lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=c1[nH]c(NC2CCC2)nc(N[C@@H]2C[C@H](CO)[C@@H](O)[C@H]2O)c1-c1nccs1
InChIKey: NUMJYVAXNJCDKI-DXLKZPDWSA-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 = 1114 nM (pChEMBL 5.949999809265137)
Descriptors: MW 393.47, ALogP 0.37, HBA 9, HBD 6, PSA 143.39, QED 0.41
Compound (SMILES): COc1n[nH]c2cc(NC(=O)N[C@H](C)c3ccccc3)nc(-c3nnc(C)o3)c12
InChIKey: LRHRZAOJPDXFKN-SNVBAGLBSA-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 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 393.41, ALogP 3.21, HBA 7, HBD 3, PSA 130.85, QED 0.47
Compound (SMILES): COCCCOc1ccc2c(c1)[C@H](N(C(=O)[C@H]1CNCCO1)C1CC1)CC2
InChIKey: ZIEDPAVQQOPNPM-WOJBJXKFSA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: Inhibitory Assay: A substrate of synthetic peptide (Nma-KHPFHLVIHK(Dnp)-NH2) and test compound were mixed, and fluorescence intensity was assayed using a fluorophotometer before staring an enzymatic reaction (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Recombinant human renin was added and the mixture was incubated at 37° C. for 1 hour, and the fluorescence intensity was measured after the reaction using a fluorophotometer (exciting wavelength: 340 nm, measuring wavelength: 460 nm). Renin activity was evaluated on the ground of fluorescence intensity which was obtained by deduction of the intensity before the reaction from the intensity after the reaction, and 50% inhibitory concentration (IC50) was calculated from renin activities under the existence of various concentration of the test compound.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 374.48, ALogP 2.07, HBA 5, HBD 1, PSA 60.03, QED 0.71
Compound (SMILES): COc1n[nH]c2cc(NC(=O)N[C@@H](c3ccccc3)[C@H](O)CO)ncc12
InChIKey: IXBNHNVZAFYSPA-HIFRSBDPSA-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 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 357.37, ALogP 1.18, HBA 6, HBD 5, PSA 132.39, QED 0.45
Compound (SMILES): COC(=O)c1ccc2c(c1)-c1cccc(c1)[C@H](N1CCC(c3c(F)ccc(Cl)c3F)=CC1=O)CCC[C@@H](C)C(=O)N2
InChIKey: PQMIPRJGMKRLRT-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 > 398.2 nM (pChEMBL 6.400000095367432)
Descriptors: MW 579.04, ALogP 7.19, HBA 4, HBD 1, PSA 75.71, QED 0.26
Compound (SMILES): CCOc1ccc(Cc2cc([C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)c3c(c2C)CCC3)cc1
InChIKey: KAXJFILXWSOBQK-RXFVIIJJSA-N
Target: Sodium/glucose cotransporter 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31639
Assay: SGLT2 Inhibition Assay: For sodium-dependent glucose transport assay, cells expressing hSGLT2 were seeded into a 96-well culture plate at a density of 5×104 cells/well in RPMI medium 1640 containing 10% fetal bovine serum. The cells were used 1 day after plating. They were incubated in pretreatment buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2, pH 7.4) at 37 °C. for 10 min. They were then incubated in uptake buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM 14C-nonlabeled AMG pH 7.4) containing 14C-labeled AMG (8 μM) and the inventive compound or dimethyl sulfoxide (DMSO) vehicle at 37 °C. for 2 h. Cells were washed twice with washing buffer (pretreatment buffer containing 10 mM AMG at room temperature) and then the radioactivity was measured using a liquid scintillation counter.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 428.53, ALogP 1.99, HBA 6, HBD 4, PSA 99.38, QED 0.56
Compound (SMILES): COc1ccc([C@H]2C[C@@H]2COc2cc(N(Cc3ccc(C)nc3C)Cc3ccc(C)nc3C)c(=O)n(C)n2)nc1
InChIKey: JTTVRMUVONKMIN-VPUSJEBWSA-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 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 540.67, ALogP 4.6, HBA 9, HBD 0, PSA 95.26, QED 0.29
Compound (SMILES): Cc1cc(-c2nc(C3CCOCC3)cnc2N)ccc1C(=O)N[C@H](CO)c1ccccc1
InChIKey: WUTFDPSGZSVLQT-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 = 0.136 nM (pChEMBL 9.869999885559082)
Descriptors: MW 432.52, ALogP 3.39, HBA 6, HBD 3, PSA 110.36, QED 0.55
Compound (SMILES): CCOC(=O)O[C@H](C)n1nnc(-c2c(-c3ccc(C(=O)N(c4ncccc4C)[C@@H]4CCCNC4)cc3)cnn2C)n1
InChIKey: ABANEIZXIXLINC-DENIHFKCSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: AlphaLISA Assay: An in-vitro AlphaLISA assay (Perkin Elmer) was developed in order to quantitate the level of PCSK9 secreted into the cell culture media following compound treatment. To detect and measure PCSK9 protein an in-house generated anti-PCSK9 antibody was coupled to AlphaLISA acceptor beads by an external vendor (PerkinElmer) and a second internally developed anti-PCSK9 antibody with an epiptope distinct from that of the acceptor beads was biotinylated in house. Streptavidin coated-donor beads (Perkin Elmer) are also included in the assay mixture which then binds the biotinylated anti-PCSK9 antibody and in the presence of PCSK9 this donor complex and acceptor beads are brought into close proximity. Upon excitation of the donor beads at 680 nm singlet oxygen molecules are released that trigger an energy transfer cascade within the acceptor beads resolving as a single peak of light emitted at 615 nm. The ability of compound to modulate PCSK9 protein levels in conditioned media by AlphaLISA was assessed in the human hepatocellular carcinoma cell line Huh7, stably over-expressing human PCSK9.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 559.63, ALogP 3.53, HBA 12, HBD 1, PSA 142.18, QED 0.32
Compound (SMILES): CC(Nc1nccc(N2C(=O)OCC2[C@@H](C)O)n1)c1cc(-c2ccc(Cl)c(Cl)c2)no1
InChIKey: UGMWZDGPRLEDLZ-NOEPWBJOSA-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 = 274 nM (pChEMBL 6.559999942779541)
Descriptors: MW 464.31, ALogP 4.32, HBA 8, HBD 2, PSA 113.61, QED 0.55
Compound (SMILES): CSc1cccc([C@@H](CO)NC(=O)c2ccc(-c3nc([C@H]4CC[C@H](O)CC4)cnc3N)cc2F)c1
InChIKey: SMASKSZEBRMKNU-OMXJDXKCSA-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 = 0.0269 nM (pChEMBL 10.569999694824219)
Descriptors: MW 496.61, ALogP 4.07, HBA 7, HBD 4, PSA 121.36, QED 0.36
Compound (SMILES): COc1cc(-c2nc(-c3cc(C)nc(CO)c3)c3n2C[C@@H](CF)OCC3)ccc1OC(F)F
InChIKey: LRHGGBPMPDFIJT-QGZVFWFLSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: [35S]GTPgammaS Binding Assay: The frozen cell membrane fraction prepared as described above was thawed before use, and the resultant was diluted with a buffer for a binding assay (final concentrations: 20 mM HEPES, 100 mM NaCl, 1 mM MgCl2, 3 uM GDP, 300 ug/mL saponin, 0.1% BSA). The compound of each example was added to a cell membrane fraction containing 1.8 to 3 ug/assay of membrane protein on a plate, followed by incubation at room temperature for 30 minutes. Thereafter, with glutamic acid (in a final concentration of 10 uM) added thereto, incubation was performed at room temperature for 15 minutes, and thereafter, [35S]GTPγS (in a final concentration of 0.8 kBq) and 588 ug WGA-SPA beads were added thereto, followed by incubation at room temperature for 1 hour. After the incubation, the plate was subjected to the centrifugal separation at 2,500 rpm and room temperature, and then, membrane cell binding radioactivity was measured with a top count. A [35S]GTPγS binding amount obtained in the presence of glutamic acid was defined as specific binding. On the basis of ratios of inhibiting the specific binding at various concentrations of the compounds of the respective examples, inhibition curves were obtained. Concentrations of the compounds of the respective examples at which the specific [35S]GTPγS binding amount was suppressed by 50% (IC50 values) were calculated on the basis of the inhibition curves.
Measurement: IC50 = 108.5 nM (pChEMBL 6.960000038146973)
Descriptors: MW 463.46, ALogP 3.93, HBA 7, HBD 1, PSA 78.63, QED 0.57
Compound (SMILES): Nc1ncc(C2CCOCC2)nc1-c1ccc(C(=O)N[C@H](CO)c2ccccc2)c(F)c1
InChIKey: JRSFMUCORNMCHO-OAQYLSRUSA-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 = 0.593 nM (pChEMBL 9.229999542236328)
Descriptors: MW 436.49, ALogP 3.22, HBA 6, HBD 3, PSA 110.36, QED 0.55
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(Cl)c(F)c2)[C@@H]3C)n1
InChIKey: WVMPHKVLGBDWHJ-MRVPVSSYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Functional Assay: Chinese Hamster Ovary recombinant cells expressing the human NK-3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. The antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound (at various concentrations) with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the concentration-dependent inhibition of the luminescence response to the addition of Neurokinin A.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 392.85, ALogP 3.11, HBA 7, HBD 0, PSA 76.8, QED 0.67
Compound (SMILES): CCNc1n[nH]c2cc(NC(=O)N[C@H](C)c3ccc(F)cc3)ncc12
InChIKey: MRVUFAQGXDMPCK-SNVBAGLBSA-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 = 1 nM (pChEMBL 9)
Descriptors: MW 342.38, ALogP 3.41, HBA 4, HBD 4, PSA 94.73, QED 0.57
Compound (SMILES): Cc1ccc(-n2nc(C3=CCN(S(C)(=O)=O)CC3)cc2NC(=O)C2=c3nccc[n+]3=NC2)cc1
InChIKey: LMEQZHHEINRNHD-UHFFFAOYSA-O
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 478.56, ALogP 0.75, HBA 7, HBD 1, PSA 115.45, QED 0.54
Compound (SMILES): COCCN1CC(Cc2ccccc2)(c2cccc(OCCNS(=O)(=O)c3cn(C)cn3)c2)C1
InChIKey: PSEPDAHDYDEDNC-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Radioligand Binding Assay: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 484.62, ALogP 2.22, HBA 7, HBD 1, PSA 85.69, QED 0.4
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(-n2ccc3cc(C#N)cnc32)cc1NC1CCOCC1
InChIKey: ANNHQLAGJSDZJL-HXUWFJFHSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 466.52, ALogP 2.72, HBA 8, HBD 3, PSA 125.09, QED 0.49
Compound (SMILES): C[C@@H](NC(=O)Nc1cc2[nH]nc(OC3CCOCC3)c2cn1)c1ccccc1
InChIKey: SACKPIJUIAZNPA-CYBMUJFWSA-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 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 381.44, ALogP 3.4, HBA 5, HBD 3, PSA 101.16, QED 0.63
Compound (SMILES): NC(=O)c1cc(-c2cn[nH]c2)c2ccc(CN3CCO[C@@H](C(F)(F)F)C3)cc2n1
InChIKey: CICUFFDWOJXMLM-QGZVFWFLSA-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 = 513 nM (pChEMBL 6.289999961853027)
Descriptors: MW 405.38, ALogP 2.49, HBA 5, HBD 2, PSA 97.13, QED 0.7
Compound (SMILES): CO[C@]12CC[C@@]3(C[C@@H]1COCc1cccc(OCC(=O)O)c1)[C@H]1Cc4ccc(O)c5c4[C@@]3(CCN1CC1CC1)[C@H]2O5
InChIKey: VGPXBTVQLHVZQY-FUYXJAFSSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Radioligand Dose-Displacement Binding Assay: Radioligand dose displacement assays used 0.4 nM [3H]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 μg membrane protein (recombinant κ opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 μl binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 μM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 2001 ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 1.11 nM (pChEMBL 8.960000038146973)
Descriptors: MW 561.68, ALogP 4.3, HBA 7, HBD 2, PSA 97.69, QED 0.44
Compound (SMILES): Cc1ccc(CNc2cc(OC[C@H]3C[C@@H]3c3ccc(C)cn3)nn(C)c2=O)nc1
InChIKey: HTYPLCLOJNPSQC-AEFFLSMTSA-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 = 0.051 nM (pChEMBL 10.289999961853027)
Descriptors: MW 391.48, ALogP 2.98, HBA 7, HBD 1, PSA 81.93, QED 0.67
Compound (SMILES): C[C@H](C(=O)O)N(Cc1csc(C(=O)O)c1)C(=O)c1ccc2cc(OC(=O)c3ccc(NC(=N)N)cc3)ccc2n1
InChIKey: DMQMZHJLQOERFT-CQSZACIVSA-N
Target: Serine protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07477
Assay: Inhibition Assay: The compound was dissolved in dimethyl sulfoxide (DMSO), and diluted to an arbitrary concentration (A). A was 100-fold diluted with a buffer (0.1 M Tris (pH 8.0), 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij38) (B). The r-h trypsin was diluted with a buffer to 0.088 ug/mL, and the m-trypsin was diluted with a buffer to 1/50 (C). The dilution ratio of the m-trypsin (1/50) was set to exhibit the same activity as the 0.088 ug/mL r-h trypsin as determined by kinetic analysis. The substrate solution of a substrate for the enzyme reaction, BZiPAR, (Rhodamine Reference Substrate) was diluted with a buffer to 5 umol/L (D). B; 5 uL, C; 5 uL, and D; 10 uL were added to a 384-plate, and incubated at room temperature for 30 minutes. The fluorescent signals were detected with Ex/Em = 497/520 using Tecan Safire Fluorometer. The compound was reviewed from 2500 nM to its 3-fold value, 0.0075 nM, at 12 concentrations, and the inhibitory rate of each compound was calculated.
Measurement: IC50 = 0.29 nM (pChEMBL 9.539999961853027)
Descriptors: MW 561.58, ALogP 3.63, HBA 8, HBD 5, PSA 196, QED 0.09
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(N[C@H]4c5ccccc5C[C@H]4O)nc23)N1
InChIKey: IFVKGXXFFHIRNY-QAIKFOBFSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 432.44, ALogP 0.95, HBA 9, HBD 5, PSA 145.57, QED 0.3
Compound (SMILES): CCC[C@H]1C(=O)NN=C(c2ccc3nc(-c4cc(C)oc4C)oc3c2)[C@@H]1C
InChIKey: UMFQZRZMBHNIPQ-IUODEOHRSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 127 nM (pChEMBL 6.900000095367432)
Descriptors: MW 365.43, ALogP 4.59, HBA 5, HBD 1, PSA 80.63, QED 0.73
Compound (SMILES): Cc1cc(C(=O)Nc2cc(C(F)(F)F)ccn2)c(F)cc1-c1nc([C@@H]2CC[C@H]3CCC(=O)N3C2)n2ccnc(N)c12
InChIKey: JGEFMLZGVNPQOV-WBVHZDCISA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 uM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 uL) was dispensed, followed by the addition of 7.5 uL of 1x kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/uL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 uL 1x kinase buffer containing 8 uM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ ID NO: 1), and 100 uM ATP. The final reaction in each well of 10 uL consists of 100 pM hBTK, 2 uM biotin-A5-peptide, and 25 uM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1x quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 8.2 nM (pChEMBL 8.09000015258789)
Descriptors: MW 567.55, ALogP 4.96, HBA 7, HBD 2, PSA 118.51, QED 0.34
Compound (SMILES): O=C1CC[C@@]2(O)[C@H]3Cc4ccc(C(=O)NCCc5ccc(-c6cccnc6)cc5)c(O)c4[C@@]2(CCN3CC2CC2)C1
InChIKey: LDQZCGLBZJGWLP-QVLFBZODSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: null: The Ki (binding affinity) for u opioid receptors was determined using a competitive displacement assay as previously described in Neumeyer (Journal of Med. Chem. 2012, p 3878), which is incorporated herein in its entirety. Briefly, membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed the cloned human u opioid receptor were incubated with 12 different concentrations of the compound set forth herein in the presence of 0.25 nM [3H]DAMGO (see Tiberi et al., Can. J. Physiol. Pharmacol. 1988, Vol. 66, p 1368, which is incorporated by reference herein in its entirety) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO (see Gulati et al., Life Sci. 1990, Vol. 47, p 159, which is incorporated by reference herein in its entirety). Nonspecific binding was measured by inclusion of 10 uM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber.
Measurement: Ki = 0.37 nM (pChEMBL 9.430000305175781)
Descriptors: MW 551.69, ALogP 4.19, HBA 6, HBD 3, PSA 102.76, QED 0.41
Compound (SMILES): C[C@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)F)n1)c1cnn(-c2ccc(Cl)cc2)c1
InChIKey: NJHKNRVBHGOLEU-IIYDPXPESA-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 = 41.5 nM (pChEMBL 7.380000114440918)
Descriptors: MW 430.87, ALogP 4.17, HBA 7, HBD 1, PSA 85.17, QED 0.63
Compound (SMILES): O=C(O)C[C@H](NC(=O)[C@@H]1CCCN(C(=O)CCC2CCNCC2)C1)c1cncc(C#Cc2cccc(OCCF)c2)c1
InChIKey: ADXRXCZYDSDWJG-UHSQPCAPSA-N
Target: Integrin alpha-IIb (SINGLE PROTEIN), Homo sapiens
UniProt: P08514
Assay: Enzyme Assay: Purified human glycoprotein IIb/IIIa (20 mM Tris-HCl, 0.1 M NaCl, 0.1% Triton X-100, 1 mM CaCl2, 0.05% NaN3, 50% Glycerol, pH 7.4) was purchased from Enzyme Research Laboratories Inc. (South Bend, Ind.). The GPIIb/IIIa receptor was diluted in phosphate-buffered saline (Dulbecco's Phosphate Buffered Saline (D-PBS (+)) with calcium and magnesium, GIBCO®, Invitrogen) with 0.01% bovine serum albumin (albumin from bovine serum-lyophilized powder, >96%, Sigma).The GPIIb/IIIa receptor was immobilized 48 hours at least (100 μL per well, 48 to maximum 96 hours) on a 96-well solid plate (Immuno Plate MaxiSorp, Nunc, Roskilde, Denmark) at 277 K to 280 K and at a concentration of 0.1 μg per well to 1 μg per well. As negative control one row of the plate (n=8) was incubated just with 2% bovine serum albumin (200 μL per well, albumin from bovine serum-lyophilized powder, >96%, Sigma, diluted in D-PBS (+)).After washing three times with the wash buffer (230 μL per well, Dulbecco's Phosphate Buffered Saline (D-PBS (−)) contains no calcium or magnesium, GIBCO®, Invitrogen) residual exposed plastic and unspecific binding sites were blocked by incubating the plate with a special blocking solution (200 μL per well, Roti®-Block, Carl Roth GmbH Co KG, Karlsruhe) containing 2% bovine serum albumin (Albumin from bovine serum-lyophilized powder, >96%, Sigma) 1 hour at room temperature.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 578.69, ALogP 3.48, HBA 6, HBD 3, PSA 120.86, QED 0.35
Compound (SMILES): O=c1ccn(C2OC(CO)[C@@H](O)[C@]23CCO3)c(=O)[nH]1
InChIKey: UTAJPKQGORLGGP-YGHADEAESA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: HCV Replicon Assay: The HCV NS5B reaction was performed in a 20 μL mixture containing varying concentrations of the test compound, 1 μM of all four natural ribonucleotides, [α-32P]UTP, 20 ng/μL of genotype 1b (−) IRES RNA template, 1 unit/μL of SUPERase·In (Ambion, Austin, Tex.), 40 ng/μL of wild type or S282T NS5B Genotype 1b, 1 mM MgCl2, 0.75 mM MnCl2, and 2 mM DTT in 50 mM Hepes buffer (pH 7.5). The reaction was quenched by adding 80 μL of stop solution (12.5 mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate) after incubating at 27° C. for 30 minutes. The radioactive RNA products were separated from unreacted substrates by passing the quenched reaction mixture through a Hybond N+ membrane (GE Healthcare, Piscataway, N.J.) using a dot-blot apparatus. The RNA products were retained on the membrane and the free nucleotides were washed out. The membrane was washed 4 times with a solution containing 0.6 M NaCl and 60 mM sodium citrate. After rinsing the membrane with water followed by ethanol, the membrane was exposed to a phosphorscreen and the products were visualized and quantified using a phosphorimager.
Measurement: EC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 270.24, ALogP -2.05, HBA 7, HBD 3, PSA 113.78, QED 0.57
Compound (SMILES): C[C@@H](NC(=O)Nc1cc2[nH]nc(N(C)C)c2cn1)c1ccccc1
InChIKey: GLYACRHXQCJXSW-LLVKDONJSA-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 = 13.81 nM (pChEMBL 7.860000133514404)
Descriptors: MW 324.39, ALogP 2.91, HBA 4, HBD 3, PSA 85.94, QED 0.69
Compound (SMILES): C[C@@H]1Cc2c(nnn2-c2ccn[nH]2)[C@H](C)N1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: IQRKGPSJDGZJEV-ZJUUUORDSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Radioligand Binding Assay: human or rat P2X7-1321 N1 cells were collected and frozen @−80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl: 10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2009, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM).
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 424.81, ALogP 3.81, HBA 5, HBD 1, PSA 79.7, QED 0.68
Compound (SMILES): NC(=O)c1ccc(N2CC[C@H](c3ccc(F)cc3)[C@H](N)C2)c2cncnc12
InChIKey: ISSHDEMQWJANGA-RHSMWYFYSA-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° 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 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 365.41, ALogP 2.19, HBA 5, HBD 2, PSA 98.13, QED 0.74
Compound (SMILES): Cc1ccc(CN2CC[C@@H](N3CC(c4ccc(O)cc4)C3)C2=O)cc1F
InChIKey: IESUXIJJIRXKNX-HXUWFJFHSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 354.43, ALogP 3.04, HBA 3, HBD 1, PSA 43.78, QED 0.92
Compound (SMILES): CCOc1ccc(/N=c2\[nH]c(=O)n(C[C@@H](C)C(=O)OC)c(=O)n2Cc2ccc(Cl)cc2)cc1C
InChIKey: AHRHHAOVZLVQPW-MRXNPFEDSA-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 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 486.96, ALogP 2.79, HBA 8, HBD 1, PSA 107.68, QED 0.49
Compound (SMILES): Cc1cc(/N=c2\[nH]c(=O)n(C[C@@H](C)C(=O)O)c(=O)n2Cc2ccc(Cl)cc2)ccc1OC(C)C
InChIKey: AFRURKYTUGHQPD-MRXNPFEDSA-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 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 486.96, ALogP 3.09, HBA 7, HBD 2, PSA 118.68, QED 0.51