Structure-guided discovery of 6-aminopyridine derivatives for group II PAK4 inhibition

p21-Activated kinase 4 (PAK4) is a Group II serine/threonine kinase implicated in tumor growth and immune evasion, and is therefore a promising target for anticancer therapy. Leveraging structure-guided design, we report a novel series of 6-aminopyridine derivatives that target a previously underexplored deep hydrophobic subpocket adjacent to Phe461, which extends from the floor pocket of PAK4. Even with a relatively concise compound set, the focused SAR study identified key substituent features required for deep-pocket engagement, and subsequent molecular docking and dynamics simulations supported an underexploited binding mode centered on the key residue Phe461. Among the synthesized compounds, 17 (PAK4 IC₅₀ 0.71 μM) and 29 (PAK4 IC₅₀ 1.88 μM) demonstrated comparable enzymatic potency, consistent with ATP-competitive inhibition, while compound 29 was effective in inhibiting cancer cell proliferation (HCT-116 GI₅₀ 23.0 μM; A549 GI₅₀ 11.3 μM). In conclusion, this study highlights the potential of targeting underexplored subpockets in PAK4 and provides new molecular insights for the discovery of PAK4 inhibitors.