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| | SHP2 Allosteric Inhibitory Enzyme Activity Assay |
| Description: | SHP2 is allosterically activated by binding of a bis-tyrosyl-phosphorylated peptide to its Src homology 2 (SH2) domain. This later activation step results in the release of the autoinhibitory interface of SHP2, which in turn activates the SHP2 protein tyrosine phosphatase (PTP) and is available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate DiFMUP in a rapid fluorescence assay format.[0511]experiment procedure:(1) Compound preparation:The compounds of the present invention (10 mM stock solution) were diluted to appropriate times with 100% DMSO, and the final test concentrations of the compounds of the present invention were 10 µM, 3.3333 µM, 1.1111 µM, 0.3704 µM, 0.1235 µM, 0.0412 µM, 0.0137 µM, 0.0046 µM, 0.0015 µM, 0.00 µM;(2) Prepare the enzyme reaction working solution:Perform SHP2 enzymatic assays in 96-well black polystyrene plates (flat bottom, low flange, non-binding surface) (Perki Elmer, Cat#6005270) at room temperature using a final reaction volume of 50 µL and the following assay buffer conditions: 60 mM HEPES, 75 mM NaCl, 75 mM KCl, 0.05% BRIJ-35, 1 mM EDTA, 5 mM DTT.(3) Enzyme-catalyzed reaction and data monitoring:Add the compound of the present invention to the corresponding 96-well plate, and set the blank test well without compound and enzyme only with buffer. Thaw SHP2 Activating Peptide (IRS1_pY1172(dPEG8)pY1222) on ice, add 25 µM to each well, then add 0.2 ng of SHP2 protein sample to the corresponding well plate and incubate at room temperature for 1 hour. The surrogate substrate DiFMUP (Invitrogen, Cat #D6567) was added to the reaction and allowed to react for 1 hour at room temperature. Fluorescence signals were monitored using a microplate reader (Envision, Perki Elmer) using excitation and emission wavelengths of 340 nM and 450 nM, respectively.(4) Data analysis:Calculation Formula:Suppression rate %=1-Conversion_sample-Conversion_min/Conversion_max-Conversion_min×100% |
| Affinity data for this assay | |
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