Pharmacophore-based high-throughput virtual screening (HTVS) to identify new c-Src kinase inhibitors with anticancer potential

c-Src is the non-receptor kinase commonly overexpressed in numerous Cancer isoforms. As potential Anticancer target, these receptors are difficult to target with drugs because of their continuous shuttling between cellular and nuclear compartments and role in relaying of vital signals for gene expression, cell growth, and survival. Besides this high structural homology to Other kinases, the involvement of compensatory pathways and the availability of multiple domains within the same proteins further complicate the targeting by drugs. The toxicity and resistance issue with the handful of c-Src inhibitors available, which are again non-selective in approach, further complicate this process. Considering the gap, we employed a drug identification strategy for a plausible c-Src inhibition and its Anticancer potential. We selected 500,000 small molecules from the ChemBridge commercial library (database) for the virtual screening. These molecules were filtered via the development of a pharmacophore model, in silico pharmacokinetics (ADME) analysis, and high-throughput virtual screening (HTVS). The top-ranked molecules based on the docking scores, which represent computational binding affinity between a protein and a ligand, were selected and eventually led to 29 best docked molecules. The visual inspection further resulted in refinement of 4 molecules (5280699, 9797370, 11200016, and 71736582), demonstrating protein-ligand interactions the most at the c-Src kinase binding site. To validate their optimal binding, we carried out 200 ns MD simulations on these four selected proteins-ligand complexes. MD analysis revealed that the inhibitors 11200016 and 71736582 were found to be exceptionally stable at the c-Src kinase binding site, meeting the essential prerequisite. The top hit, 71736582, was further corroborated biologically. 71736582 portrayed excellent Anticancer potential towards various Cancer cell lines (A549, MDAMB-231, HCT-116, DU-145, and PC-3). It was found to inhibit the c-Src-mediated kinase activity (IC₅₀: 517 nM) in comparison to the positive control, bosutinib (IC₅₀: 408 nM). The compound was also able to increase the oxidative stress and induce Apoptosis in the colorectal Cancer cells employed. The study thus may pave the way for exploration of the top identified ligands further to develop and establish their potential as c-Src kinase inhibitors with Anticancer potential.

Biological validation; Docking; Drug design; Molecular dynamics simulations; Pharmacophore; c-Src kinase.