lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(c1ccc(-c2cccs2)cc1)N1CCn2c(nnc2-c2csc(-c3ccc(Br)cc3)n2)C1
InChIKey: CYUWAUXVKXBCSE-UHFFFAOYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: 3H-SB222200 Binding Competition Assay with Human NK-3 Receptor: The ability of the compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells which express the human NK3 receptor. The membranes were incubated with 5 nM 3H-SB222200_(ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 Mm/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of the compounds of the invention. The amount of tritiated SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC50/(1+[L]/KD) where [L] is the concentration of free radioligand and KD is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 548.49, ALogP 6.22, HBA 7, HBD 0, PSA 63.91, QED 0.27
Compound (SMILES): CCc1ccc(N(CC(C)C)S(=O)(=O)c2ccc(N3CCNCC3)c(S(C)(=N)=O)c2)cc1
InChIKey: NHHKFYVVAXGSCB-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: GAL4-RORgamma Transactivation: The RORγ transactivation model was developed from the line HG5LN, which is a HeLa line that stably expresses a luciferase reporter gene controlled by a pentamer of the GAL4 recognition domain of yeast and of a β-globin promoter. The HG5LN line was stably transfected by the DNA-binding domain (DBD) of GAL4 fused to the ROR gamma ligand-binding domain (LBD). Molecules that inhibit the ROR gamma constitutive activity reduce the luciferase expression, thus leading to a reduction in the emitted luminescence.The cells are seeded in 384-well plates (5000 cells in 45 μL/well of culture medium containing 10% fetal calf serum) and incubated for 4 hours at 37° C., 5% CO2. 5 μL of the test molecules (compounds described in the tables described above) are then added to each well and the plates are incubated for 18 hours at a temperature of 37° C. under 5% of CO2. 20 μL of luciferase substrate (Promega) are added to each well and the luminescence emitted is read by a microplate reader.The luminescence units (RLU) are normalized by positive controls (POS containing a saturated concentration of benzenesulfonamide, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]) and negative controls (NEG containing DMSO): % inhibition=((RLU−NEG)*100)/(POS−NEG). The IC50 values are calculated from a 4-parameter logistic model using the XLFit software (IDBS).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 478.68, ALogP 3.55, HBA 6, HBD 2, PSA 93.57, QED 0.61
Compound (SMILES): COc1cc(C2CCN(C3CCN(CCO)CC3)CC2)ccc1Nc1ncc(Cl)c(Nc2ccccc2NS(C)(=O)=O)n1
InChIKey: AQOBZRDHNGYBNF-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibitory Ability Assay: The composition of the assay buffer used in the activity measurement was 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 7.5 mM MgCl2, 3 mM KCl, 0.01% Tween 20, 0.1% BSA, 1 mM DTT. The enzymatic reaction was carried out hereto by using the peptide substrate labeled with ATP of 50 mM concentration and biotin of 0.5 mM concentration. The analysis of an EGFR activity inhibitory effect of the compound was carried out in accordance with the analysis reaction recipe below.Component 1: 4 ml of EGFR wild-type or mutation enzymeComponent 2: 2 ml of compound solutionComponent 3: 4 ml of peptide labeled with ATP and biotinThe enzymatic reaction was initiated by mixing Component 1 and Component 2 first, and then, adding component 3. After the reaction for 2 hours at 37° C., 10 ml of a measurement solution consisting of streptavidin-XL665 provided by Cisbio and anti-phosphotyrosine antibody labeled with europium was added to the enzymatic reaction solution and reacted for 1 hour at ambient temperature. Finally, the ratio of fluorescence values at 615 nm and 665 nm was obtained by using the Envision equipment of Perkin-Elmer, and the enzymatic activity was quantitatively measured, and the inhibitory ability of the compound was confirmed. The measurement values measured at the seven concentrations of the compound were analyzed by using the Prism program (version 5.01, Graphpad Software, Inc.), and IC50 value of the compound was calculated, which is an inhibitory ability indicator.
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 630.21, ALogP 4.63, HBA 10, HBD 4, PSA 131.95, QED 0.24
Compound (SMILES): O=C(CN1C(=O)C2(CCN(C(=O)c3ccc4[nH]ncc4n3)CC2)c2c1ccc(F)c2Cl)N1CCN2CCCC2C1
InChIKey: JKOHFNOMPOZOFC-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: In Vitro Binding Assay for DDR1: An in vitro binding competition assay was performed to evaluate the effect of the compounds of present invention on DDR1 protein.Binding Competition Assay:This assay is based on the intracellular domain of the DDR1 protein which contains the kinase active site. The recombinant protein additionally carries a GST-tag that can be recognized by an Eu-labeled anti-GST antibody. A tracer compound binding to the active site is labeled with a dye so that a FRET donor acceptor pair can be formed. Excitation energy absorbed by the Europium complex (350 nm flash light or pulsed laser) is transferred to a suitable fluorescent dye, if it is in close proximity. Compounds binding competitively with the tracer molecule will displace the bound tracer molecules and reduce the FRET signal in a dose dependent manner. Due to the long lifetime of the Eu excited state, the emission of the donor and the acceptor can be measured in time-gated mode such that most of the intrinsic fluorescence contributions have already decayed. This results in high sensitivity, excellent reproducibility and high data quality. This sensitive detection method enables protein concentrations below 20 nM.Protein, tracer and labeled antibody were obtained from commercial sources. The assay was performed in 384 low volume microtiter-plates with a final volume of 15 al. Dose response curves were generated from 16 compound dilutions in DMSO as solvent compound dilutions, a solution containing protein and labeled antibody, and a solution containing the tracer which is added in the last step. The fluorescence of donor and acceptor were then measured after one hour incubation at room temperature. Every assay run was quality-controlled with dose response curves for two reference compounds.
Measurement: IC50 = 67.9 nM (pChEMBL 7.170000076293945)
Descriptors: MW 566.04, ALogP 2.58, HBA 6, HBD 1, PSA 105.74, QED 0.52
Compound (SMILES): CN1CCOc2ncc(S(=O)(=O)N3CCC4(CC3)C[C@@H](NC[C@H](O)COc3cccc(S(=O)(=O)C5CC5)c3)CO4)cc21
InChIKey: BPSWQDAREMZHQC-RTWAWAEBSA-N
Target: Beta-3 adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P13945
Assay: Homogeneous Time-Resolved Fluorescence Assay: Compounds were dissolved and serially diluted (5-fold) in DMSO to generate a 10-point dose response stock. The stock was then diluted 100-fold in assay buffer (PBS containing 1 mM IBMX) before a volume of 2.5 μL was added to the cells (the final, top concentration of compound in the dose-response is typically 10 or 100 μM). After a brief incubation, 2.5 μL of isoproterenol stock, prepared at a concentration 4 times its EC90 at the receptor of interest, was added to the wells. The EC90 for isoproterenol, a beta-adrenergic agonist, was determined in separate experiments using standard methods to measure agonist potencies.Following a 1-hour incubation at room temperature, 5 μL of cAMP-D2 Reagent diluted in Lysis Buffer was added to each well followed by 5 μL of Cryptate Reagent. Plates were further incubated at room temperature for 1 hour prior to reading. Time resolved fluorescence measurements were collected on a suitable, HTRF-capable plate reader.
Measurement: IC50 = 17500 nM (pChEMBL 4.760000228881836)
Descriptors: MW 622.77, ALogP 1.19, HBA 11, HBD 2, PSA 147.6, QED 0.39
Compound (SMILES): Cc1nc2cnc(N3CCN(C4(C(F)F)CC4)CC3)c(C#N)c2c(=O)n1CC#Cc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: SLVLRYTUPDHBNJ-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 14.3 nM (pChEMBL 7.840000152587891)
Descriptors: MW 686.14, ALogP 5.57, HBA 10, HBD 1, PSA 128.24, QED 0.23
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CCC(N3CCOCC3)CC2)nc1-c1cc2c(cc1OC(F)F)NCCO2)c1cnn2cccnc12
InChIKey: VSGMXKWZQJBTQY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip R technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip R 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.31 nM (pChEMBL 9.510000228881836)
Descriptors: MW 637.65, ALogP 2.57, HBA 12, HBD 2, PSA 140.38, QED 0.3
Compound (SMILES): CC(=O)NCC(=O)N[C@H]1CCc2cccc3c2N(C1=O)[C@H](C(=O)NCc1c[nH]nn1)C3
InChIKey: FRZDCCRVZUMYBX-HOTGVXAUSA-N
Target: Granzyme B (SINGLE PROTEIN), Homo sapiens
UniProt: P10144
Assay: Biochemical Assay: Buffer pH7.4: Prepare 1 (M) KH2PO4 and 1 (M) K2HPO4. Titrate 1(M) K2HPO4 with 1 (M) KH2PO4 to obtain pH 7.40. Dilute this buffer 10 fold in Water (30 ml buffer+270 ml of water) to obtain 100 mM phosphate buffer. Adjust pH to 7.40±0.02 using 5(N) HCl or 5(N) NaOH. NADPH Regeneration System (NRS): Prepare a solution containing 13 mM NADP, 33 mM Glucose-6-phosphate, 33 mM MgCl2and 4 U/ml Glucose-6-phosphate dehydrogenase in buffer. Liver Microsome (LM) suspension: Thaw LM vial on ice, then mix 1.0 ml LM (20 mg/ml) with 19 ml buffer [final LM Conc: 1 mg/ml] LM+NRS suspension: Mix 5.0 ml NRS with 20 ml LM suspension [final LM Conc: 0.8 mg/ml] System suitability standard: a synthesized compound having Mol wt 686.2 used as System suitability standard. Dissolve this compound in ice-cold acetonitrile to obtain concentration of 0.1 μg/ml and store at 4° C. Compound Dilution: Compound Stock: 10 mM in DMSO Sub stock (100 μM): 4 μl of 10 mM Compound Stock+398 μl Acetonitrile. Working plate (2 μM): 10 μl of 100 μM Sub stock+490 μl buffer Assay Procedure Incubate all plastic materials including tips at 37° C. overnight. Incubate LM suspension and NRS at 37° C. for 15 min before use. Add 40 μl buffer to the wells of blank plate. Add 40 μl compound (from working plate) to 0, 5, 10, 20, 30 and 60 min plates. Initiate reaction by adding 40 μl of LM+NRS suspension in each plate. Terminate reaction by adding 240 μl ice-cold acetonitrile containing system suitability standard at designated time points. For T=0 add 240 μl ice-cold acetonitrile containing system suitability standard before LM+NRS addition. Centrifuge (3500 rpm, 20 min and 15° C.) the plates. Mix 110 μl supernatant with 110 μl water and quantitate amount of Compound in the solution using LC-MS/MS.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 425.45, ALogP -1.05, HBA 6, HBD 4, PSA 149.18, QED 0.46
Compound (SMILES): N#Cc1ccc(Cn2ncc(NC(=O)Cc3ccc(C4(F)COC4)cc3)n2)cc1F
InChIKey: RPWGBVIQNKJBKK-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Measurement of Calcium Channel Flux by Means of FLIPR Assay: Stock solutions of test compounds are prepared to a concentration of 10 mM in DMSO. For the Cav3.2 assay, serial dilutions of the compounds are prepared in TEAC buffer (100 mM tetraethylammonium chloride; 20 mM Hepes; 2.5 mM CaCl2; 5 mM KCl; 1 mM MgCl2; 1% FBS; pH 7.2), for the Cav1.2 assay serial dilutions are prepared in assay buffer. Test compounds are added to the cells to give a 3-fold dilution range from 10 μM to 0.05 nM. The compounds are incubated with the cells for 3 minutes and Ca2+ entry is stimulated by adding either CaCl2 to a final concentration of 10 mM (Cav3.2 assay) or by adding KCl to a final concentration of 20 mM (Cav1.2 assay). The kinetics of fluorescence increase are recorded for every well and the area under the fluorescence trace for every compound concentration is used to generate inhibition curves using non-linear regression sigmoidal concentration-response curve analysis with in-house software. IC50 values are calculated and represent the compound concentration required to inhibit 50% of the signal that is obtained in the presence of vehicle instead of test compound. In analogy, antagonistic activities (IC50 values) of all exemplified compounds have been measured for the for the Cav3.1- and the Cav3.3-channel. Antagonistic activities (IC50 values) of all exemplified compounds are in the range of 1.7 to 970 nM with respect to Cav3.1; and in the range of 1.1 to 620 nM with respect to Cav3.3.
Measurement: IC50 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 409.4, ALogP 2.71, HBA 6, HBD 1, PSA 92.83, QED 0.68
Compound (SMILES): Cc1cc(-c2cc(Cl)ccc2OCCn2c(C)nc3cc(C(F)(F)F)cc(C#N)c3c2=O)c2scc(C(=O)NS(C)(=O)=O)c2n1
InChIKey: RQKRPCMHPWCOSD-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 29.7 nM (pChEMBL 7.53000020980835)
Descriptors: MW 676.1, ALogP 5.6, HBA 10, HBD 1, PSA 144.04, QED 0.23
Compound (SMILES): Cn1c(SCCCN2CC[C@]3(CC3c3ccc(C(F)(F)F)cc3)C2)nnc1-c1ccncn1
InChIKey: LKIZJVWLCHAZTJ-YSYXNDDBSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Functional Calcium Assay: CHO cells stably expressing human dopamine receptor type 2, long variant (hD2L), coupled to Gα16 protein (CHO-Gα16-hD2L) were seeded into black walled clear-base 384-well plates at a density of 8,000 cells per well and grown overnight at 37° C. After washing with the assay buffer (20 mM HEPES, 145 mM NaCl, 5 mM KCl, 5.5 mM glucose, 1 mM MgCl2 and 2 mM CaCl2, pH 7.4) containing 2.5 mM Probenecid, cells were incubated with the cytoplasmic Ca2+ probe Fluo-4 AM at 1 μM (final concentration), 37° C. for 60 min. Plates were washed three times as above and placed into a Fluorometric Imaging Plate Reader (FLIPR Tetra, Molecular Devices) to monitor cell fluorescence (ex=470-495 nm, em=515-575 nm) before and after the addition of different concentrations of test compounds. Compounds of invention were dissolved in DMSO and 200-fold diluted with assay buffer plus 0.01% Pluronic F-127. Cells were exposed first to test compounds for 10 min, then to a submaximal concentration of the hD2 receptor agonist dopamine (EC80, 50-140 nM). The fluorescence before compound addition (baseline) and before and after addition of agonist challenge was monitored. The peak of Ca2+ stimulation (baseline subtracted) was plotted versus the concentration of test compound and the curve fitted using a four-parameter logistic equation (XLfit) to assess the agonist/antagonist potency and maximal response.
Measurement: Ki = 288 nM (pChEMBL 6.539999961853027)
Descriptors: MW 474.56, ALogP 4.65, HBA 7, HBD 0, PSA 59.73, QED 0.37
Compound (SMILES): O=C(Nc1cn2nc(-c3c(-c4ccc(F)cc4)ncn3CCN3CCOCC3)ccc2n1)c1ccncc1
InChIKey: RTJQAQUXEUEVEQ-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours 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 unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 3.21 nM (pChEMBL 8.489999771118164)
Descriptors: MW 512.55, ALogP 3.38, HBA 9, HBD 1, PSA 102.47, QED 0.36
Compound (SMILES): CNCC(NC(=O)c1cc(-c2ccc3[nH]nc(C)c3c2)ccn1)c1cccc(OC)c1
InChIKey: MSNCQZSMEDHJLV-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Evaluation of the Inhibitory Activity Against ROCK Protein Kinase: Assay buffer solution: 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid (pH 7.5), 10 mM magnesium chloride, 1 mM ethylene glycol-bis-(2-aminoethyl)tetraacetic acid, 0.02% polyethylene glycol monododecyl ether, 0.02 mg/mL bovine serum albumin, 0.1 mM sodium vanadate, 2 mM dithiothreitol, 1% DMSO.Experimental Operation:The freshly prepared buffer solution was added to ROCK protein kinase substrate (Long S6 Kinase substrate peptide), at a concentration of 20 μM, then 1 nM ROCK protein kinase was added thereto and stirred evenly. Echo550 was used to add a series of DMSO dilutions containing the test compound (starting from 10 μM, serially diluted by 3 times), the solution was pre-incubated at room temperature for 20 minutes, then 33P-ATP (radiation intensity 10 μCi/μL) was added to initiate the reaction, and the reaction was performed at room temperature for two hours. Then the solution was filtered by P81 ion exchange paper (Whatman #3698-915) and washed with 0.75% phosphoric acid. The radiation intensity was determined by Filter-Binding method.
Measurement: IC50 = 138 nM (pChEMBL 6.860000133514404)
Descriptors: MW 415.5, ALogP 3.63, HBA 5, HBD 3, PSA 91.93, QED 0.43
Compound (SMILES): O=C(O)CCCOc1c(F)cc(-c2cccc(OCc3ccccc3)c2)cc1F
InChIKey: SYNWDXFSIHQCKH-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 398.41, ALogP 5.45, HBA 3, HBD 1, PSA 55.76, QED 0.49
Compound (SMILES): C[C@@H]1COCCN1c1cc(N2CCCC2c2cccc(F)c2)[nH]c(=O)c1
InChIKey: ZZOLVSJDRQQXEZ-IKJXHCRLSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: Biochemical Assay: Dilution series of compounds of the invention were prepared in DMSO at 100 times the final assay concentration (n1=n0/3 in 10 points). The compounds were further diluted to 4 times the assay concentration in assay buffer (Life technologies buffer Q, PV5125, diluted 5 times supplemented with 2 mM DTT and 2 mM MnCl2). 2.5 μl of the diluted compounds were added to a 384 well assay plate followed by 2.5 μl of 16.5 nM Vps34 enzyme (Life technologies, PV5126). Enzyme and compounds were pre-incubated at rt for 15 min. Then 5 μl of substrate mix containing 20 μM ATP (Life technologies, PV3227) and 200 μM PI:PS substrate (Life technologies, PV5122) in assay buffer was added to the wells containing compound and enzyme. Mixing was performed by pipetting several times. The reaction was incubated at room temperature for 1 h. Then 5 μl stop-detection mix, prepared as described in the Adapta kinase assay kit instructions (Life technologies, PV5099) containing Adapta Eu-anti-ADP antibody (2.3 nM), Alexa Fluor 647 ADP tracer (9 nM) and EDTA (30 mM) in TR-FRET buffer, was added to quench the reaction. Mixing was performed by pipetting several times. The assay plate was then incubated at room temperature for 30 min and read with Artemis micro plate reader. Percent inhibition of the compounds as compared to DMSO treated control samples was calculated. By the use of Dotmatics software compound concentration versus percent inhibition was fitted to generate IC50 values.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 357.43, ALogP 3.08, HBA 4, HBD 1, PSA 48.57, QED 0.92
Compound (SMILES): COCCN(C(=O)c1ccc(CN2C(=O)Cc3cnc(-c4ccccc4C(C)C)nc32)cc1)C(C)C
InChIKey: QHYCMIRFYDRATD-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 486.62, ALogP 4.85, HBA 5, HBD 0, PSA 75.63, QED 0.43
Compound (SMILES): N[C@H]1CCCC[C@H]1Nc1ccn2ncc(C(=O)Nc3cn(CC(F)(F)F)nc3C(F)(F)F)c2n1
InChIKey: RSUXKVHAOVSYNM-NWDGAFQWSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 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 IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 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, LJL BioSystems).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 490.41, ALogP 3.44, HBA 8, HBD 3, PSA 115.16, QED 0.47
Compound (SMILES): COCC1CCN(S(=O)(=O)c2ccc(NC(=O)NCc3cccnc3)cc2)CC1
InChIKey: JFFBMYWKPNNKOI-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 418.52, ALogP 2.45, HBA 5, HBD 2, PSA 100.63, QED 0.72
Compound (SMILES): O=C1CCC(N2Cc3c(NC=C4C(=O)CC(c5ccccc5Br)CC4=O)cccc3C2=O)C(=O)N1
InChIKey: HFBAXYPDTXAQRK-ATVHPVEESA-N
Target: Microtubule-associated protein 1 light chain 3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZQ8
Assay: Assay Targeting LC3B: By constructing a prokaryotic expression system, the LC3B protein was successfully expressed and purified, and a preliminary screening and verification platform was established using fluorescence polarization experiments to determine the activity of purchased and synthesized small compound libraries. The recombinant protein GST-LC3B (final concentration 180 nM) (SEQ ID NO: 1) and N-terminal FITC-labeled peptide (SEQ ID NO: 2, final concentration 18 nM) were placed in the FP buffer (50 mM HEPES pH 7.5, 0.1 mg/ml BSA and 1 mM DTT), to which a compound serially diluted with the FP buffer was added. Then the mixture was incubated at 25° C. in the dark. The fluorescence polarization value (PerkinElmer Envision, emission wavelength 480 nm; absorption wavelength 535 nm) was monitored, and the IC50 value was calculated using the GraphPad Prism 6.0 program.
Measurement: IC50 < 3 nM (pChEMBL 8.52299976348877)
Descriptors: MW 536.38, ALogP 3.22, HBA 6, HBD 2, PSA 112.65, QED 0.35
Compound (SMILES): O=C(O)c1ccc(-c2noc(C34CCC(OCc5c(-c6ccccc6OC(F)(F)F)noc5C5CC5)(CC3)CC4)n2)nc1
InChIKey: AOYIMGUJDVAAOL-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/ml Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 596.56, ALogP 6.82, HBA 9, HBD 1, PSA 133.6, QED 0.22
Compound (SMILES): COc1cnc(OCc2cn(-c3ccc(F)cc3C)nn2)nc1
InChIKey: BLOKUKDWVMHARP-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 2650 nM (pChEMBL 5.579999923706055)
Descriptors: MW 315.31, ALogP 2.09, HBA 7, HBD 0, PSA 74.95, QED 0.72
Compound (SMILES): COc1ccc(NC(=O)N2CCn3nc(-c4cccc(F)c4)c(C(N)=O)c3C2)cc1Cl
InChIKey: BZHYPUVEKQZEKS-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours 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 unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 443.87, ALogP 3.5, HBA 5, HBD 2, PSA 102.48, QED 0.64
Compound (SMILES): CCSC[C@H](N)[C@](O)(Cc1cc(OC2CCC3(CC2)CC3)ccn1)C(=O)O
InChIKey: PUELXDQYWRQWAN-FXAWDEMLSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97629
Assay: Inhibition of IRAP Assay: Rat epididymal fat pads were homogenized and subjected to ultracentrifugation at 100,000×g for 30 minutes to obtain microsomes containing IRAP. The microsomes (with a total protein content of 55 g/well) were mixed with a solvent (dimethyl sulfoxide; hereinafter, abbreviated as DMSO (final concentration: 0.1%)) or with each test compound (common ratio: 3; maximum concentration: 10 μM). AVP was then added to the solution to a final concentration of 25 μM, and the resulting solution was allowed to react for one hour at 37° C. An aqueous trifluoroacetic acid (hereinafter, abbreviated as TFA) solution was then added to the solution (final concentration: 1%) to stop the enzymatic reaction. Residual AVP was then determined by mass spectrometry (MALDI-MS). Based on the results, IC50 values (nM), i.e. concentrations required for 50% inhibition of decrease in AVP level in the solvent control group, of the individual test compounds were calculated by the logistic regression to evaluate inhibition of IRAP activity. As comparative examples, similar tests were performed with the compounds of the Reference Examples 1 and 2 described below: (2S,3S)-3-amino-2-hydroxy-5-methyl-2-({4-[(trans-4-methylcyclohexyl)oxy]pyridin-2-yl}methyl)hexanoic acid dihydrochloride; and (2S,3R)-3-amino-2-hydroxy-5-methyl-2-({4-[(trans-4-methylcyclohexyl)oxy]pyridin-2-yl}methyl)hexanoic acid hydrochloride, respectively.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 394.54, ALogP 2.62, HBA 6, HBD 3, PSA 105.67, QED 0.59
Compound (SMILES): O=c1c2cnc(Nc3ccc(N4CCN(CCO)CC4)cc3)nc2ccn1-c1c(Cl)cccc1Cl
InChIKey: WTOAJORRZPAAOR-UHFFFAOYSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: Wee-1 kinase Assay: In the measurement of Wee-1 activity, a commercial peptide Poly(Lys Tyr(4:1)) hydrobromide was purchased from Sigma Aldrich and used as the substrate. Activated Wee-1 kinase was purchased from Invitrogen (PV3817) and an ADP-Glo luminescent kit was purchased from Promega.All reactions took place in 60 μL volumes in reaction buffer containing 40 mM Tris-HCl and 20 mM magnesium chloride, supplemented with 0.1 mg/mL bovine serum albumin and 2 mM DTT. Compounds were serially diluted in buffer and 5 μL of each concentration pipetted into a white 384 well plate (Sigma Aldrich M6186). A 5 μL aliquot of the Wee-1 enzyme was added to each well and the plate centrifuged for 1 min to ensure mixing of the enzyme and inhibitor.The plate was incubated at room temperature for 30 minutes before the addition of 2.0 μg/mL of substrate and 30 μM ATP in a 5 μL aliquot. The plate was centrifuged for one minute and incubated for 1 h at RT.15 μL of ADP-Glo stop reagent was added to each well to quench the reaction and deplete unconverted ATP. The plate was incubated for a further 40 min in the dark at RT.30 μL of ADP-Glo kinase detection reagent was added to each well, converting ADP to ATP, catalysing the generation of luciferin by luciferase. The plate was shaken for 1 min, and incubated in the dark for an additional hour.Luminescence from each well was detected using the Biotek Synergy4 HD plate reader and the percentage inhibition of kinase activity calculated for each inhibitor tested. Positive (kinase only) and negative (no kinase) controls were added to each plate to ensure specific interaction of kinase and inhibitor. The IC50 concentration for each inhibitor was calculated by plotting the percentage kinase inhibition against concentration of inhibitor and the curve generated by non-linear regression fitting.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 511.41, ALogP 3.95, HBA 8, HBD 2, PSA 86.52, QED 0.41
Compound (SMILES): O=C(CNC(=O)c1ccc2c(c1)OCc1cnccc1-2)c1ccc(Cl)cc1
InChIKey: HXPSFPWWJYZNMK-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 1620 nM (pChEMBL 5.789999961853027)
Descriptors: MW 378.82, ALogP 3.91, HBA 4, HBD 1, PSA 68.29, QED 0.7
Compound (SMILES): C[C@@H](C(=O)O)[C@H](C)n1c(=O)[nH]/c(=N\c2ccc3cnccc3c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: GDMIIUQBNATWLG-CABCVRRESA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution 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, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 882 nM (pChEMBL 6.050000190734863)
Descriptors: MW 479.92, ALogP 3.1, HBA 7, HBD 2, PSA 122.34, QED 0.44
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCN(C(=O)N5CCCC(F)C5)CC4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: YOPDHXKOVNCMPB-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 11.7 nM (pChEMBL 7.929999828338623)
Descriptors: MW 513.58, ALogP 3.34, HBA 8, HBD 0, PSA 98.59, QED 0.42
Compound (SMILES): O=C1Nc2cc(OC(F)(F)F)ccc2Oc2ccc(-c3cccc(B(O)O)c3)cc21
InChIKey: SOLUFTUAJPZLAS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: TBD: TBD
Measurement: IC50 = 3317 nM (pChEMBL 5.480000019073486)
Descriptors: MW 415.13
Compound (SMILES): COc1cc(N2CCN(C(=O)CCC3(C)NC(=O)NC3=O)CC2)cc(OC)c1Cl
InChIKey: QIAPFAXONPZNKR-UHFFFAOYSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O75173
Assay: In Vitro Assay: TABLE VI: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID NO: 2) by human ADAMTS4.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, ⅕ dilution in DMSO further diluted 1 in 10 in water corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS) containing hADAMTS4 (0.38 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 180 min at 37° C. (Excitation 485 nm, emission 535).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 424.89, ALogP 1.38, HBA 6, HBD 2, PSA 100.21, QED 0.67