Targeted Covalent Inhibition of Grb2-Sos1 Interaction through Proximity-Induced Conjugation in Breast Cancer Cells

Targeted covalent inhibitors of protein protein interactions differ from reversible inhibitors in that the former bind and covalently bond the target protein at a specific site of the target. The site specificity is the result of the proximity of two reactive groups at the bound state, for example, one mild electrophile in the inhibitor and a natural cysteine in the target close to the ligand binding site. Only a few pharmaceutically relevant proteins have this structural feature. Grb2, a key adaptor protein in maintaining the ERIC activity via binding Sos1 to activated RTKs, is one: the N-terminal SH3 domain of Grb2 (Grb2(N-SH3)) carries a unique solvent-accessible cysteine Cys(32) close to its Sos1-binding site. Here we report the design of a peptide-based antagonist (a reactive peptide) that specifically binds to Grb2(N-SH3) and subsequently undergoes a nucleophilic reaction with Cys(32) to form a covalent bond thioether, to block Grb2-Sos1 interaction. Through rounds of optimization, we eventually obtained a dimeric reaction reactive peptide that can form a covalent adduct with endogenous Grb2 protein inside the cytosol of SK-BR-3 human breast cancer cells with pronounced inhibitory effect on cell mobility and viability. This work showcases a rational design of Grb2-targeted site-specific covalent inhibitor and its pronounced anticancer effect by targeting Grb2-Sos1 interaction.