| Summary: | The bifunctional enzyme Dihydrofolate reductase-thymidylate synthase (DHFR-TS) plays a crucial role in the survival of the <i>Leishmania</i> 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-<i>Leishmania</i> chemotherapies. In this research, using the <i>Leishmania major</i> DHFR-TS recombinant protein, enzymatic inhibitory assays were performed on four kauranes and two derivatives that had been previously tested against <i>Lm</i>PTR1. The structure <b>302</b> (6.3 µM) and its derivative <b>302a</b> (4.5 µM) showed the lowest IC<sub>50</sub> 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 <i>Lm</i>DHFR-TS, as well as the presence of the <i>p</i>-hydroxyl group of the phenylpropanoid moiety of <b>302a</b>. Finally, additional computational studies were performed on DHFR-TS structures from Leishmania species that cause cutaneous and mucocutaneous leishmaniasis in the New World <i>(L. braziliensis, L. panamensis</i>, and <i>L. amazonensis</i>) to explore the targeting potential of these kauranes in these species. It was demonstrated that structures <b>302</b> and <b>302a</b> are multi-<i>Leishmania</i> species compounds with dual DHFR-TS/PTR1 inhibitory activity.
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