In Silico and In Vitro Analysis of Ganoderic Acid A Binding to Human Monocarboxylate Transporters 1 and 4.

In recent years, the search for novel anticancer agents has increasingly focused on monocarboxylate transporters (MCTs) because of their involvement in tumor metabolism. Ganoderic acid A (GAA), a triterpenoid derived from the medicinal mushroom Ganoderma lucidum, has demonstrated anticancer potential; however, its interaction with MCT isoforms remains insufficiently characterized. In this study, we examined the interaction between GAA and MCT1 and MCT4 using complementary computational and experimental approaches. Full-length structures of MCT1 and MCT4 were predicted using AlphaFold2 and validated with the SAVES server. Molecular docking and molecular dynamics simulations using the known dual inhibitor syrosingopine as a reference indicated that GAA can associate with both MCT1 and MCT4. Cellular thermal shift assay (CETSA) and isothermal dose-response fingerprinting (ITDRF) showed that GAA thermally destabilized both MCT1 and MCT4, supporting a direct protein-compound interaction. Notably, ITDRF analysis revealed enhanced stability of a higher-molecular-weight MCT4, suggesting a biphasic binding behavior. Together, these findings indicate that GAA directly interacts with MCT1 and MCT4, and uncovers a biphasic binding pattern associated with MCT4.