Potent Inhibition of Grb2 SH2 Domain Binding by Non-Phosphate-Containing Ligands

Abstract

Development of Grb2 Src homology 2 (SH2) domain binding inhibitors has important implications for treatment of a variety of diseases, including several cancers. In cellular studies, inhibitors of Grb2 SH2 domain binding have to date been large, highly charged peptides which relied on special transport devices for cell membrane penetration. Work presented in the current study examines a variety of pTyr mimetics in the context of a high-affinity Grb2 binding platform. Among the analogues studied are new non-phosphorus-containing pTyr mimetics 23a and 23b which, when incorporated into tripeptide structures 18f and 20f, are able to inhibit Grb2 SH2 domain binding with affinities among the best yet reported for non-phosphorus-containing SH2 domain inhibitors (IC50 values of 6.7 and 1.3 microM, respectively). The present study has also demonstrated the usefulness of the Nalpha-oxalyl group as an auxiliary which enhances the binding potency of both phosphorus- and non-phosphorus-containing pTyr mimetics. When combined with the (phosphonomethyl)phenylalanine (Pmp) residue to give analogues such as L-20d, potent inhibition of Grb2 SH2 domain binding can be achieved both in extracellular assays using isolated Grb2 SH2 domain protein and in intracellular systems measuring the association of endogenous Grb2 with its cognate p185erbB-2 ligand. These latter effects can be achieved at micromolar to submicromolar concentrations without prodrug derivatization. The oxalyl-containing pTyr mimetics presented in this study should be of general usefulness for the development of other Grb2 SH2 domain antagonists, independent of the beta-bend-mimicking platform utilized for their display.

Potent Blockade of Hepatocyte Growth Factor-stimulated Cell Motility, Matrix Invasion and Branching Morphogenesis by Antagonists of Grb2 Src Homology 2 Domain Interactions

“…The Grb2 SH2 domain antagonists designated 1-4 ( Fig. 1) were synthesized and purified as described (30,34).…”

“…1. The affinity of these compounds for binding to Grb2 SH2 domains in vitro have been described (30,34). Each compound contains a common backbone structure corresponding to the invariant Grb2 SH2 domain binding motif pY-X-N, with distinct modifications to the Tyr(P)-mimetic moiety that render each resistant to phosphatases, but maintain the net charge (Ϫ2) of this moiety.…”

“…Each compound contains a common backbone structure corresponding to the invariant Grb2 SH2 domain binding motif pY-X-N, with distinct modifications to the Tyr(P)-mimetic moiety that render each resistant to phosphatases, but maintain the net charge (Ϫ2) of this moiety. Compound 4 also has these features, but differs from the others in that it has greater than 100-fold lower affinity for binding to Grb2 SH2 domains in vitro (30). The similar charge and overall structure of compound 4 to compounds 1-3 thus made it an excellent negative control throughout the course of the biological characterization of the effects of these compounds on HGF signaling.…”

“…Note that, despite its similar chemical structure, compound 4 has at least 100-fold lower affinity for Grb2 SH2 domain binding in vitro (30), and was used as a negative control throughout the biological characterization of these compounds.…”

“…These and other observations have led to the development of potent tripeptide inhibitors of SH2 domain interactions. Modification of the Tyr(P) residue to phosphonomethyl phenylala-nine, or related structures, protects this moiety from phosphatases (28,29), whereas other modifications have been identified that increase inhibitor affinity and the potential for cell membrane penetration (30).…”