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. 2020 Oct 31;11(3):750–762. doi: 10.1016/j.apsb.2020.10.021

Figure 3.

Figure 3

Scheme of the cross-validation protocol for high-throughput screening of new SHP2 inhibitors targeting the PTP active site and allosteric sites. The in-house compound library was firstly screened with wild type SHP2 (SHP2-WT) enzyme assay, which utilized the non-fluorogenic 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as substrate to characterize the phosphatase activity of SHP2. DiFMUP can be hydrolyzed by SHP2 to remove phosphate group with production of fluorogenic 6,8-difluoro-4-methylumbelliferyl hydroxid (DiFMU), whose fluorescence intensity (FI) could be monitored at 350 nm excitation and 450 nm emission. Then hit compounds would be further cross-validated with the SHP2-WT thermal shift assay and SHP2-PTP enzyme assay respectively to discriminate the new SHP2 inhibitors targeting the PTP active site and allosteric sites. (Red triangle: allosteric inhibitors; Blue circular: catalytic site inhibitors; Purple rhombus: invalid compounds).