Novel potent heterocyclic Grb2-SH2 domain antagonists as potential anti-proliferative agents.

Growth factor receptor-bound protein 2 (Grb2) plays a critical role in proliferative diseases by binding phosphotyrosine residues on target proteins leading to activation of key pathways. The phosphotyrosyl motif of Focal Adhesion Kinase (FAK) has been utilized as a pharmacophore to identify small molecules that disrupt its interaction with the Grb2 Src Homology 2 (SH2) domain. In this study, we report the hit-to-lead optimization of 11,12,479 synthesizable analogs of previously identified Grb2-SH2 antagonists via virtual screening using AutoDock Vina v1.2.0. ADMET predictions were performed using SwissADME and pkCSM. Molecular dynamics (MD) simulations were conducted using AMBER v18. The Grb2-SH2 domain was cloned, expressed as a GST fusion protein, and purified for in vitro analysis. Binding interactions of the top compounds were assessed using SPR spectroscopy and competitive ELISA. Five heterocyclic molecules with novel scaffolds demonstrated favorable AutoDock binding affinities, stable binding in MD simulations, and promising ADMET profiles. MMPBSA calculations indicated the strongest binding energy for DO71_2. Per residue decomposition confirmed the maximum contribution of charged pocket residues to the binding energy. SPR determined KD values in the nanomolar range, >50-fold better than the phosphorylated peptide substrate. KD value of the peptide substrate was similar to previously reported values. DO71_2 showed the best KD value (9.4 nM) in consonance with the in silico prediction. ELISA confirmed concentration-dependent, specific binding of all the five compounds to Grb2-SH2. Our findings highlight the potential of these non-peptidic, non-phosphorous-based Grb2-SH2 antagonists as therapeutic agents for proliferative diseases like cancer and cardiac hypertrophy.