Kaurane-Type Diterpenoids as Potential Inhibitors of Dihydrofolate Reductase-Thymidylate Synthase in New World Species.

The bifunctional enzyme Dihydrofolate reductase-thymidylate synthase (DHFR-TS) plays a crucial role in the survival of the parasite, as folates are essential cofactors for purine and pyrimidine nucleotide biosynthesis. However, DHFR inhibitors are largely ineffective in controlling trypanosomatid infections, largely due to the presence of Pteridine reductase 1 (PTR1). Therefore, the search for structures with dual inhibitory activity against PTR1/DHFR-TS is crucial in the development of new anti- chemotherapies. In this research, using the DHFR-TS recombinant protein, enzymatic inhibitory assays were performed on four kauranes and two derivatives that had been previously tested against PTR1. The structure (6.3 µM) and its derivative (4.5 µM) showed the lowest IC values among the evaluated molecules. To evaluate the mechanism of action of these structures, molecular docking calculations and molecular dynamics simulations were performed using a DHFR-TS hybrid model. Results showed that hydrogen bond interactions are critical for the inhibitory activity against DHFR-TS, as well as the presence of the -hydroxyl group of the phenylpropanoid moiety of . Finally, additional computational studies were performed on DHFR-TS structures from Leishmania species that cause cutaneous and mucocutaneous leishmaniasis in the New World , and ) to explore the targeting potential of these kauranes in these species. It was demonstrated that structures and are multi- species compounds with dual DHFR-TS/PTR1 inhibitory activity.