lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CO[C@H]1CC[C@H](n2c(=O)n(C)c3cnc(Nc4cn5ncnc5cc4C)nc32)CC1
InChIKey: ZFIZVPPSNWVMDU-HDJSIYSDSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: DNAPK Enzyme Potency Assay (DNA-PK Enz): The inhibitory activity of compounds against DNAPK was determined by TR-FRET measuring a fluorescent labelled peptide substrate converting to a phosphorylated product. fluorescently tagged peptide substrate were purchased from Thermo Fisher Scientific. 12 point half-log compound concentration-response curves, with a top concentration of 100 μM were generated from 10 mM stocks of compound solubilised in DMSO using an Echo 555 (Labcyte Inc., Sunnyvale, Calif.). All assays were preformed in white Greiner 1536 well low volume plates (Greiner Bio-One, UK), in a total reaction volume of 3 μL and 1% (v/v) final DMSO concentration. Enzymes and substrates were added separately to the compound plates and incubated at room temperature. The kinase reaction was then quenched by the addition of 3 μL of stop buffer. Stopped assay plates were read using a BMG Pherastar. IC50 values were calculated using a Genedata Screener® software (Genedata, Inc., Basel, Switzerland).Full length human DNAPK protein was purified from HeLa cell extract by ion exchange. Initially DNAPK protein was incubated with compound for 30 minutes at room temperature in reaction buffer (50 mM Hepes pH 7.5, 0.01% Brij-35, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 2 μg/ml Calf Thymus DNA). The reaction was then initiated by the addition of ATP and fluorescently tagged peptide substrate (Fluorescein-EPPLSQEAFADLWKK, Thermo Fisher Scientific). The kinase reaction (18 μM ATP, 35 μM DNAPK, 1.6 μM peptide substrate) was quenched after 40 minutes by the addition of 3 μL of stop buffer (20 mM Tris pH7.5, 0.02% sodium azide, 0.01% Nonidet-P40, 20 m EDTA, 4 nM Tb anti-phospho-p53 [Ser15] Antibody. The reaction was incubated for a further hour and the plates were read on a BMG Pherastar.Data was analysed and IC50 values were calculated using Genedata Screener® software (Genedata, Inc., Basel, Switzerland). The pIC50 values were calculated as the negative logarithm of the molar concentration of compound required for 50% reduction in measured response.
Measurement: IC50 = 0.251 nM (pChEMBL 9.600000381469727)
Descriptors: MW 408.47, ALogP 2.35, HBA 10, HBD 1, PSA 104.16, QED 0.55
Compound (SMILES): Cc1ccc(NC(=O)N2CC[C@@H](CC(F)(F)F)C2)cc1-c1cc(N2CCOCC2)nc(N2C[C@@H](N)[C@H](O)C2)c1
InChIKey: BFMKGQYOZSQRKV-CDNPAEQRSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Enzymatic RAF1 Kinase: Small molecule inhibition of RAF1 kinases was measured using ADP-Glo assay. In the assay, ADP is converted to ATP in the presence of test kinase and substrate, resulting in luciferase reaction and luminescent readout with light generated proportional to the relative kinase activity. Compounds diluted in DMSO were used in 10-point, 3-fold dose curve for both assays. Final concentrations of 3 nM RAF1 (CarnaBio, Cat. 09-125) and 30 nM MEK1 substrate (Millipore, Cat. 14-420) were incubated with 3 μM ATP, 10 mM MgCl2, 0.003% Brij-35, 2 mM DTT, 0.05% BSA, 1 mM EGTA, and 50 mM HEPES for 90 minutes at room temp prior to addition of ADP-Glo reagent (Promega, Cat. V9102) for 40 minutes, and detection reagent for 45 minutes. Luminescence was read on an Envision plate reader (PerkinElmer) and percent remaining activity was used to calculate IC50 using a four-parameter fit model using Dotmatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 548.61, ALogP 3.21, HBA 7, HBD 3, PSA 107.19, QED 0.53
Compound (SMILES): Cc1cccc2c(C(C)(C)NC(=O)[C@@H]3CNCCO3)nn(C)c12
InChIKey: JYUJCVSNMHJOMN-ZDUSSCGKSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: cAMP Assay: Compounds are dissolved and diluted in DMSO. The final test solution contains 1% DMSO. The cAMP standard (Lance cAMP 384 Kit; PerkinElmer, Cat# AD0264) is prepared in assay buffer (HBSS with 0.1% BSA, 5 mM HEPES, 0.5M IBMX, pH 7.4) containing 1% DMSO and the cAMP standard curve is included at least on one plate. Cells are centrifuged and suspended in assay buffer (incl. 1:100 diluted Alexa antibody).For the assay 5 μl of a cell suspension (approximately 5000 cells/well)¿incl. Alexa antibody (diluted 1:100) are added into a 384 well MTP microtitre plate excepting one row or column (depending on the plate layout), which is reserved for the standard curve. Then 2 μl of compound sample is added as concentration response curve (e.g. le-5M to 6e-10M), usually in triplicates. Each assay contains incubations with vehicle controls instead of compound as controls for non-inhibited cAMP generation (100% CTL; ¿high values¿) and incubations with 1 μM Somatosatin as controls for full inhibition and background (0% CTL; 'low values'). After approximately 10-15 min incubation time 3 μl Forskolin (dissolved in DMSO, final conc. 15 μM) is added. Then the plates are shaken briefly and incubated for 60 min at room temperature. After 60 min 10 μl of the detection mix is added into all wells followed by an additional incubation period of 1 h. The plates are read in a suitable plate reader.The analysis of the data is based on the "ratio" of the time-resolved fluorescence measurements of donor and acceptor fluorophore (Ex: 320 nm; Em1: 665 nm; Em2: 615 nm; ratio 665/615). From this ratio, cAMP concentrations are calculated from standard curve and the EC50 is estimated by least square curve fit program.
Measurement: EC50 = 4.1 nM (pChEMBL 8.390000343322754)
Descriptors: MW 316.41, ALogP 1.22, HBA 5, HBD 2, PSA 68.18, QED 0.89
Compound (SMILES): Cc1cc(-c2n[nH]c3cc4c(cc23)CN(C(C)c2nc(C)c(C)s2)C(=O)N4)ccn1
InChIKey: MDAOZDUHOOPNTE-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 418.53, ALogP 5.12, HBA 5, HBD 2, PSA 86.8, QED 0.48
Compound (SMILES): C[C@@H]1CC[C@@H](Oc2nccc3c2COC3=O)CN1C(=O)c1ccccc1C1(C#N)CC1
InChIKey: YOYHOMQHRBFXIN-HZPDHXFCSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Intracellular Calcium Assay : In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 417.47, ALogP 3.38, HBA 6, HBD 0, PSA 92.52, QED 0.71
Compound (SMILES): CC(=O)N[C@@H](C)/C=C(\F)[C@@H]1CC[C@@H](Oc2nc3c(F)cc(OCC(F)F)c(F)c3n2C)CO1
InChIKey: ONTRBHHRFLYYGE-DGPCQLGMSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: The Measurement of Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H].+Intensity of [AcCoA-H]) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 477.43, ALogP 3.8, HBA 6, HBD 1, PSA 74.61, QED 0.59
Compound (SMILES): COC(=O)c1ccc2c(c1)nc(Nc1ccc(C(C)C)cc1)n2C1CC(C)(C)CC(C)(C)C1
InChIKey: VHIBKBGNVOQYNJ-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μl and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μl of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/ml FMN, 2 μl/ml Decanal/Ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 447.62, ALogP 7.47, HBA 5, HBD 1, PSA 56.15, QED 0.41
Compound (SMILES): N#CCC(=O)N1CC[C@@H](Oc2ccc(-c3ccnc4[nH]c(-c5ccc(N6CCOCC6)cc5)nc34)cc2C#N)C1
InChIKey: ZWAOQFJBLRYVDG-XMMPIXPASA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKs: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKs were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2× Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 4.59 nM (pChEMBL 8.34000015258789)
Descriptors: MW 533.59, ALogP 3.89, HBA 8, HBD 1, PSA 131.16, QED 0.39
Compound (SMILES): COC1CCN(c2cnc3c(C(=O)Nc4cnccc4[C@@H]4C[C@H](C)C[C@H](N)C4)ccc(F)c3c2)CC1
InChIKey: CXJSUMQKMLNXNC-OTWHNJEPSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 491.61, ALogP 4.87, HBA 6, HBD 2, PSA 93.37, QED 0.53
Compound (SMILES): CC(=O)c1ccc(OC2CCN(C(=O)c3ccc(C(=O)NC4CCN(Cc5ccc(C#N)cc5)CC4)nn3)CC2)cc1
InChIKey: ACJLYGUYLHGBRM-UHFFFAOYSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P54646
Assay: Enzyme-Linked Immunosorbent Assay: Compounds were assayed for their ability to activate AMPK using an enzyme-linked immunosorbent assay. Reagents and procedures for measuring AMPK activation are well known and kits for AMPK activation assays are commercially available.
Measurement: EC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 566.66, ALogP 3.63, HBA 8, HBD 1, PSA 128.52, QED 0.41
Compound (SMILES): Cc1c(-c2cc3cc(NC(=O)N[C@H]4C[C@H]4C)ncc3c(N)c2F)cnc2c1NCCO2
InChIKey: OLJLOBXIXPHTEK-HWPZZCPQSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki < 0.013 nM (pChEMBL 10.88599967956543)
Descriptors: MW 422.46, ALogP 3.66, HBA 6, HBD 4, PSA 114.19, QED 0.48
Compound (SMILES): Cc1cccc(C2CCN(C(=O)c3ccc(Nc4nc5c(C6=CCN(C(=O)CCC(F)(F)F)CC6)cccn5n4)cc3)CC2)n1
InChIKey: LDVBUJQZGVKKQT-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® 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® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.69 nM (pChEMBL 9.15999984741211)
Descriptors: MW 617.68, ALogP 6.15, HBA 7, HBD 1, PSA 95.73, QED 0.27
Compound (SMILES): C=CC(=O)Nc1cc(Nc2nc(-c3cnn(C)c3)c3cc[nH]c3n2)c(OC)cc1N(C)CCN(C)C
InChIKey: RGRJZPKFWSMZRB-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition Assay: The inhibitory activities on T790M/L858R double mutant EGFR kinase (Invitrogen, PV4879) and wild-type EGFR kinase (Invitrogen, PV3872) were determined by the z′-lyte assay.The working concentrations of each component in 10 μL T790M/L858R kinase reaction system were: 25 μM ATP, 0.1 ng/μL T790M/L858R kinase, 2 μM Tyr04 substrate (Invitrogen, PV3193). The concentration of DMSO after addition of the compound prepared in the above examples of the present invention (i.e., the compound to be tested) was 2 vol %.The working concentrations of each component in 10 μL wild-type EGER kinase reaction system were: 10 μM ATP, 0.8 ng/μL wild-type EGFR kinase, 2 μM Tyr04 substrate (Invitrogen, PV3193). The concentration of DMSO after addition of the compound to be tested was 2 vol %.10 mM drug stock solution was dissolved at room temperature and diluted to a final concentration of 4-0.002 μM with 8 vol % DMSO solution. 2.5 μL of the solution of the compound to be tested and 5 μL of the mixture of T790M/L858R kinase (or wild-type EGFR kinase) and Tyr04 substrate diluted with the reaction buffer were added to each well. Then 2.5 μl of ATP was added to initiate the reaction. Wherein, ATP was replaced by the reaction buffer in C1 well, no drug was added to C2 well, and the phosphorylated substrate was added to C3 well according to the instructions. The mixture was allowed to react at 25° C. in a shaker in dark for 60 min, 5 μL of Development Reagent B (invitrogen, diluted with TR-FRET dilution buffer) was added and allowed to react at room temperature in the shaker for 60 minutes. The plate was read on a Victor X5 plate reader (PerkinElmer) and the absorbance was measured at excitation wavelengths of 405 nm and emission wavelengths of 450 nm and 520 nm (for example, C3520 nm indicates the absorbance for C3 well at 520 nm)
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 489.58, ALogP 3.23, HBA 9, HBD 3, PSA 116.23, QED 0.29
Compound (SMILES): O=C(NCc1ccc(Cl)cc1Cl)c1cnc(CN2CCN(C(=O)C3(O)CC3)CC2)s1
InChIKey: MBPKAFIDNKRGIR-UHFFFAOYSA-N
Target: Bifunctional epoxide hydrolase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34913
Assay: Biochemical Screening Assay: Compounds were tested in a biochemical screening assay using recombinant sEH purified from Sf9 insect cells and an artificial substrate, (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphtalen-2-yl)-methyl ester, Phome. The biochemical assay was performed in analogy to a fluorometric described in the literature (P. D. Jones et al., Anal. Biochem. 2005, 343, 66-75). The assay principle bases on the sEH-catalyzed hydrolysis of an artificial α-cyano-ester substrate. O-deacetylation liberates a cyanohydrin intermediate that rapidly decomposes to the highly fluorescent 6-methoxy-2-naphtaldehyde. To discriminate sample autofluorescence, the assay was carried out as a kinetic measurement with two time points. The first measurement is performed immediately before addition of the substrate and the 15 second measurement is done after completion of the assay. The assay format is either in 96- or in 384-well format.Details of the assay using a 96-well plate format are described below. 40 μl recombinant sEH enzyme and 5 μl test compound are pre-incubated for 15 minutes at 30° C. Following pre-incubation, the reaction is started by addition of 5 μl Phome. The assay mixture containing 2 nM final sEH concentration, test compound ranging in a concentration from 0.0001-10 μM and 5 μM Phome is incubated for 60 minutes at 30° C.
Measurement: IC50 = 6.37 nM (pChEMBL 8.199999809265137)
Descriptors: MW 469.39, ALogP 2.55, HBA 6, HBD 2, PSA 85.77, QED 0.68
Compound (SMILES): C#CCn1nc(C(=O)Nc2ccc(Oc3ccnc4cc(OC)c(OC)cc34)c(F)c2)c(=O)n(-c2ccc(F)cc2)c1=O
InChIKey: IHGZXZXQXYFLIX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Kinase Assay: The ability of compounds to inhibit the kinase activity of recombinant human baculovirus-expressed AXL was measured by homogeneous TRF (HTRF) using Cisbio's KinEASE assay system in white 384-well Optiplates. Assay buffer contained 1 mM DTT, 2 mM MnCl2, 2% DMSO, 50 nM supplement enzymatic buffer, and 1× enzymatic buffer. A 2× concentration of tyrosine kinase (TK) substrate-biotin/ATP mixture made in assay buffer was added to plates at 10 μL/well using the Multidrop Combi (Thermo Fisher Scientific, Waltham, Mass.). The final concentrations were 0.3 μM TK substrate-biotin, and 1.3 μM ATP. Compounds (100 nL), diluted in 100% DMSO on the Biomek FX, (Beckman Coulter, Inc., Brea, Calif.), were transferred to the assay plates using the Biomek FX pintool (2.5% final DMSO in assay). A 2× concentration (final=12 ng/mL) of GST-AXL (diluted in assay buffer) was added to plates at 10 uL/well using the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 30 minutes. A 4× stock of Streptavidin-XL665 (final=18.8 nM) and a 1:100 diluted stock of TK antibody-cryptate were made in HTRF detection buffer and mixed together just prior to adding 20 μL/well on the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 1 hour. The fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 multi-label plate reader (PerkinElmer, Waltham, Mass.) with an excitation wavelength of 337 nm (laser) and emission wavelengths of 590 and 665 nm. Raw data was expressed as the ratio of 665/590×10,000.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 585.52, ALogP 3.92, HBA 10, HBD 1, PSA 126.57, QED 0.27
Compound (SMILES): COc1c(C(C)n2nc(C(F)F)c3c(N)ncnc32)cc(Cl)c(C)c1C1CN(C(C)C)C1
InChIKey: BWPVHVGKTKQLSR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) was purchased from PerkinElmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from SigmaAldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.).The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI31(6. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (PerkinElmer).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 464.95, ALogP 4.73, HBA 7, HBD 1, PSA 82.09, QED 0.57
Compound (SMILES): Cn1cc(C(=O)Nc2ccc(-c3n[nH]c(=O)c4ccccc34)cc2)c2ccccc21
InChIKey: ILOVBIWKAGJFHZ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
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 < 100 nM (pChEMBL 7)
Descriptors: MW 394.43, ALogP 4.33, HBA 4, HBD 2, PSA 79.78, QED 0.48
Compound (SMILES): C[C@H]1Cn2c(-c3cccc(C(F)(F)F)n3)nnc2-c2nnc(-c3cccc(C(F)(F)F)c3Cl)n21
InChIKey: LAOIMXUAJWKNLY-VIFPVBQESA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z1M0
Assay: P2X7 FLIPR Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 = 698 nM (pChEMBL 6.159999847412109)
Descriptors: MW 499.81, ALogP 5.53, HBA 7, HBD 0, PSA 74.31, QED 0.34
Compound (SMILES): CC(C)NC(=O)c1ccccc1NCc1ccc(-c2noc(C(F)(F)F)n2)cn1
InChIKey: VBZWOKNCCLBJOI-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Assay: HDAC1 and 6 reagents: FLAG-tagged HDACs 1 and 6 were prepared in-house by protein expression in HEK293F cells followed by anti-FLAG affinity purification. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. In addition to the above buffer ingredients, TCEP [CalBiochem, catalog #580561, 500 mM stock] was added at a final concentration of 0.5 mM to the buffer for the HDAC6 assays. HDAC1, 2, 3, and 6 enzymes were run at the final concentrations of 0.3 nM, 1.5 nM, 0.3 nM, and 1.333 nM, respectively. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-104], used to evaluate enzyme activity, was added at the final concentrations of 20 uM, 40 uM, 20 uM, and 2.5 uM for HDACs 1, 2, 3, and 6. To enable detection of the signal, Developer [BioMol Research Laboratories, catalog #KI-105] was added at a 1:250 dilution to the stop solution, which also included 10 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 405.38, ALogP 3.9, HBA 6, HBD 2, PSA 92.94, QED 0.65
Compound (SMILES): CCN1C[C@@]2(C[C@@H]2CO)n2cc(C(=O)NCc3ccc(F)cc3F)c(=O)c(O)c2C1=O
InChIKey: IPMFRVJDCBDKPF-XUSGNXJCSA-N
Target: Protein Nef (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: P03406
Assay: Measurement of Antiviral (HIV-1) Activity: The medium (40 μL), a test substance (10 μL) diluted with the medium, and a 1×105 cells/mL MT-4 cell suspension (50 μL) wherein HIV-1 NL4-3 strain was infected with MOI (infection multiplicity) 0.05 were added to each well of a 96-well black plate (manufactured by Corning Incorporated), and the mixture was cultured at 37° C. for 5 days.medium composition: RPMI1640, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin.Then, Cell Titer-Glo (manufactured by Promega Corporation, 100 μL) was added to each well, and the mixture was stood at room temperature for 10 min, and the luminescence intensity was measured.
Measurement: EC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 433.41, ALogP 0.95, HBA 6, HBD 3, PSA 111.87, QED 0.65
Compound (SMILES): O=C(NCc1cccs1)[C@@H]1CN(C(=O)Oc2ccccc2Cl)CCN1C(=O)[C@H](NC1CCCCC1)C1CCN(S(=O)(=O)c2cn[nH]c2)CC1
InChIKey: QCSYYRIREALFHR-MFMCTBQISA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the IC50, the inhibitor concentration causing a 50% decrease in Factor XIIa protease activity.Factor XIIa activity determinations were made in 50 mM HEPES buffer containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific) at pH 7.4. Determinations were made using purified human Factor XIIa at a final concentration of 500 pM (Sekisui Diagnostics) and the synthetic substrate, n-Acetyl-Lys-Pro-Arg-AFC, TFA salt (Sigma #C6608) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. IC50 was determined as the concentration of I yielding Vi=Vo/2.
Measurement: IC50 = 21.1 nM (pChEMBL 7.679999828338623)
Descriptors: MW 732.33, ALogP 3.84, HBA 9, HBD 3, PSA 157.04, QED 0.28
Compound (SMILES): NC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(Cl)c2-n2cncn2)c2ccc(-c3ccnnc3)cc12
InChIKey: LEGRAROFLIPOMP-VGSWGCGISA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 588.99, ALogP 2.4, HBA 10, HBD 2, PSA 166.81, QED 0.29
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H](C(=O)Nc3nc(Br)ccc3CN(C)C)C[C@@]3(C)C[C@@H]23)c2c(Cl)cc(-c3cnc(C)nc3)cc12
InChIKey: YHRIETOONINXBR-JLELKNTQSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 680.01, ALogP 4.9, HBA 9, HBD 1, PSA 126.21, QED 0.21
Compound (SMILES): Cc1nc(N)ccc1CNC(=O)[C@H](C)NC(=O)[C@H]1Cc2c([nH]c3ccccc23)CN1
InChIKey: JRUFTDZUIWKWRT-SCLBCKFNSA-N
Target: Mannan-binding lectin serine protease 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00187
Assay: Enzymatic Assay for MASP-2: The assay was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50 values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed., John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 406.49, ALogP 1.29, HBA 5, HBD 5, PSA 124.93, QED 0.44
Compound (SMILES): C=CCn1c(=O)c2cnc(Nc3ccc(N4CCNCC4)cc3)nc2n1-c1cccc(N=S(C)(C)=O)c1
InChIKey: DQBSGRUCTNXDOR-UHFFFAOYSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: Kinase Activity Assay: 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.
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 518.65, ALogP 3.28, HBA 10, HBD 2, PSA 109.44, QED 0.36
Compound (SMILES): O=S(=O)(CF)c1ccc(Oc2cc(F)c(F)c(F)c2)c2c1[C@H](O)C(F)(F)C2
InChIKey: GMCWHPWLKUAKDH-HNNXBMFYSA-N
Target: Vascular endothelial growth factor A, long form (SINGLE PROTEIN), Homo sapiens
UniProt: P15692
Assay: ELISA Assay: Four hours later, serial dilutions of 10× compound stocks were made in growth medium from 500×DMSO stocks, and 20 μL of those 10× stocks were added to each well to make final concentrations as follows (μM): 20, 6.67, 2.22, 0.74, 0.25, 0.082, 0.027, 0.009, 0.003, 0.001, and 0. Each concentration had duplicated wells. About 20 hours later, medium was removed by suction and each well was supplied with 180 μL of growth medium. About 20 μl freshly-made 10× compound stocks were added to each well. About 24 hours later, cell culture medium was removed for the determination of VEGFA concentration using an ELISA kit purchased from R&D systems by following the manufacturer's suggested method. The EC50 was calculated by GraphPad Prism using the dose-response-inhibition (four parameter) equation. The cell seeded plate was then subjected to CellTiter-Glo luminescence cell viability assay (Promega) by adding 50 μL of Celltiter Glo reagent into each well and shaking the plate for 8 minutes at 550 rpm (Thermomixer R, Eppendorf) then read luminescence signal in plate reader (3 second delay, 0.5 second/well integration time, Synergy 2 multi Detection Microplate reader) immediately.
Measurement: EC50 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 412.31, ALogP 3.82, HBA 4, HBD 1, PSA 63.6, QED 0.61
Compound (SMILES): C=CC(=O)N1CCC[C@@H](n2cc(C(N)=O)c(-c3ccc(C(=O)N4CCCCC4)cc3)n2)C1
InChIKey: CLOYRPHRGGDCJN-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK Assay: TBDAn HTRF assay (Cisbio KinEASE-TK cat #62TKOPEC) was performed to quantitate the ability of test compounds to inhibit BTK mediated phosphorylation of substrate. Assays were assembled in 384 well plates where 6 nM of full-length human His-tagged BTK (Life Technologies cat # PV3587) and test compound at varying concentrations were preincubated for 15 minutes at 28° C. Then, 1 uM of TK substrate-biotin and 30 uM ATP were added and incubated for an additional 30 minutes at 28° C. Phospohrylation was detected by adding 62.5 nM Streptavidin-XL665 and TK-Antibody Cryptate diluted 1:100 in HTRF detection buffer (Cisbio cat #62SDBRDF) and incubated for 60 minutes at RT. The plate was read on an Envision plate reader and the fluorescence is measured at 620 nm (cryptate) and 665 nm (XL665). A ratio is calculated (665/620) and converted to POC relative to control and blank wells.
Measurement: IC50 = 8200 nM (pChEMBL 5.090000152587891)
Descriptors: MW 435.53, ALogP 2.62, HBA 5, HBD 1, PSA 101.53, QED 0.73
Compound (SMILES): O=C(CN1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O)NCc1ccc(C(=O)NC2CC2)cc1
InChIKey: AOIBQXDKYJFFLY-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 575.07, ALogP 3.55, HBA 7, HBD 3, PSA 125.55, QED 0.36
Compound (SMILES): Cc1nc(C2CCN(c3ncnc4[nH]c(-c5cccc(C(F)(F)F)c5)cc34)CC2)[nH]c1-c1ccccc1
InChIKey: KTHNMXLUYJNKEN-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: Assays for biochemical cell-based activity of c-kit kinase are known in the art, for example, as described in U.S. Pat. Nos. 7,498,342 and 7,846,941, the disclosures of which are hereby incorporated by reference as it relates to such assays. The c-kit (or kinase domain thereof) is an active kinase in AlphaScreen. IC50 values are determined with respect to inhibition of c-Kit kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested were dissolved in DMSO to a concentration of 30 mM. These were diluted 30 μl into 130 μl of DMSO (4 mM) and 1 μl was added to an assay plate. These were then serially diluted 1:3 (50 μl to 100 μl DMSO) for a total of 8 points. Plates were prepared such that each kinase reaction is 30 μl in 1× kinase buffer (50 mM HEPES, pH 7.2, 5 mM MgCl2, 5 mM MnCl2, 0.01% NP-40, 0.2% BSA), 5% DMSO and 10 μM ATP. Substrate was 100 nM biotin-(E4Y)3 (Open Source Biotech, Inc.). C-kit kinase was at 0.1 ng per sample. After incubation of the kinase reaction for 1 hour at room temperature, 5 μl of donor beads (Streptavidin coated beads (Perkin Elmer Life Science) final concentration 1 μg/ml) in stop buffer (50 mM EDTA in 1× kinase buffer) was added, the sample was mixed and incubated for 30 minutes at room temperature before adding 5 ml of acceptor beads (PY30 coated beads (Perkin Elmer Life Science) final concentration 1 μg/ml) in stop buffer. The samples were incubated for 60 minutes at room temperature and the signal per well was read on AlphaQuest reader. Phosphorylated substrate results in binding of the PY30 antibody and association of the donor and acceptor beads such that signal correlates with kinase activity.
Measurement: Ki = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 502.54, ALogP 6.73, HBA 4, HBD 2, PSA 73.49, QED 0.29
Compound (SMILES): COc1ccccc1C1C2=C(CCCC2=O)Nc2n[nH]c(C)c21
InChIKey: WSYAZSOYXLAXAQ-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Caliper Assay: A solution of 4× inhibitor (5 μL), 4× substrate/ATP-Metal solution (5 μL), and 2× Kinase solution (10 μL) was prepared with assay buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH 7.5) and mixed/incubated in 384 well plates for 1 or 5 hours depending on the kinase being examined, at room temperature. A solution of termination buffer (QuickScout Screening assist MSA; Carna Biosciences) (60 μL) was added to each well. The entire reaction mixture was then applied to a LabChip3000 system (Caliper Life Science) and the product and substrate peptide peaks were separated and quantified. Evaluation of kinase activity was then determined based on ratio of calculated peak heights of product (P) and substrate (S) peptides (P/(P+S)).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 309.37, ALogP 3.29, HBA 4, HBD 2, PSA 67.01, QED 0.89
Compound (SMILES): Cc1nc2c(Nc3ccc([C@H]4CCC(F)(F)CO4)cc3S(C)(=O)=O)cc(NC(=O)C3CC3)nc2[nH]1
InChIKey: YTHXBYFKPHJKTH-QGZVFWFLSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Binding Assay: Binding constants for compounds of the present invention against the JH2 domain were determined by the following protocol for a KINOMEscan® assay (DiscoveRx). A fusion protein of a partial length construct of human TYK2 (JH2domain-pseudokinase) (amino acids G556 to D888 based on reference sequence NP_003322.3) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The TYK2 (JH2domain-pseudokinase) fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd = 0.75 nM (pChEMBL 9.119999885559082)
Descriptors: MW 505.55, ALogP 4.25, HBA 7, HBD 3, PSA 126.07, QED 0.46