Potent inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands

Development of Grb2 Src homology 2 (SH2) domain binding inhibitors has impo rtant implications for treatment of a variety of diseases, including severa l cancers. In cellular studies, inhibitors of Grb2 SH2 domain binding have to date been large, highly charged peptides which relied on special transpo rt devices for cell membrane penetration. Work presented in the current stu dy examines a variety of pTyr mimetics in the context of a high-affinity Gr b2 binding platform. Among the analogues studied are new norm-phosphorus-co ntaining pTyr mimetics 23a and 23b which, when incorporated into tripeptide structures 18f and 20f, are able to inhibit Grb2 SH2 domain binding with a ffinities among the best yet reported for non-phosphorus-containing SH2 dom ain inhibitors (IC50 values of 6.7 and 1.3 mu M, respectively). The present study has also demonstrated the usefulness of the Na-oxalyl group as an au xiliary which enhances the binding potency of both phosphorus- and non-phos phorus-containing pTyr mimetics. When combined with the (phosphonomethyl)ph enylalanine (Pmp) residue to give analogues such as L-20d, potent inhibitio n of Grb2 SH2 domain binding can be achieved both in extracellular assays u sing isolated Grb2 SH2 domain protein and in intracellular systems measurin g the association of endogenous Grb2 with its cognate p185(erbB-2) ligand. These latter effects can be achieved at micromolar to submicromolar concent rations without prodrug derivatization. The oxalyl-containing pTyr mimetics presented in this study should be of general usefulness for the developmen t of other Grb2 SH2 domain antagonists, independent of the beta-bend-mimick ing platform utilized for their display.