The Discovery of Small ERK5 Inhibitors via Structure-Based Virtual Screening, Biological Evaluation and MD Simulations

ERK5, a member of the MAP kinase family, has been implicated in several cancer types due to its role in regulating cell proliferation, survival, and migration. In this study, structure-based virtual screening was employed, followed by cell assays, and molecular dynamics simulations to identify novel ERK5 inhibitors. A commercially available library of 1.6 million compounds was subjected to a three-stage docking process (HTVS, SP, and XP), using the docking module in Schrodinger Maestro, yielding 40 candidates with superior docking scores compared to the co-crystallized ligand. These compounds were then tested for antiproliferative activity using an MTT assay in A549 and H292 lung cancer cell lines. Among the hits, STK038175, STK300222, and GR04 showed significant activity with IC₅₀ values of ranging from 10 to 25 µM. Western blot analysis revealed that STK300222 at 50 µM reduced the phosphorylation of ERK5 downstream targets similarly to a known inhibitor, while wound healing assays confirmed a dose-dependent decrease in cell migration. Molecular dynamics simulations of 200 ns further demonstrated that all three compounds form stable complexes with ERK5 that are comparable to the co-crystallized ligand in 5BYZ. The MD simulations also revealed strong electrostatic and solvation interactions observed for STK300222 and GR04 particularly. Furthermore, by calculating the MM-GB/SA scores from the MD trajectories, the binding affinities of the three hits, along with the co-crystallized ligand in 5BYZ, were re-scored. Although the co-crystallized ligand had the highest MM-GB/SA score at -38.96 Kcal mol^-1, STK300222 had a comparable score of -35.45 Kcal mol^-1. These results highlight STK300222 and GR04 as promising candidates for further optimization and in vivo validation as ERK5 inhibitors.