Exploring the pKa, Molecular Binding, and Caspase-3 Activation Potential of Coumarin Derivatives

Caspase-3 is a central executioner protease in apoptosis, and its activation by small molecules is considered a promising anticancer strategy. Coumarin derivatives are known for their diverse biological activities, but the influence of structural modifications, particularly hydroxyl group positioning, on acidity constants (pKa) and binding affinity to caspase-3 remains underexplored. This study aimed to investigate how the electronic and spatial configuration of hydroxyl substituents in coumarin derivatives modulates their pKa behavior and binding potential to caspase-3, with the ultimate goal of identifying promising scaffolds for caspase-3 activation. A series of twenty-one structurally diverse coumarin derivatives was analyzed. pKa values were determined experimentally by UV–Vis spectrophotometry and theoretically predicted using MarvinSketch software. Molecular docking (AutoDock) and molecular dynamics simulations (GROMACS) were conducted, followed by MM/PBSA binding free energy calculations to evaluate interactions with caspase-3 (PDB ID: 3DEI). Experimental and theoretical pKa₁ values exhibited strong concordance (R² = 0.93), while discrepancies were observed in pKa₂ values, especially among piperazine- and piperidine-substituted derivatives. Molecular docking identified Compound 13 as the most potent derivative (ΔGbinding = –9.3 kcal/mol), with its 7-position hydroxyl group forming strong hydrogen bonds and electrostatic interactions particularly with Arg207 and His121. In contrast, derivatives with the hydroxyl at position 6 showed weakened interactions and reduced affinity. The position-dependent ionization states significantly influenced the molecular interaction profiles. Conclusion: These findings provide mechanistic insight into how small changes in hydroxyl positioning affect ionization and caspase-3 binding, offering valuable guidance for pKa-optimized design of novel small-molecule caspase-3 activators with potential anticancer applications.