Discovery of Novel Pyridin-2-yl Urea Inhibitors Targeting ASK1 Kinase and Its Binding Mode by Absolute Protein–Ligand Binding Free Energy Calculations

Apoptosis signal-regulating kinase 1 (ASK1), a key component of the mitogen-activated protein kinase (MAPK) cascades, has been identified as a promising therapeutic target owing to its critical role in signal transduction pathways. In this study, we proposed novel pyridin-2-yl urea inhibitors exhibiting favorable physicochemical properties. The potency of these compounds was validated through in vitro protein bioassays. The inhibition (IC₅₀) of compound <b>2</b> was 1.55 ± 0.27 nM, which was comparable to the known clinical inhibitor, Selonsertib. To further optimize the hit compounds, two possible binding modes were initially predicted by molecular docking. Absolute binding free energy (BFE) calculations based on molecular dynamics simulations further discriminated the binding modes, presenting good tendency with bioassay results. This strategy, underpinned by BFE calculations, has the great potential to expedite the drug discovery process in the targeting of ASK1 kinase.