lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Nc1ncnn2c(C3CCC(O)CC3)cc(-c3ccc(NC(=O)c4cccn(-c5ccccc5)c4=O)cc3F)c12
InChIKey: ONSJKYVPJNEILC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: TAM Enzymatic Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LLIMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 538.58, ALogP 4.54, HBA 8, HBD 3, PSA 127.54, QED 0.3
Compound (SMILES): Cc1cc(COc2cccc(-c3[nH]ncc3Oc3ccc(C#N)cc3)c2)on1
InChIKey: PKZCZYPUOVZFMD-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition Assay: G-402 cells can be obtained from ATCC (CRL-1440) and were originally derived from a renal leiomyoblastoma.The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 0.032 nM (pChEMBL 10.489999771118164)
Descriptors: MW 372.38, ALogP 4.62, HBA 6, HBD 1, PSA 96.96, QED 0.53
Compound (SMILES): CC(C)c1c(-c2cc(Cl)ncc2C#N)[nH]c2ccc(C3CCNCC3)cc12
InChIKey: PRDFESRDFZXCRA-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 481 nM (pChEMBL 6.320000171661377)
Descriptors: MW 378.91, ALogP 5.35, HBA 3, HBD 2, PSA 64.5, QED 0.6
Compound (SMILES): COc1ccc(NC(=O)N[C@@H]2CCCN(c3ccc(-c4ccccc4NS(C)(=O)=O)cc3)C2=O)c(F)c1
InChIKey: KDHTXCZCRZTGAE-XMMPIXPASA-N
Target: fMet-Leu-Phe receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21462
Assay: cAMP Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays. A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 flurorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm.
Measurement: EC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 526.59, ALogP 4.19, HBA 5, HBD 3, PSA 116.84, QED 0.43
Compound (SMILES): CC(C)CO[C@H]1CCCC[C@@H]1N1CCN(c2cccc3c2cnn3-c2ccccc2F)C1=O
InChIKey: VQEMSPFCVDYFKS-DQEYMECFSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y9
Assay: FLIPR Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.8 (Essen, Ann Arbor, Mich.) were quickly thawed and plated at 22,500 cells/well in growth medium [MEM (Invitrogen #11095) with 10% FBS (Invitrogen #10082), 1 mM sodium pyruvate (Invitrogen, #C11360), 10 units/mL penicillin/10 U/mL streptomycin/29.2 μg/mL glutamine ((PSG 1%, Invitrogen #10378), 400 μg/mL zeocin (Invitrogen #R250) in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 μL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 μM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 μL assay buffer containing 2.5 μM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 60 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR®TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics.
Measurement: IC50 = 10800 nM (pChEMBL 4.96999979019165)
Descriptors: MW 450.56, ALogP 5.39, HBA 4, HBD 0, PSA 50.6, QED 0.51
Compound (SMILES): COc1cc(Nc2ccc3ncc(N4CCOCC4)nc3c2C#N)ccc1OCc1cnn(C)c1
InChIKey: JRRVOKVKPMWSTL-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kinase Assay: c-KIT: Inhibition of c-KIT kinase activity by a test compound disclosed herein was estimated by radiometric assay. Standard assay conditions were 20 ng of recombinant c-Kit (SignalChem) with 2 μg of substrate Poly-(Glu 4:Tyr 1) (sigma) in the assay buffer in kinase reaction buffer (10 mM MOPS pH 7.0, 0.21 mM EDTA, 0.5% glycerol, 0.001% Brij-35, 0.01 2-mercaptoehtanol, 1 mg/ml BSA, 10 mM MgCl2, 10 mM MnCl2, 10 μM ATP, 0.1 μCi per well [33P]-ATP, in the presence of test compound (diluted in final concentration of 4% DMSO) or DMSO control, with a final volume of 25 μl for 60 minutes at 30° C. The reaction was stopped by adding 5 μl of 3% phosphoric acid solution. Total reaction solution was then harvested onto a filter plate (UniFilter-96 GF/B, PerkinElmer), and washed 20 times for 5 min with dH2O. 30 μl of MicroScint-20 Cocktail (PerkinElmer) was added to dried plate. The plate was sealed and counted using a TopCount scintillation detector (PerkinElmer). The readout from control reaction (complete reaction mixture) was designated as 0% inhibition and the readout for the reaction without enzyme as 100% inhibition. The IC50 values of quinoxaline compounds of this invention against c-KIT kinase were determined using the GraphPad Prism 5 software.
Measurement: Ki = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 471.52, ALogP 3.4, HBA 10, HBD 1, PSA 110.35, QED 0.43
Compound (SMILES): CC(NC(=O)c1c(N)nn2cccnc12)c1cc(Cl)c2cn[nH]c2c1-c1ccccc1
InChIKey: GZPIIBURDJFNSH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3K-delta Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (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) Recombinant Human Protein was purchased from Eurofins (St Charles, Mo.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one 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 2%. The PI3Kδ assay was 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, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 3.4 nM PI3Kδ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 431.89, ALogP 4, HBA 6, HBD 3, PSA 113.99, QED 0.4
Compound (SMILES): Cc1ccc(-c2ncccn2)c(C(=O)N2CC3CC(Nc4cnc(C(F)(F)F)cn4)C2C3)n1
InChIKey: CGNHJOCYDMNLSG-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 163 nM (pChEMBL 6.789999961853027)
Descriptors: MW 455.44, ALogP 3.37, HBA 7, HBD 1, PSA 96.79, QED 0.64
Compound (SMILES): CCOc1ncccc1C(=O)N1C[C@H](n2cc(C(C)(C)O)cn2)CC[C@H]1C
InChIKey: BJKWQCPVCKGDDF-GDBMZVCRSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: FLIPR Ca2+ Flux Assay: 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.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 372.47, ALogP 2.77, HBA 6, HBD 1, PSA 80.48, QED 0.87
Compound (SMILES): Cn1c(SCCCN2CC3CCN(c4c(F)cccc4F)C3C2)nnc1-c1ccc(=O)[nH]c1
InChIKey: LGCNQKJCXSSMEC-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Functional Calcium Assay: Functional Calcium Assay at hD2 recombinant receptor. 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 = 794 nM (pChEMBL 6.099999904632568)
Descriptors: MW 472.57, ALogP 3.14, HBA 7, HBD 1, PSA 70.05, QED 0.42
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1ncc2c(n1)N(CC(F)F)C(=O)N(c1cc(OC)cc(OC)c1Cl)C2
InChIKey: CXDBMIJORVIMIH-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme: A 10× stock solution of FGFR4-WT (PR4380C or P3054), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 10, was prepared as described below. Alternatively, a 10× stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 10, was prepared as described below. A solution of 1.4×ATP (AS001A) and 5× Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM P3-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 μL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 μl of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 μL of 1.4×ATP. The reactions were monitored every 71 seconds for 240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to −60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 544.95, ALogP 5.23, HBA 7, HBD 2, PSA 108.92, QED 0.36
Compound (SMILES): FC(F)(F)COc1cc(-c2noc(C3CC3)n2)ncc1Cl
InChIKey: HMUPXUIISJZXTJ-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Radioligand Binding Assay: The affinity of the compounds of the invention for cannabinoid CB1 receptors was determined using recommended amounts of membrane preparations (PerkinElmer) of human embryonic kidney (HEK) cells expressing the human CNR1 or CNR2 receptors in conjunction with 1.5 or 2.6 nM [3H]-CP-55,940 (Perkin Elmer) as radioligand, respectively. Binding was performed in binding buffer (50 mM Tris, 5 mM MgCl2, 2.5 mM EDTA, and 0.5% (wt/vol) fatty acid free BSA, pH 7.4 for CB1 receptor and 50 mM Tris, 5 mM MgCl2, 2.5 mM EGTA, and 0.1% (wt/vol) fatty acid free BSA, pH 7.4 for CB2 receptor) in a total volume of 0.2 ml for 1 h at 30° C. shaking. The reaction was terminated by rapid filtration through microfiltration plates coated with 0.5% polyethylenimine (UniFilter GF/B filter plate; Packard). Bound radioactivity was analyzed for Ki using nonlinear regression analysis (Activity Base, ID Business Solution, Limited), with the Kd values for [3H]CP55,940 determined from saturation experiments. The compounds of formula (I) show an excellent affinity for the CB2 receptor with affinities below 10 μM, more particularly of 1 nM to 3 μM and most particularly of 1 nM to 100 nM.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 319.67, ALogP 3.6, HBA 5, HBD 0, PSA 61.04, QED 0.86
Compound (SMILES): CCc1ccn(Cc2cccc(N)c2C)c(=O)c1
InChIKey: CWLNKRWVJHVVKW-UHFFFAOYSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADPH] FabI (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q6GI75
Assay: Jump dilution assay: 10 μM saFabI, 15 μM inhibitor, and 500 μM NADPH were preincubated overnight at room temperature followed by a 1:200 dilution into reaction buffer (50 mM potassium phosphate, pH 7.5, 150 mM NaCl, 1 M potassium glutamate, 8 v/v % glycerol) containing 1.5 mM trans-2-butenoyl-CoA and 350 μM NADPH.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 242.32, ALogP 2.35, HBA 3, HBD 1, PSA 48.02, QED 0.84
Compound (SMILES): C=CC(=O)N1CCN(CCCn2c(=O)c(-c3cc(OC)cc(OC)c3Cl)cc3cnc(N[C@H]4CCOC4)nc32)CC1
InChIKey: XFGMNTTVFYLJIS-FQEVSTJZSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Activity Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of FGFR family (FGFR1, FGFR2, FGFR3, FGFR4) kinase activity of a compound of Formula (III). Serial dilutions of test compounds were incubated with either human recombinant FGFR1 (0.5 nM), FGFR2 (0.1 nM, FGFR3 (0.9 nM), or FGFR4 (2 nM), ATP (FGFR1: 100 μM; FGFR2: 75 μM; FGFR3: 120 μM; FGFR4: 250 μM) and a phosphoacceptor peptide substrate FAM-KKKKEEIYFFF-CONH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper LabChip 3000. Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 583.09, ALogP 3.05, HBA 10, HBD 1, PSA 111.05, QED 0.36
Compound (SMILES): N#CC1(C(=O)N2CC3CCc4cc(C(F)(C(F)(F)F)C(F)(F)F)ccc4C3(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: FPABLRXQWBFMDY-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 271 nM (pChEMBL 6.570000171661377)
Descriptors: MW 592.51, ALogP 5.49, HBA 4, HBD 0, PSA 78.24, QED 0.35
Compound (SMILES): CN1C(N)=N[C@](C)(c2cc(/C=C(\F)c3cnc(OCC#CCF)cn3)ccc2F)CS1(=O)=O
InChIKey: SIQONXZDOCTZEC-OIOSCTRGSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE1 Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 45K cells/well in 384 well plates (Corning/BioCoat 354663). The test compounds were then added to cells in 22-point dose response concentrations with the starting concentration being 62.5 μM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.625%. The cells were cultivated overnight at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. After 24 h of incubation with the test compounds, the conditioned media was collected and the Aβ40 levels were determined using HTRF (Homogeneous Time Resolved Fluorescence). The IC50 of the compound was calculated from the percent of control or percent inhibition of Aβ 40 as a function of the concentration of the test compound.The HTRF to detect Aβ40 was performed in 384 well plates (Costar 3658). The antibody pair that were used to detect Aβ 40 from cell supernatants were ConfAb40 antibody (Amgen in-house) and biotinylated 6E10 (BIOLEGEND). As an alternative to ConfAb40, a commercially available antibody, Anti-beta Amyloid 1-40 antibody [BDI350] from Abcam, Cambridge, Mass. 02139-1517 (Product code: ab20068), may be used in this assay. The concentrations were 0.35 μg/mL of ConfAb40 antibody and 1.33 μg/mL of 6E10-biotinylated antibody, as well as 4.5 μg/mL of Streptavidin Allophycocyanin Conjugate (ThermoFisher Scientific) in HTRF Buffer (1M Hepes pH 7.5, 1M NaCl, 1% BSA, 0.5% Tween 20).The conditioned media was incubated with above antibodies and Streptavidin Allophycocyanin Conjugate for 30-60 minutes at 23° C. The final readout was performed on Envision from PerkinElmer.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 479.48, ALogP 2.24, HBA 7, HBD 1, PSA 110.77, QED 0.66
Compound (SMILES): Nc1nn2cc(F)cnc2c1C(=O)Nc1cnccc1CN1CCCC1=O
InChIKey: MPFFRVKAAMTQRD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Table 5: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [y-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide(ASELPASQPQPFSAKKK) (SEQ ID NO:1).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).
Measurement: Ki = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 369.36, ALogP 1.22, HBA 7, HBD 2, PSA 118.51, QED 0.71
Compound (SMILES): CC(CC(=O)N1CCN(c2ccc(C#N)cn2)CC1)c1nc2ccccc2c(=O)[nH]1
InChIKey: HBVQNSQGWJOPQF-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: Scintillation Proximity Assay: The scintillation proximity assay (SPA) is designed to measure PARP activity using purified recombinant PARP-1 enzyme and is ideal for high throughput screening of small molecular inhibitors for drug discovery. Here, recombinant human PARP-1 or PARP-2 enzyme was incubated with substrate mix (NAD, 3H-NAD and biotinylated-NAD) and the [3H] and biotin-labeled ADP-ribose polymers were captured using Streptavidin-conjugated PVT SPA beads. In the absence of enzyme inhibition, 100% signal was obtained. Inhibitors are identified by a decrease in signal when PARP-1 or PARP-2 mediated poly-ADP ribose polymer formation is reduced.
Measurement: IC50 = 3817 nM (pChEMBL 5.420000076293945)
Descriptors: MW 402.46, ALogP 2.03, HBA 6, HBD 1, PSA 105.98, QED 0.72
Compound (SMILES): COC(=O)N1CCc2nn(C(=O)C3CCNc4ccccc43)c(N)c2C1
InChIKey: DECRNXFJLJALHT-UHFFFAOYSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Counterscreens for Selectivity: Thrombin. In a 96-well white opaque plate, 80 μl of assay buffer was added to each well. Test compounds were added, and serially diluted as above, to generate a concentration range of approximately 1-100 μM. Human alpha-thrombin (Enzyme Research Labs) was diluted in assay buffer to a final concentration of 12.5 nM. 80 μl of this enzyme solution was added to the assay wells. The enzyme/compound mixtures were incubated for 10 minutes at room temperature. Fluorogenic substrate (Boc-Val-Pro-Arg-7-amido-4-methylcoumarin; Sigma) was added to assay wells at a final concentration of 20 μM. The assay plate was spun for 1 minute at 1500 g and then read at 37° C. in a SpectraMax M2 plate reader (λex=380 nm and λem=460 nm). Activity was quantified as the rate of change in fluorescence, which corresponds to the rate of substrate cleavage. IC50 values were determined as the concentration of inhibitor that produced 50% of the rate of change of control wells without any inhibitor. For compounds with activity <1 μM, the assay was repeated with a lower concentration range, typically from 10-1000 nM.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 355.4, ALogP 1.83, HBA 7, HBD 2, PSA 102.48, QED 0.81
Compound (SMILES): O=S(=O)(c1cc(-c2cnc(NC3CC3)nc2)cc(N2CCOCC2)c1)C1CCOC1
InChIKey: RUNUGPBGQHZGLN-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: pAKT Protocol: Inhibition of the PI3K-AKT-mTOR pathway was measured by quantifying the loss of (Ser-473) pAKT using AlphaScreen (Perkin Elmer). B103 (Rat Neuroblastoma) cells were seeded in serum containing medium (High Glucose DMEM (-Phenol Red)+10% FBS+2× Glutamax+1 mM Sodium Pyruvate+10 mM HEPES+1× Non-Essential Amino Acids+1× Pen/Strep) on a 96-well tissue culture treated plate and grown for 20 hours. Cells were then serum starved in serum free medium (High Glucose DMEM (-Phenol Red)+1× Glutamax+1 mM Sodium Pyruvate+1× Pen/Strep) for 6 hours prior to a 2-hour pretreatment with inhibitors of the pathway, including reference inhibitor LY294002. These inhibitors were prepared at a 200× final concentration as a 6-point, 1:3 serial dilution in DMSO series, with DMSO as the 7th point. The inhibitors were then diluted in experimental medium (High Glucose DMEM (-Phenol Red)+1× Glutamax+1 mM Sodium Pyruvate+1× Pen/Strep+25 mM HEPES+0.1% BSA) and combined with the cells at 1× final concentration in 0.5% DMSO. The cells were then stimulated for 20 minutes with (2.5 μg/mL) insulin, an activator of the PI3K-AKT-mTOR pathway and a demonstrated (Ser-473) pAKT agonist. Cells were promptly lysed using Perkin Elmer proprietary lysis buffer and the (Ser-473) pAKT and total AKT contained in the lysate was measured by AlphaScreen. In AlphaScreen, donor beads were coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads were coated with Protein A to immobilize the other antibody. In the presence of target protein, the two antibodies bring the donor and acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of target protein present in the sample. For each inhibitor tested: the ratio of measured (Ser-473) pAKT/totalAKT was plotted in GraphPad Prism as a 7-point, non-linear regression, 4-parameter curve with bottom constrained to reference control bottom and unconstrained top anchored to DMSO.
Measurement: IC50 = 5810 nM (pChEMBL 5.239999771118164)
Descriptors: MW 430.53, ALogP 2.12, HBA 8, HBD 1, PSA 93.65, QED 0.74
Compound (SMILES): COc1ccc(C(=O)Nc2ccc(F)c([C@]34CCC[C@H]3S(=O)(=O)N(C)C(=N)N4)c2)nc1
InChIKey: YDWKWRWGRZUAPK-YLJYHZDGSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: BACE-2 Assay: The compounds of the invention were assessed for their ability to inhibit BACE-1 using the following assay. The resulting values are reported in the tables above.Inhibitor IC50s at purified human autoBACE-2 were determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay was initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1× BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm was collected for 400 ms following a 50 s delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data was normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values. IC50 values were determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively. Similar IC50s were obtained when using raw RFU data. The Ki values were calculated from the IC50 using the Cheng-Prusoff equation.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 447.49, ALogP 2.03, HBA 6, HBD 3, PSA 124.48, QED 0.66
Compound (SMILES): Cn1ccnc1CN1CCC(n2cnc(-c3ccc(F)cc3)c2-c2ccc3nc(NC(=O)c4ccnc(F)c4)cn3n2)CC1
InChIKey: JJHQTEOODOGIKR-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
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 = 0.265 nM (pChEMBL 9.579999923706055)
Descriptors: MW 594.63, ALogP 4.76, HBA 10, HBD 1, PSA 111.06, QED 0.27
Compound (SMILES): CCS(=O)(=O)c1ccccc1-c1cnc(N)c(-c2nnc(-c3ccccc3)o2)n1
InChIKey: QUEYXALCERUNGS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK) (SEQ ID NO:1)).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).
Measurement: Ki = 2550 nM (pChEMBL 5.590000152587891)
Descriptors: MW 407.46, ALogP 3.24, HBA 8, HBD 1, PSA 124.86, QED 0.53
Compound (SMILES): CCNC(=O)c1noc(-c2cc(C(C)C)c(O)cc2O)c1-c1ccc(CN2CCN(C)CC2)cc1
InChIKey: LRQGHEGCJYDULB-UHFFFAOYSA-N
Target: ATP-dependent molecular chaperone HSP82 (SINGLE PROTEIN), Saccharomyces cerevisiae S288c
UniProt: P02829
Assay: Malachite Green ATPase Assay: 1. Dilute test compounds to 500 uM in AR water (DMSO concentration will be 2.5%). Transfer 2.5 ul of these compounds directly from the daughter plate to the assay plate, giving a final assay concentration of 100 uM. To obtain 12 point IC50 values, perform serial dilutions 1:2 to produce a range of assay concentrations from 100 uM to 97.6 nM (2.5% DMSO), and transfer 2.5 uM of each concentration into the assay plate. Column 1 in the assay plate contains no compound, as a negative control. An additional row with no compound is also used as a background.2. Prepare ATP by diluting 100 mM stock to 925 uM with assay buffer, and aliquot 5 ul of diluted ATP to each well including controls (final assay concentration 370 uM).3. Add 5 ul of buffer to background row.4. Dilute enzyme preparation to 1.05 uM with assay buffer, and aliquot 5 uM into each compound well and to the negative control column.5. Collect the reagents to the bottom of the well, cover plate with plate seal and incubate overnight at 37deg C.6. First thing in the morning prepare the Malachite Green Reagent. Add 2 parts of Malachite Green Solution, 1 part of Polyvinyl Alcohol Solution, 1 part of Ammonium Molybdate Solution, and 2 parts of AR water. 7. Invert to mix, and leave for approximately 1 hour until the colour turns from brown to golden yellow.8. Add 40 uM of Malachite Green Reagent to each well, allow 5 mins for colour to develop.9. Add 5 ul of Sodium Citrate Reagent to each well (see comment 2)10. Re-cover with plate seal and shake on plate shaker for at least 15 mins.11. Measure Absorbance at 620 nM using a suitable plate reader (e.g. Victor, Perkin Elmer Life Sciences, Milton Keynes, UK). Under these conditions, the control absorbance is 0.9 to 1.4, and the background is 0.2-0.35 giving a signal to noise ratio of 12. The Z factor calculated from data obtained using these conditions is between 0.6
Measurement: IC50 < 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 478.59, ALogP 4.04, HBA 7, HBD 3, PSA 102.07, QED 0.47
Compound (SMILES): CCCn1cc(-c2ccc(F)cc2)c(=O)c2c(Nc3ccc(Oc4ccnc5cc(OCC(C)(C)O)ccc45)c(F)c3)nccc21
InChIKey: AWBXMXCPZREFGT-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition Assay: A study on inhibition of protein kinases activity at in vitro biochemical levelMaterial and method: kinases such as c-Met, VEGFR-2, Axl and RET, obtained from Invitrogen; HTRF KinEASE; TK kit (Cisbio Company); 384-well plate (Greiner Company); ATP (Sigma Company), MgCl2 (Sigma Company); PHERAstar FS multifunctional plate reader (BMG Company); conventional centrifuge (StaiteXiangyi Company); incubator (Binder Company). Dilution and storage of compounds: depending on solubility, the test compounds are prepared as stock solutions of 0.5-10 mmol/L with dimethyl sulphoxide (DMSO), and aliquots of the stock are stored at −20° C.;Preparation of working solution of the compounds: Before test, aliquots of the compounds are removed from the refrigerator, and diluted with pure DMSO to 50× of the level needed; and then the compounds are diluted with deionized water to 4× of the level needed;Preparation of 1.33× enzymatic buffer: 5× enzymatic buffer (from HTRF kit) is diluted to 1.33× with deionized water, and appropriate components of the final level 1.33× are added: 1.33 mmol/L dithiothreitol (DTT) and 1.33 mmol/L MgCl2;Preparation of kinase working solution: Met is diluted to 2× final level required, 0.2 ng/μL, with 1.33× enzymatic buffer;Preparation of substrate working solution: the substrate labeled with biotin (from HTRF kit) and ATP (10 mM) are diluted with 1.33× enzymatic buffer to prepare a mixture at 4× of the final level required;Preparation of the testing working solution: Streptavidin-XL665, 16.67 μmol/L is diluted with HTRF test buffer to 4× of the final level required, and then mixed with antibody-Cryptate of the same volume (both from the HTRF kit).Enzymatic reaction step: 4 μl of kinase working solution is added to each well of the low volume 384-well plate. Meanwhile, 4 μL 1.33× enzymatic buffer is added as the negative control. 2 μL compound working solution is added into each well, and 2 μL 8% DMSO in water is added as zero compound level control (i.e., positive control). At 25° C. (or 30 C), incubate for 5-10 min; add 2 μL substrate working solution into each well to initiate the enzymatic reaction. Allow to react while shaking at 25° C. (or 30° C.) for 15-60 min.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 622.67, ALogP 7.99, HBA 8, HBD 2, PSA 98.5, QED 0.16
Compound (SMILES): COCCOc1cc(-c2cn[nH]c2)ccc1C(=O)N[C@H](C)c1cccc(C(=O)NC2CCCC2)c1
InChIKey: MAUISDMNPMEWQF-GOSISDBHSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Kinase Inhibition Assay: The following assay protocol is for measuring the phosphorylation of a peptide substrate (FAM-KKLRRTLSVA-OH wherein FAM is carboxyfluorescein). The peptide is >98% purity by Capillary Electrophoresis. The peptide is phosphorylated by the protein kinase ROCK1 or ROCK2. The ROCK1 or ROCK2 enzyme, substrate, and cofactors (ATP and Mg2+) are combined in a well of a microtiter plate and incubated for 3 hours at 25° C. in the presence or absence of an inhibitor compound. At the end of the incubation, the reaction is quenched by the addition of an EDTA-containing buffer. The substrate and product are separated and quantified electrophoretically using the microfluidic-based LABCHIP® 3000 Drug Discovery System from Caliper Life Sciences (Hopkinton, Mass.).The components of the assay mixture are:100 mM HEPES, pH 7.50.1% BSA0.01% Triton X-1001 mM DTT10 mM MgCl210 μM Sodium Orthovanadate10 LM Beta-Glycerophosphate5 μM ATP (for ROCK1) or 7 μM ATP (for ROCK2)1% DMSO (from compound)1.25 μM FAM-KKLRRTLSVA-OH3 nM ROCK1 or 2.5 nM ROCK2 enzymeSubstrate and product peptides present in each sample are separated electrophoretically using the LABCHIP® 3000 capillary electrophoresis instrument. As substrate and product peptides are separated two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured reflecting enzyme activity. Capillary electrophoregramms (RDA acquisition files) are analyzed using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). The kinase activity in each sample is determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product peptide and S is the peak height of the substrate peptide. For each compound, enzyme activity is measured at various concentrations (12 concentrations of compound spaced by 3× dilution intervals).
Measurement: IC50 = 1.02 nM (pChEMBL 8.989999771118164)
Descriptors: MW 476.58, ALogP 4.27, HBA 5, HBD 3, PSA 105.34, QED 0.38
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCN(Cc5ccc(Cl)cc5)CC4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: RPWABDWRYSLHIF-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 = 71.4 nM (pChEMBL 7.150000095367432)
Descriptors: MW 509.02, ALogP 4.64, HBA 8, HBD 0, PSA 78.28, QED 0.34
Compound (SMILES): CS(=O)(=O)N1CCC(c2cc(C(F)(F)F)cn3cncc23)CC1
InChIKey: YBYWCWYVNZIBKC-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: TDO and IDO Biochemical Coupled Assay: Recombinant human IDO or TDO was incubated in 50 mM KPO4 (pH 7.0), 0.5 mM EGTA, 0.5 mM EDTA, 0.05% Triton X100, 20 mM ascorbate, 10 μM methylene blue, 500 U/ml catalase, 50 μg/ml KynB (kynurenine formamidase). TDO assays were carried out in the presence of 330 μM L-tryptophan, while IDO assays had the addition of 45 μM L-tryptophan. After incubation for 17 minutes at room temperature the reactions were stopped by the addition of Erhlich's reagent and incubated at room temperature for 5 minutes before the fluorescence was read (Ex 475, Em530).
Measurement: IC50 = 17400 nM (pChEMBL 4.760000228881836)
Descriptors: MW 347.36, ALogP 2.49, HBA 4, HBD 0, PSA 54.68, QED 0.84