Development of a Peptide Inhibitor Targeting the C‐SH2 Domain of the SHP2 Phosphatase

Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) mediates important signal transduction upon cell surface receptor stimulation, regulating multiple cellular functions. In addition to the catalytically active phosphotyrosine (pTyr) phosphatase domain, SHP2 contains two regulatory pTyr-binding domains: the N-SH2 and C-SH2 domains. While the role of the N-SH2 domain is well understood, the role of the C-SH2 domain is less clear. To support studies on the involvement of the domains in SHP2 function, herein, the development of a peptide inhibitor containing a nonhydrolysable pTyr mimetic, which selectively binds to the C-SH2 domain of SHP2 and blocks its protein-protein interactions, is described. Incorporation of the pTyr mimetic l-O-malonyltyrosine (l-OMT) results in robust binding affinity to the C-SH2 domain, while the widely used pTyr mimetic phosphonodifluoromethyl phenylalanine (F₂Pmp) abolishes binding, showing that this mimetic is not a general binder of SH2 domains, which challenges existing notions. The C-SH2 inhibitor peptide (CSIP) is stable, selective, cell permeable, and noncytotoxic. CSIP enriches the toolbox of inhibitors with different modes of action targeting SHP2, and will support studies to better understand SHP2 regulation and interactions, which can ultimately inform new drug discovery efforts.