lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCCCNC(=O)c1cnc(-n2ncc3cc(C#N)c(N)nc32)cc1NC1COC1
InChIKey: PPYTUSPPPLLRKK-UHFFFAOYSA-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 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 406.45, ALogP 1.61, HBA 9, HBD 3, PSA 143.77, QED 0.5
Compound (SMILES): CCOc1cc(CC(=O)O)ccc1-c1ccc(F)c2c1CN(C(=O)NCc1ccccc1)CC2
InChIKey: SUWHCVAIXFTABB-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 462.52, ALogP 4.79, HBA 3, HBD 2, PSA 78.87, QED 0.53
Compound (SMILES): CCCCC1NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@@H](CCCNC(=N)N)NC(=O)[C@H](Cc2c(C)cc(O)cc2C)NC1=O
InChIKey: JOJDGSFKFOEAPA-FPOJHQRRSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P33533
Assay: Displacement of [3H]DSLET from DOR in rat brain membrane measured after 2 hrs
Measurement: Ki = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 607.76, ALogP 1.2, HBA 6, HBD 8, PSA 198.53, QED 0.11
Compound (SMILES): CCOC(=O)C(C)NC(=O)N1CC(CC#N)(n2cc(C(N)=O)c(Nc3ccccc3)n2)C1
InChIKey: LIQWGCWZZCEOEP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 439.48, ALogP 1.31, HBA 8, HBD 3, PSA 155.37, QED 0.52
Compound (SMILES): O=C(c1n[nH]c2c1CN(C(=O)N1CCCC1)C2)N1CCC(c2ccc(F)c(F)c2C(F)(F)F)CC1
InChIKey: BUTDCAJURAHJIA-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: Scintillation Proximity Binding Assay: Untagged human RBP4 purified from urine of tubular proteinuria patients was purchased from Fitzgerald Industries International. It was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit from Pierce following the manufacturer's recommendations. Binding experiments were performed in 96-well plates (OptiPlate, PerkinElmer) in a final assay volume of 100 ul per well in SPA buffer (IX PBS, pH 7.4, 1 mM EDTA, 0.1% BSA, 0.5% CHAPS). The reaction mix contained 10 nM 3H-Retinol (48.7 Ci/mmol; PerkinElmer), 0.3 mg/well Streptavidin-PVT beads, 50 nM biotinylated RBP4 and a test compound. Nonspecific binding was determined in the presence of 20 uM of unlabeled retinol. The reaction mix was assembled in the dark under dim red light. The plates were sealed with clear tape (TopSeal-A: 96-well microplate, PerkinElmer), wrapped in the aluminum foil, and allowed to equilibrate 6 hours at room temperature followed by overnight incubation at +4° C. Radiocounts were measured using a TopCount NXT counter (Packard Instrument Company).
Measurement: IC50 = 20.39 nM (pChEMBL 7.690000057220459)
Descriptors: MW 497.47, ALogP 4.26, HBA 3, HBD 1, PSA 72.54, QED 0.63
Compound (SMILES): O=C(O)CCc1ccc(-c2c(/C=C/c3ccc4ccccc4n3)nc3c(N4CCOCC4)ccnn23)cn1
InChIKey: RRLJCFFOSBAPLP-PKNBQFBNSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 506.57, ALogP 4.36, HBA 8, HBD 1, PSA 105.74, QED 0.35
Compound (SMILES): COc1cc(OC)cc(N(CCN2C[C@@H](C)O[C@@H](C)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: ZGRYRAJCDPWOIP-AMDBCLDASA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 245.47 nM (pChEMBL 6.610000133514404)
Descriptors: MW 502.62, ALogP 4.29, HBA 9, HBD 0, PSA 77.77, QED 0.35
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2ccccc2)C(=O)N[C@@H](Cc2c[nH]c3ccccc23)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: GAEPCQZQRDPARJ-NOCHOARKSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 617.75, ALogP 4.41, HBA 5, HBD 5, PSA 138.34, QED 0.13
Compound (SMILES): O=C(NCC1CCN(C(=O)C2CCOC2)C1)c1ccc(NC(=O)N2Cc3ccncc3C2)cc1
InChIKey: WQFJKOHQSONASL-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 2.77 nM (pChEMBL 8.5600004196167)
Descriptors: MW 463.54, ALogP 2.24, HBA 5, HBD 2, PSA 103.87, QED 0.71
Compound (SMILES): COc1cc2cncc(Cc3ccc4ccccc4n3)c2cc1OC
InChIKey: MSLBBVBQDGUFJE-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Diaxonhit Phosphodiesterase Assay: The phosphodiesterase assay was developed using the LANCE cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffer).
Measurement: IC50 = 208 nM (pChEMBL 6.679999828338623)
Descriptors: MW 330.39, ALogP 4.39, HBA 4, HBD 0, PSA 44.24, QED 0.56
Compound (SMILES): COc1cc2cnc(C)c(Cc3ccc4ccccc4c3)c2cc1OC
InChIKey: OMHKVMSICNZNLK-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Diaxonhit Phosphodiesterase Assay: The phosphodiesterase assay was developed using the LANCE cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffer).
Measurement: IC50 = 1069 nM (pChEMBL 5.96999979019165)
Descriptors: MW 343.43, ALogP 5.3, HBA 3, HBD 0, PSA 31.35, QED 0.5
Compound (SMILES): CN(C)c1cc(Nc2nccc(C(F)(F)F)n2)cc(-c2ccc([C@H](O)C(F)F)cc2)c1
InChIKey: JXYKDCXSYMYBIE-SFHVURJKSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 438.4, ALogP 5.27, HBA 5, HBD 2, PSA 61.28, QED 0.51
Compound (SMILES): O=C(OC1CCN(c2ccc(CO)nc2)CC1)N1CCN(C2CCC2)CC1
InChIKey: QJZBRZHFIJZDCP-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: LANCE Ultra cAMP Assay: The potency of compounds of the invention as H3 receptor antagonists can be assessed by measuring the blockade of (R)-alpha-methylhistamine-mediated cAMP production utilizing a LANCE Ultra cAMP kit (PE #TRF0263) in CHO cells expressing human H3 receptors (GenBank: BC096840; Strausberg R L et al, Proc. Natl. Acad. Sci. U.S.A. 99(26), 16899-16903; 2002).Protocol:1. Preparation of the stimulation buffer (30 ml): 29.4 ml HBSS (GIBCO #14025), 150 ul 1 M HEPES (GIBCO #15630), 30 ul 500 mM IBMX (CALBIOCHEM #410957) and 400 ul 7.5% BSA (GIBCO #10438-026).2. Preparation of assay plate: Different concentrations of the compounds of the invention (0.01-1000 nM), H3 positive controls and cAMP calibration standards; 3 mM Forskolin (CALBIOCHEM #344270); 5 uM (R)-alpha-methylhistamine (H3 receptor agonist); 1% DMSO (SIGMA #D2650); total volume: 95 nl.3. Preparation of the cell solution: Collect cells with stimulation buffer, final density: 100,000 cells/ml.
Measurement: Ki = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 374.49, ALogP 1.85, HBA 6, HBD 1, PSA 69.14, QED 0.87
Compound (SMILES): Cc1cc(C#Cc2cccc(F)c2)cnc1N1CC(C)(C)N(C)C1=O
InChIKey: YJGLEGKIKAJLCG-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Intracellular Ca2+ Mobilization Assay: A monoclonal HEK-293 cell line stably transfected with a cDNA encoding for the human mGlu5a receptor was generated; for the work with mGlu5 Positive Allosteric Modulators (PAMs), a cell line with low receptor expression levels and low constitutive receptor activity was selected to allow the differentiation of agonistic versus PAM activity. Cells were cultured according to standard protocols (Freshney, 2000) in Dulbecco's Modified Eagle Medium with high glucose supplemented with 1 mM glutamine, 10% (vol/vol) heat-inactivated bovine calf serum, Penicillin/Streptomycin, 50 μg/ml hygromycin and 15 μg/ml blasticidin (all cell culture reagents and antibiotics from Invitrogen, Basel, Switzerland). bout 24 hrs before an experiment, 5×104 cells/well were seeded in poly-D-lysine coated, black/clear-bottomed 96-well plates. The cells were loaded with 2.5 μM Fluo-4AM in loading buffer (1×HBSS, 20 mM HEPES) for 1 hr at 37 °C. and washed five times with loading buffer. The cells were transferred into a Functional Drug Screening System 7000 (Hamamatsu, Paris, France), and 11 half logarithmic serial dilutions of test compound at 37 °C. were added and the cells were incubated for 10-30 min. with on-line recording of fluorescence. Following this pre-incubation step, the agonist L-glutamate was added to the cells at a concentration corresponding to EC20 (typically around 80 μM) with on-line recording of fluorescence; in order to account for day-to-day variations in the responsiveness of cells, the EC20 of glutamate was determined immediately ahead of each experiment by recording of a full dose-response curve of glutamate. Responses were measured as peak increase in fluorescence minus basal (i.e. fluorescence without addition of L-glutamate), normalized to the maximal stimulatory effect obtained with saturating concentrations of L-glutamate.
Measurement: EC50 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 337.4, ALogP 3.58, HBA 2, HBD 0, PSA 36.44, QED 0.75
Compound (SMILES): CCCN(CC1CC1)c1cc(C(=O)Nc2ccc(S(=O)(=O)NCCOC)cc2C)ncn1
InChIKey: PQDPFEDCPQNXSS-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: 35S-GTPgammaS Binding: Measurements of 35S-GTPgammaS Binding: Membranes (1 to 10 ug protein) prepared as described above, were incubated in 96-well Scintiplates (PerkinElmer) with test compounds diluted in DMSO, in 180 ul of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 2 ug/well Saponin, 0.2% fatty acid free BSA (Assay buffer), 140 mM NaCl and 1.7 uM GDP. The assay was initiated with the addition of 20 ul of 1.5 nM [35S]-GTPgammaS (1100 Ci/mmol; GE Healthcare) in assay buffer. After 60 min incubation at 30° C. on a shaker, plates were centrifuged for 10 min at 2000 RPM. Supernatant was discarded and membrane bound radioactivity was measured on a PerkinElmer 1450 MicroBeta counter. Triplicate samples were averaged and expressed as % response relative to S1P activation in absence of compound (n=2).
Measurement: EC50 = 202 nM (pChEMBL 6.699999809265137)
Descriptors: MW 461.59, ALogP 2.59, HBA 7, HBD 2, PSA 113.52, QED 0.47
Compound (SMILES): NCC1CCN(c2c(N)cccc2NC(=O)c2cnn3cccnc23)CC1
InChIKey: NJRLVIMZYCFPQE-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: In vitro IRAK-1 and IRAK-4 Assay: Purified recombinant IRAK-4 protein was incubated with 250 uM synthetic peptide(KKARFSRFAGSSPSQSSMVAR) in 30 ul of kinase buffer including (20 mM MOPS pH7.2, 25 mM beta glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 50 uM ATP, 20 mM MgCl2, 10 uCi gamma-33P, 0.1% BSA) for the indicated time. For purified recombinant IRAK-1 protein kinase assay, 50 uM ATP was used. A 25 ul aliquot of the reaction mixture was transferred on to p81 phosphocellulose squares (Upstate Biotechnology, Lake Placid, N.Y.). The assay squares were washed three times with 0.75% phosphoric acid and once with acetone. Enzyme activity was measured by determining the bound radioactivity by liquid scintillation counting.
Measurement: IC50 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 365.44, ALogP 1.74, HBA 7, HBD 3, PSA 114.57, QED 0.61
Compound (SMILES): COc1ccc(Nc2n[nH]c3cccnc23)cc1OC(F)F
InChIKey: CMXBVWMFNJBRCT-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Receptor Activity Assay: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGluR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured.
Measurement: EC50 = 885 nM (pChEMBL 6.050000190734863)
Descriptors: MW 306.27, ALogP 3.31, HBA 5, HBD 2, PSA 72.06, QED 0.76
Compound (SMILES): CC(C)[C@H](C(=O)O)N(CCNC(=O)c1cccc(C(=O)NCCN([C@@H](C(=O)N(C)C)C(C)C)S(=O)(=O)c2ccc(-c3ccccc3)cc2)c1)S(=O)(=O)c1ccc(-c2ccccc2)cc1
InChIKey: QGMPTSWHSCMOJI-NDOUMJCMSA-N
Target: Interstitial collagenase (SINGLE PROTEIN), Homo sapiens
UniProt: P03956
Assay: Inhibition of p-aminophenylmercuric acetate activated human rheumatoid synovial fibroblast MMP-1 pretreated for 4 hrs followed by Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 fluorogenic substrate addition measured every 15 secs for 20 mins by fluorimetric assay
Measurement: IC50 = 2050 nM (pChEMBL 5.690000057220459)
Descriptors: MW 910.13, ALogP 6.08, HBA 8, HBD 3, PSA 190.57, QED 0.08
Compound (SMILES): c1cc(N2CCNCC2)nc(-n2ncc3cnc(-c4cncc(N5CCC5)n4)cc32)c1
InChIKey: XKCUAIJHVDEELV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase Binding 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 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL 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. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.02 nM (pChEMBL 10.699999809265137)
Descriptors: MW 413.49, ALogP 1.89, HBA 9, HBD 1, PSA 87.89, QED 0.54
Compound (SMILES): CCCNC(=S)Nc1cc(-n2cccn2)ncn1
InChIKey: ACVXSTOIRNZAGI-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P37136
Assay: Inhibition of rat cortex AChE using acetylthiocholine iodide as substrate measured after 20 mins by Ellman's method
Measurement: IC50 = 22000 nM (pChEMBL 4.659999847412109)
Descriptors: MW 262.34, ALogP 1.36, HBA 5, HBD 2, PSA 67.66, QED 0.81
Compound (SMILES): COc1cc(C(=O)N2CCC(O)C(C)C2)cc2nc(N[C@@H](CF)c3cccc(Cl)c3)oc12
InChIKey: JUKYMUDXJNGELI-SZSCYUJKSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Inhibition Assay: To determine the thrombin inhibition of the substances listed above, a biochemical test system is constructed in which the conversion of a thrombin substrate is used for determining the enzymatic activity of human thrombin. Here, thrombin cleaves aminomethylcoumarin, which is measured fluorescently, from the peptic substrate. The determinations are carried out in microtitre plates.Substances to be tested are dissolved in various concentrations in dimethyl sulphoxide and incubated for 15 min with human thrombin (0.06 nmol/l dissolved in 50 mmol/l of Tris buffer [C,C,C-tris(hydroxymethyl)aminomethane], 100 mmol/l of sodium chloride, 0.1% BSA [bovine serum albumin], pH 7.4) at 22° C. The substrate (5 umol/l Boc-Asp(OBzl)-Pro-Arg-AMC from Bachem) is then added. After 30 min of incubation, the sample is excited at a wavelength of 360 nm and the emission is measured at 460 nm. The measured emissions of the test batches with test substance are compared to the control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide) and the IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 461.92, ALogP 4.46, HBA 6, HBD 2, PSA 87.83, QED 0.56
Compound (SMILES): CC(C)n1nccc1-c1ccc(OCC(O)CN2CCc3ccccc3C2)cc1
InChIKey: XWUXXJWRAYNNSP-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 10001 nM (pChEMBL 5)
Descriptors: MW 391.52, ALogP 3.93, HBA 5, HBD 1, PSA 50.52, QED 0.66
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccnc(C(F)(F)F)c4)cn3CCNCCCCl)CC2)c1Br
InChIKey: KIOZCJFPASJAIE-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 587.88, ALogP 4.7, HBA 8, HBD 2, PSA 97.78, QED 0.28
Compound (SMILES): O=C(NCC1CCO1)c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1
InChIKey: MOUFNZPMTXIXBE-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 392 nM (pChEMBL 6.409999847412109)
Descriptors: MW 403.36, ALogP 3.96, HBA 5, HBD 1, PSA 73.34, QED 0.7
Compound (SMILES): CC[C@H](C)[C@H](NC(=O)OC)C(=O)N1CCC[C@@H]1c1nc2ccc(-c3ccc4cc(-c5cnc([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)[C@H](C)OC)[nH]5)ccc4c3)cc2[nH]1
InChIKey: MWXCHVLPDKGEDG-JOWBIXCGSA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Transient Transfection Assay: In a transient set-up, a Huh-7 lunet hepatoma cell line was transiently transfected with an autonomously replicating RNA encoding a bi-cistronic expression construct. This construct comprises a firefly luciferase reporter gene preceding the NS3-NS5B subgenomic region of HCV (genotype 1a H77 or 1b Con1). Translation of the HCV subgenomic region is mediated by an internal ribosome entry site of encephalomyocarditis virus. The construct is furthermore flanked by 5' and 3' untranslated regions of HCV (genotype 1a H77 or 1b Con 1, respectively), which allow for replication of the RNA. In addition to the wild-type constructs, site-directed mutations were introduced into the transient HCV genotype 1b replicon in the gene encoding for the non-structural protein 5A (NS5A). More precisely, amino acid residues 28, 30, 31 and 93 in NS5A were independently altered. Cells were plated in 384 well plates in the presence of test and control compounds, which were added in various concentrations. Following an incubation of two days, replication of the HCV subgenomic replicon RNA was measured by assaying luciferase activity (using standard luciferase assay substrates and reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager).
Measurement: EC50 = 336 nM (pChEMBL 6.46999979019165)
Descriptors: MW 792.94, ALogP 6.63, HBA 9, HBD 4, PSA 183.87, QED 0.11
Compound (SMILES): COc1ccc(C#CCCN)cc1
InChIKey: VVEBXHIYHLNGCX-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Bos taurus
UniProt: Q9TTK6
Assay: Inhibition Assay: In the following Examples, 0.9 mL aliquots of a solution of candidate inhibitor in 100 mM potassium phosphate buffer, pH 7.2 was mixed bovine plasma amine oxidase (0.1 mL, about 8 AM) and incubated at 30° C. aerobically. Aliquots (0.1 mL) were periodically withdrawn using disposable calibrated Drummond micropipettes and diluted with 1.0 mL of benzylamine (5 mM in 50 mM sodium phosphate buffer, pH 7.2) in a 1 cm quartz cuvette (1.5 mL volume). The rate of oxidation of benzylamine to benzaldehyde was measured by recording the increase in absorbance at 250 nm for 1 minute and compared to the rate of benzylamine oxidation in a companion control solution of enzyme without inhibitor. The concentration of active BPAO was estimated from the rate of benzylamine oxidation. Inhibitory potency values (IC50 values) and partition values for inactivation of BPAO were measured at various times as in indicated in the Tables.
Measurement: IC50 = 8400 nM (pChEMBL 5.079999923706055)
Descriptors: MW 175.23, ALogP 1.4, HBA 2, HBD 1, PSA 35.25, QED 0.69
Compound (SMILES): C=CCN1CCN(c2cncc(-c3cn(-c4cc(NC(=O)c5ccc(C(C)(C)C)o5)ccc4C)nn3)c2)CC1
InChIKey: INXTYEBZZFBLQE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: B-RAF (V600E) Kinase Assay: In a dilution series 10 uL/well of test substance solution are placed in a multiwell plate. The dilution series is selected so that generally a range of concentrations of 2 uM to 0.119 nM or 0.017 nM is covered. If necessary the initial concentration of 2 uM is changed to 50 uM, 10 uM or 0.4 uM or 0.2857 uM and further dilution is carried out accordingly. The final concentration of DMSO is 5%. 10 uL/well of the B-Raf (V600E)-kinase solution are pipetted in (containing 0.5 ng B-Raf (V600E)-kinase (e.g. from Upstate) in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.286 mM sodium orthovanadate, 10% glycerol, 1 mg/mL bovine serum albumin, 1 mM dithiothreitol) and the mixture is incubated for 1 h at RT under with shaking. The kinase reaction is started by the addition of 20 uL/well ATP solution [final concentration: 250 uM ATP, 30 mM Tris-HCl pH 7.5, 0.02% Brij, 0.2 mM sodium orthovanadate, 10 mM magnesium acetate, 0.1 mM EGTA, phosphatase cocktail (Sigma, #P2850, dilution recommended by the manufacturer)] and 10 uL/well MEK1 solution [containing 50 ng biotinylated MEK1 (prepared from purified MEK1 according to standard procedure, e.g. with EZ-Link Sulpho-NHS-LC-Biotin reagent, Pierce, #21335)] and carried out for 60 min at RT with constant shaking. The reaction is stopped by the addition of 12 uL/well of a 100 mM EDTA solution and incubation is continued for a further 5 min. 55 uL/well of the reaction solution are transferred into a streptavidin-coated plate (e.g. Streptawell HighBond, Roche, #11989685001) and shaken gently for 1 h at RT, in order to bind biotinylated MEK1 to the plate. After elimination of the liquid the plate is washed five times with 200 uL/well of 1Ã¿PBS and 100 uL/well solution of primary antibody plus europium-labelled secondary antibody [Anti Phospho-MEK (Ser217/221), Cell Signaling, #9121 and Eu-N1 labelled goat-anti-rabbit antibody, Perkin Elmer, #AD0105] is added, the primary antibody is diluted 1:2000 and the secondary antibody is diluted to 0.4-0.5 ug/mL in Delfia Assay Buffer (Perkin Elmer, #1244-111). After 1 h shaking at RT the solution is poured away and washed five times with 200 uL/well Delfia Wash Buffer (Perkin Elmer, #4010-0010/#1244-114). After the addition of 200 uL/well Enhancement Solution (Perkin Elmer, #4001-0010/#1244-105) the mixture is shaken for 10 min at RT and then measured in a Wallac Victor using the program "Delfia Time Resolved Fluorescence (Europium)". IC50 values are obtained from these dosage-activity curves using a software program (GraphPadPrizm).
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 525.66, ALogP 5.09, HBA 8, HBD 1, PSA 92.32, QED 0.34
Compound (SMILES): C=CS(=O)(=O)Nc1cccc(Nc2ncnc3cc(OC)c(OC)cc23)c1
InChIKey: XWEWXOXVGICYJP-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: In vitro Kinase Assay: In vitro kinase assays using LRRK WT and LRRK G2019S.
Measurement: IC50 = 213 nM (pChEMBL 6.670000076293945)
Descriptors: MW 386.43, ALogP 3.28, HBA 7, HBD 2, PSA 102.44, QED 0.64
Compound (SMILES): CCC(=O)Nc1cc2c(Nc3ccc(Oc4cccnc4)cc3)ncnc2cc1OC
InChIKey: VIVGTLWPQZQFPV-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Irreversible inhibition of GST-tagged ERBB1 (unknown origin) (Met-668 to Ala-1211 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 531 nM (pChEMBL 6.28000020980835)
Descriptors: MW 415.45, ALogP 4.92, HBA 7, HBD 2, PSA 98.26, QED 0.44
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)cc3F)ncnc2cc1OCC1CN(C)CCO1
InChIKey: PKMKYIJSZRWDDY-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 432.88, ALogP 3.88, HBA 7, HBD 1, PSA 68.74, QED 0.63
Compound (SMILES): O=C(CCCc1c[nH]nn1)NC1CCN(C(=O)/C=C/c2ccc(Cl)cc2Cl)CC1
InChIKey: GPXXBCUCRBYWJT-FNORWQNLSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibition Assay: ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.
Measurement: IC50 = 77 nM (pChEMBL 7.110000133514404)
Descriptors: MW 436.34, ALogP 3.25, HBA 4, HBD 2, PSA 90.98, QED 0.65
Compound (SMILES): CCNc1nc2ccccc2n1-c1nc2c(c(C(C)(C)S(C)(=O)=O)n1)OC[C@@H]1COCCN21
InChIKey: GZMCDKYNELBFSQ-AWEZNQCLSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: LanthaScreen Kinase Activity Assay: The enzymatic mTOR activity was measured using a LanthaScreen kinase activity assay (Invitrogen). The enzyme was purchased from Invitrogen (PV4754), as well as the GFP-labelled substrate (4EBP1-GFP; PV4759) and the Tb-antip4EBP1(pThr46) antibody (PV4757). The assay was performed in 50 Mm HEPES buffer, pH 7.5, containing 1.5 mM MnCl2, 10 mM MgCl2, 1 mM EGTA, 2.5 mM DTT and 0.01% Tween-20. The concentration of the assay components were the following: 0.24 nM mTOR kinase, 400 nM 4EBP1-GFP, 10 mM ATP and serial dilutions of the compound (inhibitor) to be evaluated. After 1 h incubation at room temperature, 20 mM EDTA was used to stop the reaction and terbium-labelled antibody (4 nM) added to detect phosphorylated product. The antibody associates with the phosphorylated product resulting in an increased TR-FRET value. The TR-FRET value (a dimensionless number) was calculated as the ratio of the acceptor signal (GFP, emission at 520 nm) to the donor signal (terbium, emission at 495 nm).
Measurement: IC50 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 472.57, ALogP 2.12, HBA 10, HBD 1, PSA 111.47, QED 0.6
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)c1ccc(CNC(=O)CN2CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC2)cc1)C(C)C)C(N)=O
InChIKey: KPCUGRDVNGRIAQ-CADXXNFBSA-N
Target: Gastrin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30550
Assay: Displacement of [125I]-BBN from GRPR in human PC3 cells after 1 hr by compuGamma counting or microplate scintillation counting method
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 1459.7
Compound (SMILES): c1ccc(COc2ccc3[nH]nc(-c4ccccc4)c3c2)cc1
InChIKey: PDNMCKCPAMGRPP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 10000 nM (pChEMBL 5)
Descriptors: MW 300.36, ALogP 4.81, HBA 2, HBD 1, PSA 37.91, QED 0.59
Compound (SMILES): CC(C)Oc1ccc2[nH]nc(-c3cc(N4CCC(C(C)(C)O)CC4)ncn3)c2c1
InChIKey: LATMCOBVHRMHJQ-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 5.4 nM (pChEMBL 8.270000457763672)
Descriptors: MW 395.51, ALogP 3.79, HBA 6, HBD 2, PSA 87.16, QED 0.68
Compound (SMILES): COc1cc(-c2cnn(C)c2)ccc1Nc1ncc2ccnc(N3CCS(=O)(=O)CC3)c2n1
InChIKey: ODLORASFJWJPOG-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Enzyme Inhibition Assay: The enzyme reaction (total volume 10 μl) was carried out in black 384-well low volume plates containing full length MPS1 (12.5 nM or 3 nM), fluorescent labelled peptide [known as H236, which has the sequence: 5FAM-DHTGFLTEYVATR-CONH2] (5 μM), ATP(10 μM), either DMSO (1% v/v) or the test compound (in the range 0.25 nM-100 μM in 1% DMSO) and assay buffer (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovandate, 10 μM MgCl2, 1 μM DTT, Roche protease inhibitor). The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μl) containing 20 mM EDTA, 0.05% (v/v) Brij-35, in 0.1M HEPES-buffered saline (Free acid, Sigma, UK). The plate was read on a Caliper EZ reader II (Caliper Life Sciences).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 465.54, ALogP 2.41, HBA 10, HBD 1, PSA 115.13, QED 0.47
Compound (SMILES): O=C(NCC(c1cnc(C2CC2)nc1)N1CCC(F)(F)CC1)c1c(F)cccc1Cl
InChIKey: IHDBOBNECYYTAS-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 438.88, ALogP 4.35, HBA 4, HBD 1, PSA 58.12, QED 0.73
Compound (SMILES): O=C(OCC12CCN(CC1)CC2)c1c[nH]c2cc(Cl)ccc12
InChIKey: FLSBENCAGMDPGD-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Radioligand Binding Assay: Protocol 1: The assay was conducted following the literature reference Meyer E. M., et al., Analysis of 3-(4-Hydroxy, 2-Methoxybenzylidene) Anabaseine Selectivity and Activity at Human and Rat Alpha-7 Nicotinic Receptors, J. Pharmacol. Exp. Ther., 287: 918-925, 1998. The assay was conducted using the following materials: receptor source: rat brain, radioligand: [125I] α-Bungarotoxin (2200 Ci/mmol) with a final concentration of 1 nM, non-specific determinant: Methyllycaconitine (MLA) [1 μM], reference compound: Methyllycaconitine (MLA), and positive control: Methyllycaconitine (MLA). Incubation Conditions: Reactions were carried out in 20 mM HEPES (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2 and 1.2 mM MgSO4 at 37° C. for 90 minutes. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined by liquid scintillation counting and compared to control values in order to ascertain any interactions of test compound with the nicotinic, ganglionic binding site.
Measurement: Ki = 398 nM (pChEMBL 6.400000095367432)
Descriptors: MW 318.8, ALogP 3.46, HBA 3, HBD 1, PSA 45.33, QED 0.88