| Assay Method Information | |
| | Fluorescence Intensity-Based Assay |
| Description: | To test the synthesized compounds for their potential to inhibit SHP2 activity, a fluorescence intensity-based assay using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as the substrate was adapted for three recombinant SHP2 constructs: 1) the SHP2 catalytic domain (SHP2cat; residues 248-527), 2) the full-length SHP2-E76K oncogenic mutant, and 3) the full-length SHP2 wild-type (SHP2-WT). The recombinant SHP2 proteins were expressed and purified. A dually phosphorylated peptide derived from the insulin receptor substrate 1 (IRS-1) served as a surrogate binding protein and was used to activate SHP2-WT. The constitutively active E76K mutant did not require activation. Similarly, the SHP2cat construct, which lacks the SH2 domains, did not need to be activated. Michaelis-Menten experiments to determine the DiFMUP Michaelis-Menten constant (Kmn) for each SHP2 construct were performed and yielded the following values: SHP2-WT, Km=60 μM; SHP2-E76K, Km=20 μM; SHP2cat, Km=20 μM. Relative maximum rates (Vmax) of the DiFMUP reactions were as follows: SHP2-WT, Vmax=871 AFU/min; SHP2-E76K, Vmax, =2730 AFU/min; SHP2cat, Vmax=2912 AFU/min. IC50 values for each compound were determined from initial rates in 10-point dose-response assays using DiFMUP at a concentration corresponding to its Km value for the respective SHP2 construct. |
| Affinity data for this assay | |
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