Mechanistic insights into carbonic anhydrase IX inhibition by coumarins from Calendula officinalis: in vitro and in silico approaches †

Given the critical role of carbonic anhydrase IX (CA IX) in various pathological conditions, there is a significant demand for new inhibitors to enhance patient outcomes and clinical management. In this study, we examined the inhibitory effectiveness of five coumarins derived from Calendula officinalis against CA IX using in vitro assays and computational modeling. Among the coumarins tested, xeroboside and isobaisseoside were identified as the most potent inhibitors. Kinetic studies indicated that xeroboside and isobaisseoside exhibit a mixed inhibition mode. Molecular docking analysis showed that the tested coumarins exhibit binding affinities and extensive polar interactions with CA IX. These coumarins demonstrated significant hydrophobic interactions and occupied the same binding site as acetazolamide (AAZ). Molecular dynamics (MD) indicated that xeroboside and isobaisseoside exhibited consistent trajectories and notable energy stabilization during their interaction with CA IX. MM/PBSA calculations showed that xeroboside displayed the lowest binding free energy (−27.26 ± 2.48 kJ mol−1). Potential Energy Landscape (PEL) analysis revealed distinct and stable conformational states for the CA IX–ligand complexes, with xeroboside exhibiting the most stable and lowest energy configuration. These computational findings are consistent with the experimental results, highlighting the potential efficacy of xeroboside and isobaisseoside as CA IX inhibitors. In conclusion, Calendula officinalis-derived coumarins are promising candidates as effective CA IX inhibitors.