Structure-based discovery of novel ROCK2 inhibitors using integrated virtual screening, molecular dynamics, metadynamics and absolute binding free energy calculations.

UNLABELLED: Breast cancer remains one of the most prevalent and deadly malignancies in women globally, with metastasis and invasion contributing significantly to its poor prognosis. Rho-associated kinase 2 (ROCK2), a key regulator in the Ras homolog family member (Rho) GTPase signaling pathway, is implicated in tumor progression and cytoskeletal remodeling, making it an attractive therapeutic target. This study presents a structure-based computational strategy to identify and characterize novel ROCK2 inhibitors. An integrated pipeline involving virtual screening of over 1.5 million compounds focused on heterocyclic scaffolds chosen for their structural compatibility. Promising candidates were refined using MM/GBSA rescoring and molecular dynamics simulations. Conformational stability was assessed through principal component analysis and free energy landscape mapping, while well-tempered metadynamics simulations provided detailed free energy surface profiles. Binding affinities were estimated via Absolute Binding Free Energy Perturbation (ABFEP) calculations. Two lead compounds, ASN52 and Selles74, were identified with synthetically tractable heterocyclic cores and demonstrated strong, stable interactions within the ROCK2 active site. ASN52 exhibited a pronounced energetic minimum at 4 Å (- 11 kcal/mol), indicating a highly favorable binding conformation. ABFEP binding free energies of - 6.7 ± 1.27 kcal/mol (ASN52) and 6.15 ± 1.78 kcal/mol (Selles74) further supported their potential as effective ROCK2 inhibitors. These findings provide a computational foundation for the rational design and preclinical development of ROCK2-targeted therapeutics. The approach and results are applicable to the discovery of both small-molecule and peptidomimetic inhibitors, supporting broader efforts in structure-guided drug design targeting kinases involved in cancer and other diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-026-00714-4.