Structural and functional implications of MIT2 and NT2 mutations in amodiaquine and piperaquine resistant Plasmodium...

Long-acting drugs, amodiaquine (AQ), lumefantrine (LM), and piperaquine (PQ), are vital components of artemisinin-based combination therapies (ACTs) for malaria treatment. However, the emergence of partial artemisinin-resistant parasites poses significant challenges, particularly in malaria-endemic regions. Despite extensive research, parasite's resistance mechanisms to these drugs still need complete elucidation. This study investigated the genetic basis of resistance to AQ, LM, and PQ using Plasmodium berghei, focusing on selected genes encoding transport proteins in Plasmodium species. In silico bioinformatics tools were used to map genes encoding transport proteins, their ligand-binding sites, and their conservation across different Plasmodium species. PCR amplification and sequence analysis were employed to examine single nucleotide polymorphisms (SNPs) in the genes encoding the selected transporters in AQ, LM, and PQ-resistant P. berghei. The structural impacts of the mutations were evaluated using AlphaFold, ITASSER, UCSF Chimera, and MOTIF Finder. Genes encoding CorA-like Mg2+ transporter protein (MIT2), nucleoside transporter 2 (NT2), ABC Transporter G family member 2 (ABCG2), and novel putative transporter 1 (NPT1) transport proteins with notable conserved motifs and ligand-binding motifs in Plasmodium species were selected and examined. In AQ-resistant (AQ^R) parasites, a non-synonymous mutation (I433∗) was found in MIT2. PQ-resistant (PQ^R) parasites possessed a non-synonymous mutation (D511H) in NT2 and a silent mutation in the NPT1 protein. No mutations were observed in the targeted regions of the transporters in LM-resistant (LM^R) parasites, nor in the ligand-binding motifs of ABCG2 across all resistant strains. These findings suggest that selection pressure from AQ and PQ leads to mutations in MIT2 and NT2. Further investigation is required to understand how these mutations affect drug susceptibility on a functional level.