDROYEGLSA-N
Target: Insulin-degrading enzyme (SINGLE PROTEIN), Homo sapiens
UniProt: P14735
Assay: IDE Activity Assay: Macrocycles were assayed for IDE inhibition by monitoring cleavage of a substrate peptide containing a fluorophore-quencher pair such that cleavage of the model substrate results in increased fluorescence. Although our selection did not explicitly select for inhibition (only binding is required for enrichment), all four strongly enriched macrocycles inhibited IDE activity with IC50<10uM or equal to 10uM. The most potent macrocycle, trans-A12-B8-C6-D5 (6b), inhibited IDE with an IC50 of 140 nM. The three-dimensional conformation adopted by these compounds appears to be important to their inhibitory activity, as the trans-olefin isomers of macrocycles 3, 5, and 6 exhibit >10-fold stronger potency than the corresponding cis-olefin macrocycle isomers (Table 1). Compounds 1 and 4, which contained glycine (A2) instead of D-benzoylphenylalanine (A12) at the A position were inactive against IDE.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 716.84, ALogP 0.7, HBA 8, HBD 7, PSA 225.89, QED 0.19
Compound (SMILES): CSc1cccc([C@@H](CO)NC(=O)c2ccc(-c3nc(C4CCOCC4)cnc3N)cc2F)c1
InChIKey: RKUQEKVAKZGSGS-JOCHJYFZSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.01 nM (pChEMBL 11)
Descriptors: MW 482.58, ALogP 3.94, HBA 7, HBD 3, PSA 110.36, QED 0.44
Compound (SMILES): COc1ccc([C@H]2C[C@@H]2COc2cc(NCc3cnn(C)c3N(C)C)c(=O)n(C)n2)nc1
InChIKey: JQXSFFSRQUNMBM-PBHICJAKSA-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).
Measurement: Ki = 42.6 nM (pChEMBL 7.369999885559082)
Descriptors: MW 439.52, ALogP 1.78, HBA 10, HBD 1, PSA 99.33, QED 0.54
Compound (SMILES): Cc1cc(N=S2(=O)CCC2)cc2ncnc(Nc3ccc(F)cc3O[C@H](C)C(=O)NC(C(F)(F)F)C(F)(F)F)c12
InChIKey: JVNFBEPYSAUPEH-CYBMUJFWSA-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 = 1 nM (pChEMBL 9)
Descriptors: MW 593.53, ALogP 5.7, HBA 7, HBD 2, PSA 105.57, QED 0.34
Compound (SMILES): O=c1[nH]c(N2CCOCC2)nc(N[C@@H]2CCCNC2)c1-c1ccc2ccccc2n1
InChIKey: HBCQWZVJTOBKNR-MRXNPFEDSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IMAP technology (Molecular Devices): A 20 ul reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 uM 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 ul 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, LJL BioSystems).
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 406.49, ALogP 1.99, HBA 7, HBD 3, PSA 95.17, QED 0.61
Compound (SMILES): Nc1n[nH]c2cc(NC(=O)N[C@H](CO)c3ccccc3)ncc12
InChIKey: JIXRLALPFQBVPI-GFCCVEGCSA-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. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 312.33, ALogP 1.4, HBA 5, HBD 5, PSA 128.95, QED 0.5
Compound (SMILES): CCOc1ccc(/N=c2\[nH]c(=O)n(C[C@@H](C)C(=O)O)c(=O)n2Cc2ccc(Cl)cc2)cc1C
InChIKey: WZNRHGGFRRUHKW-OAHLLOKOSA-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 = 198 nM (pChEMBL 6.699999809265137)
Descriptors: MW 472.93, ALogP 2.7, HBA 7, HBD 2, PSA 118.68, QED 0.52
Compound (SMILES): CN(C)CCCOc1ccc(-c2ccc3ncc4c(c3c2)n([C@@H]2CCOC2)c(=O)n4C)c(F)n1
InChIKey: YEFCFNFQLXFZGI-QGZVFWFLSA-N
Target: Serine-protein kinase ATM (SINGLE PROTEIN), Homo sapiens
UniProt: Q13315
Assay: Cellular Assay: Assay a) HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 3500 cells/well in 40 μl EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compounds of Formula (I) in 100% DMSO were added to assay plates by acoustic dispensing. After 1 h incubation at 37° C. and 5% CO2 plates (up to 6 at a time) were irradiated using the X-RAD 320 instrument (PXi) with equivalent to Ë¿600 cGy. Plates were returned to the incubator for a further 1 h. Then cells were fixed by adding 20 μl of 3.7% formaldehyde in PBS solution and incubating for 20 minutes at r.t. before being washed with 50 μl/well PBS, using a Biotek EL405 plate washer. Then 20 μ 0.1% Triton X100 in PBS was added and incubated for 20 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50 μl/well PBS, using a Biotek EL405 plate washer. Phospho-ATM Ser1981 antibody (Millipore #MAB3806) was diluted 10000 fold in PBS containing 0.05% polysorbate/Tween and 3% BSA and 20 μl was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and then 20 μl of secondary Ab solution, containing 500 fold diluted Alexa Fluor® 488 Goat anti-rabbit IgG (Life Technologies, A11001) and 0.002 mg/ml Hoeschst dye (Life technologies #H-3570), in PBS containing 0.05% polysorbate/Tween and 3% BSA, was added. After 1 h incubation at r.t., the plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and plates were sealed and kept in PBS at 4° C. until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10× objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405 nm) and secondary antibody staining of pSer1981 (488 nm).
Measurement: IC50 = 1.44 nM (pChEMBL 8.84000015258789)
Descriptors: MW 465.53, ALogP 3.38, HBA 8, HBD 0, PSA 74.41, QED 0.31
Compound (SMILES): N#CC1(NC(=O)[C@@H]2C[C@@H](S(=O)(=O)c3ccc(Cl)cn3)CN2C(=O)C2(c3ncc(Cl)cc3F)CC2)CC1
InChIKey: XIVTVSMXEWBQIK-WBVHZDCISA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Enzyme Inhibition Assay: Enzyme activity is measured by observing the increase in fluorescence intensity caused by cleavage of a peptide substrate containing a fluorophore whose emission is quenched in the intact peptide.Assay buffer: 100 mM potassium phosphate pH 6.5, EDTA-Na 5 mM, Triton X-100 0.001%, DTT 5 mM.Enzymes (all at 1 nM): human and mouse Cathepsin S, Cat K, Cat B, Cat L.Substrate (20 μM): Z-Val-Val-Arg-AMC, except for Cat K which uses Z-Leu-Arg-AMC (both from Bachem).Z=Benzyloxycarbonyl.AMC=7-Amino-4-Methyl-Coumarin.DTT=dithiothreitol.Final volume: 100 μL.Excitation 360 nm, Emission 465 nm.Enzyme is added to the substance dilutions in 96-well microtitre plates and the reaction is started with substrate. Fluorescence emission is measured over 20 minutes, during which time a linear increase is observed in the absence of inhibitor.
Measurement: IC50 = 2.996 nM (pChEMBL 8.520000457763672)
Descriptors: MW 552.42, ALogP 2.57, HBA 7, HBD 1, PSA 133.12, QED 0.58
Compound (SMILES): Cc1cc2c3c([nH]c2cc1Cl)C(C)(C)c1cc(OC[C@@H](O)[C@H](O)CO)ccc1C3=O
InChIKey: PUAVYRMIRKEKNE-RTBURBONSA-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 = 1664.82 nM (pChEMBL 5.78000020980835)
Descriptors: MW 429.9, ALogP 3.09, HBA 5, HBD 4, PSA 102.78, QED 0.5
Compound (SMILES): COc1cccc(-c2nc(C(=O)Nc3cnn(C)c3[C@@H]3CC[C@@H](N)[C@H](F)CO3)cs2)c1F
InChIKey: NFLPKRRWALYAGV-XNJGSVPQSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO. PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000® (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
Measurement: Ki = 0.036 nM (pChEMBL 10.4399995803833)
Descriptors: MW 463.51, ALogP 3.46, HBA 8, HBD 2, PSA 104.29, QED 0.6
Compound (SMILES): Cc1nc([C@H](NC(=O)c2cccc(-c3ccc(F)c(Cl)c3)n2)C2CC2)no1
InChIKey: AMDKVUYZLMDDAS-QGZVFWFLSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 10.1 nM (pChEMBL 8)
Descriptors: MW 386.81, ALogP 4.11, HBA 5, HBD 1, PSA 80.91, QED 0.71
Compound (SMILES): Cc1ccc2sc(-c3c(N[C@@H]4CCCNC4)nc(N4CCOCC4)[nH]c3=O)nc2c1
InChIKey: TZUXTMLEAKQRKO-CQSZACIVSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IMAP technology (Molecular Devices): A 20 ul reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 uM 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 ul 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, LJL BioSystems).
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 426.55, ALogP 2.36, HBA 8, HBD 3, PSA 95.17, QED 0.59
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4C(NC(C(=O)O)c4ccccc4)c4ccccc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: CWRICNOKPCBBSF-UJVLHDJOSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Radioligand Dose-Displacement Binding Assays (mu): Radioligand dose-displacement binding assays for mu-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2,1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hr at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 ul/well), and plates were counted using a Packard Top-Count.
Measurement: Ki = 278.49 nM (pChEMBL 6.550000190734863)
Descriptors: MW 620.79, ALogP 6.08, HBA 6, HBD 2, PSA 80.26, QED 0.28
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@@H]3C[C@@H]3C[C@H]2C(=O)NC[C@@H]2C(C)(C)C2(Cl)Cl)c2ccc(OCc3ncccn3)cc12
InChIKey: PSNBKXYTIBAZFE-ZINXYGQCSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Complement Factor D Assay: Method 1: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter.
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 585.49, ALogP 3.54, HBA 8, HBD 1, PSA 119.31, QED 0.3
Compound (SMILES): O=C(N[C@H]1CC[C@H](c2cccc(F)c2F)[C@H](F)c2cccnc21)c1cnc2c(c1)C[C@@]1(C2)C(=O)Nc2ncccc21
InChIKey: OSDLWDUUSYIRGY-PLRKAVCOSA-N
Target: Calcitonin gene-related peptide type 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q16602
Assay: Recombinant Receptor Functional Assay C: RECOMBINANT RECEPTOR FUNCTIONAL ASSAY (Assay C): Cells were resuspended in DMEM/F12 (Hyclone) supplemented with 1 g/L BSA and 300 uM isobutyl-methylxanthine. Cells were then plated in a 384-well plate (Proxiplate Plus 384; 509052761; Perkin-Elmer) at a density of 3,500 cells/well and incubated with antagonist for 30 min at 37° C. Human α-CGRP was then added to the cells at a final concentration of 1 nM and incubated an additional 20 min at 37° C. Following agonist stimulation, the cells were processed for cAMP determination using the two-step procedure according to the manufacturer's recommended protocol (HTRF cAMP dynamic 2 assay kit; 62AM4PEC; Cisbio).
Measurement: IC50 = 570 nM (pChEMBL 6.239999771118164)
Descriptors: MW 555.56, ALogP 5.2, HBA 5, HBD 2, PSA 96.87, QED 0.34
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N2[C@@H](COC)CC[C@@H]2COC)n2c1COCC2)c1ccccc1
InChIKey: DRFFKJUBHQARRC-KCZVDYSFSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 820 nM (pChEMBL 6.090000152587891)
Descriptors: MW 469.58, ALogP 3.17, HBA 6, HBD 1, PSA 82.03, QED 0.61
Compound (SMILES): C[C@@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(-c2ccc(Cl)cc2)no1
InChIKey: HUKBLSVUBDBPMC-JTNHKYCSSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 213 nM (pChEMBL 6.670000076293945)
Descriptors: MW 430.85, ALogP 3.06, HBA 9, HBD 2, PSA 126.5, QED 0.61
Compound (SMILES): N#Cc1ccc2c(c1)C(=O)N[C@H](C(=O)NC1(C#N)CC1)Cc1ccc(c(Cl)c1)OC/C=C/CO2
InChIKey: COFICJUHEUQSGC-ZGTHZTNPSA-N
Target: Procathepsin L (SINGLE PROTEIN), Homo sapiens
UniProt: P07711
Assay: Cathepsin Enzyme Inhibition Assay: Enzyme activity is measured by observing the increase in fluorescence intensity caused by cleavage of a peptide substrate containing a fluorophore whose emission is quenched in the intact peptide. Assay buffer: 100 mM potassium phosphate pH 6.5, EDTA-Na 5 mM, Triton X-100 0.001%, DDT 5 mM. Enzymes (all at 1 nM): human and mouse Cathepsin S, Cat K, Cat B, Cat LSubstrate (20 uM): Z-Val-Val-Arg-AMC, except for Cat K which uses Z-Leu-Arg-AMC (both from Bachem). Z=Benzyloxycarbonyl. AMC=7-Amino-4-Methyl-Coumarin. Final volume: 100 uL. Excitation 360 nm, Emission 465 nm. Enzyme is added to the substance dilutions in 96-well microtitre plates and the reaction is started with substrate. Fluorescence emission is measured over 20 minutes, during which time a linear increase is observed in the absence of inhibitor.
Measurement: IC50 = 6.9 nM (pChEMBL 8.15999984741211)
Descriptors: MW 476.92, ALogP 3.05, HBA 6, HBD 2, PSA 124.24, QED 0.64
Compound (SMILES): Cc1cc(-c2n[nH]c3ccc(C(=O)N[C@@H]4CCC[C@@](O)(Cn5cncn5)C4)cc23)ccn1
InChIKey: OHAWTTBQBDLXCC-JPYJTQIMSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Mus musculus
UniProt: P63085
Assay: Active ERK2 (aERK) Assay: Activated ERK2 activity was also determined in the IMAP assay format using the procedure outlined above. 1 μl of 25× compound was added to 14 μl of kinase buffer containing 0.25 ng fully phosphorylated, active Mouse ERK2 protein. Following a 30 minute incubation, the reactions were initiated by addition of 10 μl of kinase buffer containing 1 μM ERK2 IMAP substrate peptide (0.9 μM unlabeled IPTTPITTTYFFFK-CONH2 and 100 nM IPTTPITTTYFFFK(5-carboxyfluorescein)-CONH2) and 30 μM ATP. Reactions proceeded for 30 minutes before termination by addition of 60 μl IMAP detection beads in binding buffer. Plates were read as above after 30 minute binding equilibration. Active compounds were reconfirmed in an independent assay.
Measurement: IC50 = 99.9 nM (pChEMBL 7)
Descriptors: MW 431.5, ALogP 2.63, HBA 7, HBD 3, PSA 121.61, QED 0.45
Compound (SMILES): C=CC[C@@H](N)c1ccc(Cl)c(C(=O)c2ccc(N)nc2)c1F
InChIKey: CFAIQVFFEUCTRK-GFCCVEGCSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 319.77, ALogP 3.26, HBA 4, HBD 2, PSA 82, QED 0.65
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)NCC(C)(C)O)n2c1COCC2)c1ccccc1
InChIKey: ZTQHLADDVSZZGX-QGZVFWFLSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 2500 nM (pChEMBL 5.599999904632568)
Descriptors: MW 399.49, ALogP 2.4, HBA 5, HBD 3, PSA 92.59, QED 0.67
Compound (SMILES): CO[C@H]1CC[C@H](c2cnc(N)c(-c3ccc(C(=O)N[C@H](CN)c4cccc(Br)c4)c(F)c3)n2)CC1
InChIKey: ZYBUNYMKRZVXEV-YIFZBPKDSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.0689 nM (pChEMBL 10.15999984741211)
Descriptors: MW 542.45, ALogP 4.73, HBA 6, HBD 3, PSA 116.15, QED 0.4
Compound (SMILES): CN(C)CC(C)(C)COC(=O)N1CC[C@@H](n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)[C@H](C#N)C1
InChIKey: DELNJGJNUBNCFS-OXQOHEQNSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 485.56, ALogP 2.98, HBA 8, HBD 2, PSA 129.51, QED 0.59
Compound (SMILES): Cn1cc(CNc2cc(OC[C@H]3C[C@@H]3c3ccc(OC(F)F)cn3)nn(C)c2=O)cn1
InChIKey: PVWFZGSZWVFUEM-HIFRSBDPSA-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).
Measurement: Ki = 0.552 nM (pChEMBL 9.260000228881836)
Descriptors: MW 432.43, ALogP 2.3, HBA 9, HBD 1, PSA 96.09, QED 0.55
Compound (SMILES): C[Si](C)(C)CCNC(=O)[C@@H]1C[C@H]2C[C@H]2N1C(=O)Cn1nc(C(N)=O)c2cccnc21
InChIKey: WZWXJGLRBCOCKR-YUELXQCFSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: In Vitro Assay: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter.
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 428.57
Compound (SMILES): COC(=O)c1ccc2c(c1)-c1cccc(c1)[C@H](N1CCC(c3c(Br)ccc(Cl)c3F)=CC1=O)CCC[C@@H](C)C(=O)N2
InChIKey: BSNSKODJGQKGRG-DNOBIOAJSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: In Vitro Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki > 624.8 nM (pChEMBL 6.203999996185303)
Descriptors: MW 639.95, ALogP 7.81, HBA 4, HBD 1, PSA 75.71, QED 0.23
Compound (SMILES): O=C(Nc1ccc(SC(F)(F)Cl)cc1)c1cnc(N2CC[C@@H](O)C2)c(-c2cncnc2)c1
InChIKey: SYYIIGJFTFOOLB-MRXNPFEDSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radio ABL1 (64-515) Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 uL of the compound pre-diluted with 10 uL of ATP (20 uM ATP with 0.1 uCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 uL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 mM at room temperature, the reaction was stopped by the addition of 50 uL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3x with 0.5% H3PO4, followed by addition of 30 uL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 477.92, ALogP 4.24, HBA 7, HBD 2, PSA 91.24, QED 0.4
Compound (SMILES): CCNC(=O)C(C)c1ccc(O[C@@H]2CCN(c3ccc(OC4CCCCC4)nc3)C2)cc1
InChIKey: HLUKSXWXECATJD-JKSFWZLDSA-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 = 583 nM (pChEMBL 6.230000019073486)
Descriptors: MW 437.58, ALogP 4.69, HBA 5, HBD 1, PSA 63.69, QED 0.65
Compound (SMILES): C#CCN(Cc1ccc(OC)cc1)c1cc(OC[C@H]2C[C@@H]2c2ccc(OC)cn2)nn(C)c1=O
InChIKey: WCCCVEUVNRVULZ-KNQAVFIVSA-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).
Measurement: Ki = 7.9 nM (pChEMBL 8.100000381469727)
Descriptors: MW 460.53, ALogP 3.01, HBA 8, HBD 0, PSA 78.71, QED 0.43
Compound (SMILES): Cc1cc(N=S2(=O)CCC2)cc2ncnc(Nc3ccc(F)cc3O[C@H](C)C(=O)NCCF)c12
InChIKey: NENATNOETYQYFC-OAHLLOKOSA-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 489.55, ALogP 4.18, HBA 7, HBD 2, PSA 105.57, QED 0.49
Compound (SMILES): Nc1nc([C@@H]2CC[C@H]2c2nc3c(cnn3C3CCC(F)(F)CC3)c(=O)[nH]2)cs1
InChIKey: OIJUAOFHVQHSKX-GHMZBOCLSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Scintillation Proximity Assay: The PDE9A2 enzymatic activity assay was run as scintillation proximity assay (SPA), in general according to the protocol of the manufacturer (GE Healthcare, former Amersham Biosciences, product number: TRKQ 7100). As enzyme source, lysate (PBS with 1% Triton X-100 supplemented with protease inhibitors, cell debris removed by centrifugation at 13.000 rpm for 30 min) of SF 9 cell expressing the human PDE9A2 was used. The total protein amount included in the assay varied upon infection and production efficacy of the SF9 cells and lay in the range of 0.1-100 ng. In general, the assay conditions were as follows: total assay volume: 40 microlitre; protein amount: 0.1-50 ng; substrate concentration (cGMP): 20 nanomolar; ~1 mCi/l; incubation time: 60 min at room temperature; final DMSO concentration: 0.2-1%. The assays were run in 384-well format. The test reagents as well as the enzyme and the substrate were diluted in assay buffer. The assay buffer contained 50 mM Tris, 8.3 mM MgCl2, 1.7 mM EGTA, 0.1% BSA, 0.05% Tween 20; the pH of assay buffer was adjusted to 7.5. The reaction was stopped by applying a PDE9 specific inhibitor (e.g. compounds according to WO 2004/099210 or WO 2004/099211, like one of the enantiomers of example 37, e.g. 1-(2-Chlorophenyl)-6-[2R)-3,3,3-trifluoro-2-methyl-propyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one) in excess.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 406.46, ALogP 3.57, HBA 7, HBD 2, PSA 102.48, QED 0.69
Compound (SMILES): COc1ccc(C[C@@H](NC(=O)CCc2nc[nH]c2C)C(=O)N2CC(OCC3CC3)(c3ccccc3C)C2)cc1
InChIKey: NIJVIWSFUPHAMB-MUUNZHRXSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: Transactivation Assay: The products are dissolved at 10 mM in DMSO. They are tested as a response dose at 0.1% of DMSO final. The range comprising 10 points and a zero starts at 10 μM with four-fold dilutions. To test agonists, the products are tested alone. To determine the antagonist behaviour, the products of interest are tested in the presence of 1 nM NDP-MSH (reference agonist). The cells are inoculated at a rate of 5000 cells per well (384-well plate) in serum-free DMEM medium and incubated overnight at 37° C. and 5% CO2. The products and the reference ligand (NDP-MSH) are added the following day and the plates are reincubated for 6 hours at 37° C. and 5% CO2. After adding the lysis buffer containing luciferin, the plates are read in a Top-Count machine.
Measurement: EC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 530.67, ALogP 3.86, HBA 5, HBD 2, PSA 96.55, QED 0.37
Compound (SMILES): COCCOc1n[nH]c2cc(NC(=O)N[C@@H](c3ccc(F)cc3)C3(O)CCC3)nc(CO)c12
InChIKey: QGTYPACXDKHGOD-IBGZPJMESA-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 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 459.48, ALogP 2.39, HBA 7, HBD 5, PSA 141.62, QED 0.31