Discovery of N -Acylhydrazone Derivatives as ROCK Inhibitors: A Journey from Virtual Screening and Structure-Based De Novo Design to the Identification of ROCK2 Selective Inhibitors and Beyond

Rho-associated coiled-coil containing kinases (ROCK1 and ROCK2) are central regulators of actin cytoskeleton organization and cell contractility under physiological conditions. Dysregulation of ROCK signaling contributes to aberrant cell migration, invasion, and tissue remodeling, positioning these kinases as attractive therapeutic targets in cancer and fibrotic diseases. In this work, we report the discovery of N-acylhydrazone (NAH) derivatives as potent and selective ROCK inhibitors, integrating structure-based virtual screening (SBVS), de novo design, and biological evaluation. Initial hit identification from the LASSBio Chemical Library revealed three inhibitors (LASSBio-1828 (1), LASSBio-1829 (2), and LASSBio-1919 (3)), which guided the rational design of a virtual library of 321 NAH analogues. Docking-based prioritization, synthesis, and SAR exploration yielded compounds with low nanomolar potency, among which LASSBio-2360 (12), LASSBio-2380 (17), and LASSBio-2382 (18) exhibited dual ROCK1/2 inhibition (IC50 values in the 1–15 nM range), while LASSBio-2389 (21) showed remarkable ROCK2 selectivity (IC50 = 0.051 μM; 21-fold vs ROCK1 - IC50 = 1.143 μM) and minimal inhibition of other related kinases at 500 nM. Molecular dynamics simulations demonstrated that 21 stabilizes the DFG-out conformation of ROCK2, providing a structural rationale for isoform selectivity. In vitro studies using MDA-MB-231 triple-negative breast cancer cells confirmed that compounds 12, 17, 18, and 21 inhibit migration more effectively than fasudil and comparably to belumosudil. Altogether, this work identifies NAH as a privileged scaffold for ROCK inhibition, delineates the molecular determinants of ROCK2 selectivity, and highlights new chemical leads for the development of antimetastatic and antifibrotic agents targeting the Rho/ROCK pathway.