Nonhydrolyzable phosphotyrosyl mimetics for the preparation of phosphatase-resistant SH2 domain inhibitors.

Src homology 2 (SH2) domains participate in protein tyrosine kinase (PTK)-mediated cellular signal transduction through their ability to bind with high affinity to phosphotyrosyl (pTyr)-bearing protein sequences. Although peptides containing pTyr competitively inhibit the binding between phosphoproteins and cognate SH2 proteins in a sequence-specific manner, such peptides are rapidly dephosphorylated by cellular phosphatases. We now describe our efforts to develop SH2 inhibitory peptides containing phosphatase-resistant pTyr surrogates. The parent compound, (phosphonomethyl)phenylalanine (Pmp), is a phosphonate-based mimetic of pTyr in which the phosphate ester oxygen (> COPO3H2) has been replaced by a methylene unit (> CCX2PO3H2, X2 = H2). Pmp analogues bearing fluorine (X2 = H, F or X2 = F2) or hydroxyl (X2 = H, OH) substituents on the phosphonate alpha-methylene carbon have been prepared and incorporated into peptides for use as SH2 domain inhibitors. In an assay using the C-terminal SH2 domain of phosphatidylinositol (PI) 3-kinase, peptides having a GXVPML sequence [where X = pTyr, Pmp, hydroxy-Pmp (HPmp), monofluoro-Pmp (FPmp), and difluoro-Pmp (F2Pmp)] exhibited binding potency in the order HPmp < Pmp < FPmp < F2Pmp = pTyr. Distinct peptide sequences which bind selectively with Src and Grb2 SH2 domains were also prepared with pTyr and F2Pmp. The F2Pmp peptides bound with high (0.2- to 5-fold) relative affinity, compared to analogous pTyr peptides. We conclude that peptides containing F2Pmp bind to SH2 domains with high affinity and specificity and, being resistant to cellular phosphatases, should provide a generally useful tool for disrupting SH2 domain-mediated signaling pathways in intact cells.