An ADP-Glo (Promega) detection kit assay was used to determine the kinetic inhibition parameters, i.e., the inhibition constant (Ki), of each inhibitor. Similar to the Cab1 kinetics study protocol, assays were conducted at rising PA concentrations (1–800 μM),and 1000 μM ATP with selected constant inhibitors concentrations (DMSO final concentration 0.5%). Ki was determined by Lineweaver–Burk plot analysis using GraphPad Prism.
Adp glo
Manufactured by Promega
Sourced in United States
The ADP-Glo is a luminescent assay that quantifies the amount of ADP produced in kinase reactions. The assay converts ADP to ATP, which is then measured using a luciferase/luciferin reaction.
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Lab products found in correlation
Adenosine diphosphate (adp), by Promega (2 mentions)
Kinase detection reagent, by Promega (2 mentions)
Cdk cyclind3, by Promega (2 mentions)
Enspire multimode plate reader, by PerkinElmer (2 mentions)
Pemigatinib, by Abcam (2 mentions)
Xfluor software, by Tecan (1 mentions)
Half area 96 well microplates, by Corning (1 mentions)
Adenosine triphosphate (atp), by Promega (1 mentions)
Adp glo reagent, by Promega (1 mentions)
Selectscreen biochemical kinase profiling service, by Thermo Fisher Scientific (1 mentions)
Clariostar plate reader, by BMG Labtech (1 mentions)
Victor2 microplate reader, by PerkinElmer (1 mentions)
Anti flag conjugated magnetic beads, by Merck Group (1 mentions)
Lxs196, by MedChemExpress (1 mentions)
Adenosine triphosphate (atp), by Abcam (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Cellular calcineurin phosphatase activity assay kit, by Abcam (1 mentions)
Ni nta purification system, by Thermo Fisher Scientific (1 mentions)
Reduced triton x 100, by Merck Group (1 mentions)
Cytation 3, by Agilent Technologies (1 mentions)
25 protocols using adp glo
1
Kinetic Inhibition of Cab1 Enzyme
2
Gyrase and Topoisomerase Enzyme Assay
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GyrA and gyrB genes were amplified from M. tuberculosis genomic DNA and the recombinant proteins with an N-terminal (His)6 tag were prepared from E. coli cells following standard procedures. The enzyme reactions contained 100 mM Tris–HCl (pH 7.6), 6 mM MgCl2, 20 mM KCl, 1% dimethyl sulfoxide, 0.05 mg/ml of bovine serum albumin, 5 μg/ml PBR322, 700 μM ATP, and 2 μM Gyrase B or Topoisomerase (GyrA2GyrB2). ATP hydrolysis during the reaction was monitored by determining the increase in ADP using ADPGlo (Promega, Inc.).
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GyrA and gyrB genes were amplified from M. tuberculosis genomic DNA and the recombinant proteins with an N-terminal (His)6 tag were prepared from E. coli cells following standard procedures. The enzyme reactions contained 100 mM Tris–HCl (pH 7.6), 6 mM MgCl2, 20 mM KCl, 1% dimethyl sulfoxide, 0.05 mg/ml of bovine serum albumin, 5 μg/ml PBR322, 700 μM ATP, and 2 μM Gyrase B or Topoisomerase (GyrA2GyrB2). ATP hydrolysis during the reaction was monitored by determining the increase in ADP using ADPGlo (Promega, Inc.).
3
Quantifying LonP1 and Proteasome Inhibition
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ADP-Glo (Promega) assays were performed according to the manufacturer's protocols. Purified wild-type or mutant LonP1 (400 nM monomer), the 26S proteasome (3 nM), or no enzyme controls were preincubated in 96-well plates (60 min, 25 °C) with ten different concentrations of CDDO and its derivatives, TP-82, enoxolone (5 μM) or DMSO vehicle control in buffer (40 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 5% DMSO). The 26S and 20S proteasome were also assayed at 0, 0.75, 1.5, 3, 6, and 12 nM in the absence of CDDO derivatives. UltraPure ATP (1 mM final) was added, and reactions (50 μl final) were incubated for 60 min at 25 °C, then 5 μl was transferred in quadruplicate to a 384-well plate. Assay reagents were used to quench further ATPase activity and generate luminescence signals proportional to the concentration of ADP formed. Background (no enzyme control) luminescence values were subtracted, and resultant values were reported as a percentage of the no drug control (100%). Data were fit to four-parameter dose–response curves using GraphPad Prism. The error bars represent the standard deviation (SD) of four replicate reactions from at least three independent experiments.
4
Recombinant human ALK kinase assay
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N-terminal 6His tagged recombinant human ALK expressed in baculovirus Sf21 was purchased from EMD Millipore (Billerica, MA, USA) (purity ≥60% by sodium dodecyl sulfate polyacrylamide gel electrophoresis) and aliquoted to 1 μL fractions when it was used the first time (to avoid multiple freeze/thaws for subsequent experiments). BNP7787 was prepared by a proprietary method (purity >97%, no mesna was detected by mass spectroscopy). Kinase inhibitor, PF02341066 (crizotinib), was purchased from Selleck Chemicals, LLC. (Houston TX, USA). Polyglutamate-tyrosine (PolyGT) substrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kinase assay buffer was prepared and consisted of 20 mM HEPES, 0.1% Brij 96, 10 mM NaF, 1 mM Na3VO4, and 10 mM MnCl2 adjusted to a final pH of 7.5. Half-area 96-well microplates were purchased directly from Corning Incorporated (Corning, NY, USA). ADP-Glo reagents were purchased from Promega (Madison WI, USA) and consisted of ADP, ATP, ADP-Glo, kinase detection reagent buffer, and kinase detection substrate. All other reagents were purchased from Sigma-Aldrich Co (St Louis, MO, USA). A Tecan Ultra microplate reader with XFluor software (V4.51; Tecan [Morrisville, NC, USA]) and RdrOle software (V4.50; Tecan) were used in this study.
5
Abl and RIPK2 Kinase Assays
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For ADPGlo (Promega) assays, 1 ng of Abl or 10 ng of RIPK2 was diluted in reaction buffer (40 mM Tris-HCl [pH 7.5], 20 mM MgCl2, 0.5 mM DTT, and 0.01% BSA) supplemented with 50 μM ATP and a 10-point dose range of inhibitors. Detailed information is provided in the Supplemental Experimental Procedures .
6
Kinase Inhibitory Activity Profiling
7
PI3K-p110β Lipid Kinase Activity Assay
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Lipid kinase activity of commercially available purified PI3K-p110β and PI3K-p110βE1051K complexes was determined using an ADP-Glo (Promega, Madison, WI, USA) assay performed at ProQinase GmbH. Briefly, PI3K complexes (GST-His6-p110β+myc-p85α) were co-expressed from PIK3CB and PIK3R1 baculoviral vectors in Sf9 cells and purified by GST-affinity chromatography using standard techniques. Proteins were mixed with ATP in an assay buffer (50 mM HEPES-NaOH, pH 7.5, 1 mM EGTA, 100 mM NaCl, 3 mM MgCl2, 0.03% CHAPS, 2 mM DTT and PIP2:PS substrate). To determine Km and Vmax, the ATP concentration was incrementally increased. For enzyme inhibition assays, compounds were added at a range of concentrations in 10% DMSO in the presence of [ATP] at the predetermined enzyme Km and 100 μM substrate. Lipid kinase reactions were incubated at 30 °C for 40 min prior to the addition of ADP-Glo reagent and incubated for another 40 min at room temperature. Kinase detection reagent was then added and incubated for another 60 min at room temperature. Luminescence proportional to the amount of ADP generated in the reaction was quantified using a Victor2 microplate reader (Perkin Elmer, Boston, MA, USA).
8
PAK5 Kinase Activity Assay
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PAK5 kinase activity was measured using ADP-Glo following the Promega protocol. Briefly, FLAG-PAK5 was immunoprecipitated from hMELT stable cell lines using anti-FLAG conjugated magnetic beads (Sigma #M8823). After washing the beads three times with 150 mM RIPA for 5 minutes on a rotator, purified FLAG-PAK5 was incubated with 0.2 mg/ml AKT (PKB) substrate (CKRPRAASFAE; SignalChem #A05-58), 50 µM UltraPure ATP, and Kinase Reaction Buffer A (40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA) for 15 minutes at room temperature. The kinase reaction was quenched with ADP Glo Reagent for 40 minutes and then Kinase Detection Reagent was added for 30 minutes at room temperature. Luciferase activity was measured using a Veritas™ Microplate Luminometer with 1 second of integration. Raw Luciferase measurements were normalized to the amount of FLAG-PAK5 immunoprecipitated, which was quantified from immunoblots using Image Studio Software. p-values were calculated using multiple t-tests and significance determined by FDR (Q = 5%).
9
Comparative CDK6 Inhibition Assay
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Example 4
In 96 semi area plate (VWR 33501-814), we added substrates, enzyme, and inhibitors for a total volume of 15 μl per well. The mixture contains 0.1 ug/ul histoneH1, 250 uM ATP, CDK/cyclinD3 60 ng (Promega V4511), and tested inhibitor C2 (Abemaciclib) or C3 with final concentration of 0 nM, 5 nM, 10 nM, 20 nM, 39 nM, 78 nM, 156 nM, 313 nM, 625 nM, 1.25 μm, 2.50 μM, 5 μM, 10 μM. The mixtures were incubated for 60 min at RT. Kinase reactions were stopped by adding 15 μl ADP-Glo (Promega V9101) for 40 min RT, followed by adding 30 μl Kinase Detection Reagent (Promega V9101) for 30 min RT. Luminescence signal from the plate was recorded with EnSpire Multimode Plate Reader (PerkinElmer) (integration time 0.5 second).
CDK inhibition curves were prepared for C2 (Abemaciclib) (
These results indicate that C3 has a similar CDK6 inhibitory activity as C2 (Abemaciclib).
10
Synthesis and Characterization of BRG1/BRM Inhibitor
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Example 6
BRG1/BRM Inhibitor Compound A has the structure:
Compound A was synthesized as shown in Scheme 3 below.
The ATPase catalytic activity of BRM or BRG-1 in the presence of Compound A was measured by the in vitro biochemical assay using ADP-Glo™ (Promega, V9102) described above. Compound A was found to have an IP50 of 10.4 nM against BRM and 19.3 nM against BRG1 in the assay.
Example 14
Procedure: MP41 uveal melanoma cells were made resistant to the PKC inhibitor (LXS196; Med Chem Express), by long-term culture in growth media (see Table 2) containing increasing concentrations of the compound, up to 1 μM. After 3 months, sensitivity of the parental MP41 cells and the PKC inhibitor (PKCi)-resistant cells to the PKC inhibitor (LXS196) or the BRG1/BRM ATPase inhibitor (Compound B) was tested in a 7-day growth inhibition assay as described above in Example 6.
Results: While the PKCi-resistant cells could tolerate growth at higher concentrations of LXS196 than could the parental MP41 cell line (
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