PfDHFR Resistance Mutations in Mali: Impact on Protein Structure and Identification of Novel Natural Inhibitors

Abstract

Malaria drug resistance remains one of the greatest challenges to the malaria control program in Mali. Mutation in the Plasmodium falciparum Dihydrofolate Reductase (PfDHFR), a key enzyme in the folate biosynthesis pathway and primary target of antifolate drugs, are knows to induces resistance to pyrimethamine and Proguanil. Four-point mutations: N51I, C59R, S108N, and I164L are particularly associated with antifolate resistance. This study aims to characterize resistance-associated PfDHFR mutations circulating in three sentinel sites in Mali (Dioro, Missira, and Sélingué) and identify potential inhibitors effective against mutant PfDHFR variants through virtual screening of African natural compound libraries. A total of 47 Pfdhfr were analyzed to identify Single-Nucleotide Polymorphisms (SNPs) using Geneious Prime. Homology-based 3D protein structures of mutant PfDHFR using PRIMO server, and structural visualization as well as molecular analyses were performed using UCSF Chimera and AutoDock vina. Among the 47 PfDHFR sequences analyzed, 22 exhibited the triple mutations N51I, C59R, and S108N, will the I164L mutation was absent in all samples across the three sentinel sites. Structural modeling of these mutants revealed restricted access and crowding in the PfDHFR active site, consistent with known antifolate resistance mechanisms. Virtual screening identified several promising inhibitors, among which compound SANC00747 exhibiting exceptional binding affinity (-14.046 kcal/mol), highlighting the potential of African natural products as resistance-breaking candidates. This study demonstrates the prevalence of key antifolate-resistance mutations in Mali and reveals promising natural compounds capable of effectively targeting mutant PfDHFR. These findings underscore the value of African natural products as a rich reservoir for developing next-generation antimalarial therapies.