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- ChEMBL_462553 (CHEMBL928479) Inhibition of PKB
- ChEMBL_603272 (CHEMBL1049496) Inhibition of PKB
- ChEMBL_304608 (CHEMBL828495) Inhibition of PKB beta
- ChEMBL_623291 (CHEMBL1113529) Inhibition of PKB by radioactive kinase assay
- ChEMBL_162071 (CHEMBL766867) Inhibition of Akt (proto-oncogenic serine/threonine PKB) kinase
- ChEMBL_464979 (CHEMBL947151) Inhibition of phosphorylation of T308-PKB in human U87MG cells by ELISA
- ChEMBL_2336715 Inhibition of PKB phosphorylation in human U87 cells incubated for 1 hrs by ELISA
- ChEMBL_2336713 Inhibition of PKB phosphorylation in human BT-20 cells incubated for 1 hrs by ELISA
- ChEMBL_608352 (CHEMBL1074386) Inhibition of PKB in human PC3M cells assessed as GSK3-beta phosphorylation by ELISA
- ChEMBL_608354 (CHEMBL1074388) Inhibition of PKB in human U87MG cells assessed as GSK3-beta phosphorylation by ELISA
- ChEMBL_2336714 Inhibition of PKB phosphorylation in human SK-OV-3 cells incubated for 1 hrs by ELISA
- ChEMBL_1672661 (CHEMBL4022690) Inhibition of human AKT1/PKB using phospho-AKT peptide substrate after 1 hr by scintillation counting method
- ChEMBL_1854925 (CHEMBL4355654) Inhibition of C-terminal His4-tagged Aurora A (unknown origin) using PKB-GSK2 biotinylated peptide as substrate incubated for 90 mins by ELISA
- ChEMBL_2516981 Inhibition of human PKB expressed in baculovirus at pH 7.2 measured after 30 mins in presence of gamma-[33P]-ATP by scintillation counter analysis
- ChEMBL_787183 (CHEMBL1918853) Inhibition of GST-tagged PKB/Akt1 incubated for 5 mins before eNOS substrate and ATP addition measured after 30 mins by kinase assay
- ChEMBL_1822731 (CHEMBL4322495) Inhibition of mTORC2 in human A2058 cells assessed as reduction in PKB phosphorylation at S473 residues incubated for 1 hr by Western blot analysis
- ChEMBL_2014679 (CHEMBL4668257) Inhibition of mTORC2 in human A2058 cells assessed as reduction in PKB phosphorylation at S473 residue incubated for 1 hr by Western blot analysis
- ChEMBL_1280702 (CHEMBL3097335) Inhibition of PI3Kgamma in mouse RAW264.7 cells assessed as C5a-mediated PKB/Akt phosphorylation preincubated for 30 mins followed by C5a stimulation measured after 5 mins
- ChEMBL_885414 (CHEMBL2216155) Inhibition of PI3Kgamma in mouse RAW264.7 cells assessed as inhibition of C5a-induced PKB/Akt phosphorylation incubated for 30 mins prior to C5a-induction measured after 5 mins by immunostaining method
- ChEMBL_776271 (CHEMBL1914068) Inhibition of PIK3CA-mediated cell signaling in PTEN-deficient human U87MG cells assessed as inhibition of insulin-induced pAkt/PKB phosphorylation at Thr308 treated for 15 mins before insulin challenge measured after 5 mins by immunoblotting
- ChEMBL_776272 (CHEMBL1914069) Inhibition of PIK3CA-mediated cell signaling in PTEN-deficient human U87MG cells assessed as inhibition of insulin-induced pAkt/PKB phosphorylation at Ser473 treated for 15 mins before insulin challenge measured after 5 mins by immunoblotting
- PKB In Vitro Enzyme Assay The purified PKB beta enzyme was assayed with a peptide substrate and test compound in the presence of 30 uM ATP/ [gamma-33P]ATP in 96-well plates. IC50 is the concentration of inhibitor that inhibits 50% of kinase activity that catalyzes the transfer of the terminal phosphate from [gamma-33P] labeled ATP to substrate peptide. IC50 values were determined from an eight-point concentration range run in duplicate and calculated in GraphPad Prism 4.00.
- PKB In Vitro Kinase Assay An ELISA-based assay has been developed for the serine/threonine kinase, PKB-alpha. The assay utilizes an N-terminally biotinylated substrate peptide. When this substrate is phosphorylated by the kinase, the product is recognized by a substrate/sequence-specific antibody, which binds to particular phosphorylated serine residues. The amount of phosphorylated peptide was measured by the enzyme-catalyzed ABTS conversion and photometrical measurement at 405 nm. Calculation of IC50 values was performed with ActivityBase.
- ChEMBL_662256 (CHEMBL1252348) Inhibition of PKB/Akt activation in human overnight starved MCF7 cells assessed as phosphorylated Akt at Ser473 level at 50 ug/ml after 1 hr followed by treated with 1 ug/mL of insulin for 15 mins by Western blotting
- Akt1 Inhibitory Activity Assay In vitro kinase assays were carried out using HTScan PKB/Akt1 Kinase Assay kit (Cell Signaling Technology). Active recombinant Akt1 kinase (GST fusion protein, 4 ng) in 8 µL of 2.5× kinase buffer [62.5 mM Tris-HCl (pH 7.5), 25 mM MgCl2, 12.5 mM β-glycerophosphate, 0.25 mM Na3VO4, 5 mM dithiothreitol (DTT)] was mixed with 2 µL of dimethyl sulfoxide (DMSO) vehicle or each of the compound (indicated concentrations), incubated at room temperature for 5 min, and 10 µL of ATP/substrate cocktail (20 mM ATP, 3 mM eNOS served as substrate) was added.After incubation at room temperature for 30 min, add 20 µL of 50mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) and terminate the reaction. Then, PKB/Akt1 kinase activity was analyzed according to the manufacturer's instructions.
- Human Orexin 2 Receptor Ca2+ Mobilization Assay PFSK-1 cells endogenously expressing the human orexin 2 receptor were grown to confluency in RPMI1640 (Hyclone, cat #30027.02), 10% FBS, 1× pen-strep. Cells were seeded on to 384-well Packard viewplates at a density of 5,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin B, 100 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
- Human Orexin 1 Receptor Ca2+ Mobilization Assay CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
- Luminex Assay The cellular assay for the determination of the PDK1 kinase activity is carried out as a Luminex assay in the 96-well format. PC3 cells are sown at 20,000 cells per well in 100 ul of medium (45% of RPMI1460/45% of Ham's F12/10% of FCS) and incubated on the following day for 30 min with a serial dilution of the test substance (7 concentrations) under serum-free conditions. The cells are subsequently lysed using 90 ul of lysis buffer (20 mM tris/HCl pH 8.0, 150 mM NaCl, 1% of NP40, 10% of glycerol, 1% of phosphatase inhibitor I, 1% of phosphatase inhibitor II, 0.1% of protease inhibitor cocktail III, 0.01% of benzonase) per well, and the lysates are separated off from insoluble cell constituents by means of centrifugation through a 96-well filter plate (0.65 um). The lysates are incubated overnight at 4° C. with shaking with Luminex beads to which an anti-total PKB antibody is coupled. The detection is carried out on the following day by addition of a phospho-T308-PKB antibody and a species-specific peroxidase-labelled secondary antibody.
- In Vitro AKT1 Kinase Assay Following addition of compound or control to the assay plate, 6p1 peptide mix containing 3 μM substrate (5-FAM-GRPRTSSFAEG-CONH2; CRB) and 40 μM ATP in Kinase base buffer (100 mM Hepes pH 7.5, 0.015% Brij-35) and 6p1 enzyme mix containing 8 nM AKT1/PKBα active enzyme (Upstate Biotechnology, Cat No. 14-276), 8 mM DTT and 20 mM MgCl2 in kinase base buffer was added. All buffers were made up with 18MΩ water. The plates were sealed and incubated at room temperature for 50 minutes. The reaction was stopped by the addition of 10 μl stop buffer (100 mM Hepes pH 7.5, 0.015% Brij-35 solution, 0.1% coating reagent #3, 40 mM EDTA, 5% DMSO) to each well (N.B. plates can be frozen after stopping and read later).
- Kinase Inhibition Assays The aim of this experiment is to detect the inhibitory activity of the compounds of the present invention against in vitro protein kinases using isotope labeling (labeled gamma phosphate groups on ATP). Kinase inhibition profiles were determined using KinaseProfiler services provided by Euro fins, and ATP concentrations used are the Km of corresponding kinases. In this study, we examined Abl (T315I) (h), ALK (h), ARK5 (h), Axl (h), Blk (h), Bmx (h), BTK (h), B-Raf (H), ckit (h), cSRC (h), CDK7, CHK1 (h), c-RAF (h), DDR2 (h), EGFR (h), EphA1 (h), EphA2 (h), EphA8 (h (H), FGF (h), Ft (h), Ft (h), F (h), F (h), Ft (h), Hb (h), ErbB2 (h), FAK (h) (H), JK3 (h), IKKalpha (h), IKKbeta (h), Itk (h), JAK3 (h), JNK11 (h), KDR (h), Lyn (h), MAPK1 (h), MEK1 (h (H), PKA (h), PKB [alpha](h), PKB [beta] (h), PKC [alpha] (h), Ret (H), RIPK2 (h), Src (1-530) (h), TAK1 (h), TBK1 (h), Tec (h) activated, Tie2 (h), TrkA (h), ULK3 (h) Yes (h), PI3 Kinase a (h) and other kinase in vitro inhibitory activity. The kinase inhibitory activity of the test compound is expressed as IC50 (half inhibitory concentration) or the inhibitory rate of the test compound at a concentration of 10 ??M for kinase activity. IC50 values can be obtained by calculating the inhibition rate of the kinase activity by the test compound at a series of different concentrations. The assay was as follows: In a reaction tube, the buffer (8 mM MOPS, pH 7.0, 0.2 mM EDTA, 10 mM MnCl2), the kinase to be tested (5-10 mU), the substrate to be tested kinase, 10 mM acetic acid Magnesium and gamma 33P-ATP solutions, as well as different concentrations of the test compound. MgATP was then added to the reaction to initiate the enzymatic reaction and incubated at room temperature for 40 minutes. The reaction was finally quenched with 5 ul of 3% phosphate buffer and 10 uL of the reaction solution was titrated onto a filtermat A membrane, washed three times with 75 mM phosphate solution for 5 minutes each, and washed again with methanol. Finally, the filtermat A film is dried and scintigraphized, and the size of the scintillation count reflects the degree to which the substrate is phosphorylated, thereby demonstrating that the kinase activity is inhibited. Among them, the percentage of the remaining active protein=the active protein of the experimental group the active protein of the control group 100%.
- AKT/PKB Kinase Assay In order to measure AKT inhibition in the Caliper Life Sciences LC3000, a TTP Mosquito liquid handling instrument was used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction, the following components were added to a final volume of 12.5 μl: 0.1 ng/μl His-AKT (Full Length) (Invitrogen, Part # P2999, Lot #641228C); 160 μM ATP (Fluka, 02055); 1 mM DTT (Sigma, D0632); 1 mM MgCl2 (Sigma, M1028); 1 μM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service; 100 mM HEPES pH 7.5 (Calbiochem, 391338); and 0.015% Brij-35 (Sigma, B4184).The reaction was incubated for 90 min at 25° C., and then stopped by the addition of 70 μl of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)). The plate was read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure −2.3 psi, upstream voltage −500, and downstream voltage −3000. These conditions cause unphosphorylated substrate and phosphorylated product peptide to resolve as separate peaks allowing direct measurement of percentage of conversion of substrate to product. The percent conversion was plotted against concentration of inhibitor to produce a sigmoidal dose response curve, from which an IC50 was calculated.
- In Vitro AKT1 Kinase Assay This assay detects inhibitors of AKT1 (PKBα) kinase activity using Caliper LabChip LC3000. The Caliper off-chip incubation mobility shift assay uses a microfluidic chip to measure the conversion of a fluorescent labelled peptide to a phosphorylated product by a respective kinase. The complete enzyme reaction is carried out in microtitre plates and then quenched. The resulting stopped solutions are serially "sipped" through a capillary onto the chip, where the peptide substrate and phosphorylated product are separated by electrophoresis. They are then detected via laser-induced fluorescence. Substrate and product are separated into two peaks by the application of a high electric field and directly detected using fluorescence. The signature of the fluorescent signal reveals the extent of the reaction.For Echo dosing the solvent was 100% DMSO. A master plate was prepared with 40 ul of 10 mM stock from our Primary Liquid Store in quadrant 1 of a Labcyte 384 well plate. A 1 in 100 dilution was made from quadrant 1 into quadrant 2 by removing 0.4 ul and adding it to 39.6 ul of DMSO. Subsequent 1 in 100 dilutions were made into quadrant 3 from quadrant 2 and quadrant 4 from quadrant 3. Multiple 2.5 nl droplets were dispensed from each quadrant of the master plate using ECHO dosing technology (Labcyte Inc. Sunnyvale, Calif., USA) to generate the dose range that was required in the test. The dose range most commonly used was as follows: 100 uM, 30 uM, 10 uM, 3 uM, 1 uM, 0.3 uM, 0.1 uM, 0.03 uM, 0.01 uM, 0.003 uM, 0.001 uM, 0.0001 uM. Each well was backfilled with Dimethyl Sulphoxide (DMSO) to a total volume of 120 nl, such that when the enzyme and substrate mix was added the final DMSO concentration was 1%. DMSO was added to max control wells as 120 nl, minimum control wells were treated with 120 nl of compound at a concentration that inhibited the enzyme activity 100%. Following addition of compound or control to the assay plate, 6 ul peptide mix containing 3 uM substrate (5-FAM-GRPRTSSFAEG-CONH2; CRB) and 40 uM ATP in Kinase base buffer (100 mM Hepes pH 7.5, 0.015% Brij-35) and 6 ul enzyme mix containing 8 nM AKT1/PKBα active enzyme (Upstate Biotechnology, Cat No. 14-276), 8 mM DTT and 20 mM MgCl2 in kinase base buffer was added. All buffers were made up with 18M water. The plates were sealed and incubated at room temperature for 50 minutes. The reaction was stopped by the addition of 10 ul stop buffer (100 mM Hepes pH 7.5, 0.015% Brij-35 solution, 0.1% coating reagent #3, 40 mM EDTA, 5% DMSO) to each well (N.B. plates can be frozen after stopping and read later). The plates were then analysed using the Caliper LabChip LC3000 Drug Discovery System (Caliper Life Sciences, 1 Wellfield, Preston Brook, Runcorn, WA7 3AZ) using the following separation conditions; -1.8 PSI, -500 upstream voltage, -1700 downstream voltage, sample sip time of 0.2 sec, post sample sip time of 30 sec and a final delay of 120 sec. Integration of the substrate and product peaks was carried out using Caliper LabChip software and IC50 curves were calculated using Origin software (OriginLab Corporation, Northampton, Mass., USA).
- In Vitro Kinase Assay For Echo dosing the solvent was 100% DMSO. A master plate was prepared with 40 ul of 10 mM stock from our Primary Liquid Store in quadrant 1 of a Labcyte 384 well plate. A 1 in 100 dilution was made from quadrant 1 into quadrant 2 by removing 0.4 ul and adding it to 39.6 ul of DMSO. Subsequent 1 in 100 dilutions were made into quadrant 3 from quadrant 2 and quadrant 4 from quadrant 3.Multiple 2.5 nl droplets were dispensed from each quadrant of the master plate using ECHO dosing technology (Labcyte Inc. Sunnyvale, Calif., USA) to generate the dose range that was required in the test. The dose range most commonly used was as follows: 100 uM, 30 uM, 10 uM, 3 uM, 1 uM, 0.3 uM, 0.1 uM, 0.03 uM, 0.01 uM, 0.003 uM, 0.001 uM, 0.0001 uM. Each well was backfilled with Dimethyl Sulphoxide (DMSO) to a total volume of 120 nl, such that when the enzyme and substrate mix was added the final DMSO concentration was 1%. DMSO was added to max control wells as 120 nl, minimum control wells were treated with 120 nl of compound at a concentration that inhibited the enzyme activity 100%.Following addition of compound or control to the assay plate, 6 μl peptide mix containing 3 μM substrate (5-FAM-GRPRTSSFAEG-CONH2; CRB) and 40 μM ATP in Kinase base buffer (100 mM Hepes pH 7.5, 0.015% Brij-35) and 6 μl enzyme mix containing 8 nM AKT1/PKBα active enzyme (Upstate Biotechnology, Cat No. 14-276), 8 mM DTT and 20 mM MgCl2 in kinase base buffer was added. All buffers were made up with 18MΩ water. The plates were sealed and incubated at room temperature for 50 minutes. The reaction was stopped by the addition of 10 μl stop buffer (100 mM Hepes pH 7.5, 0.015% Brij-35 solution, 0.1% coating reagent #3, 40 mM EDTA, 5% DMSO) to each well (N.B. plates can be frozen after stopping and read later).