Genetic diversity of Plasmodium falciparum equilibrative nucleoside transporters PfENT1 and PfENT4: Implications for purine-based antimalarial drug development

The emergence and spread of Plasmodium falciparum resistance to current antimalarial drugs underscores the urgent need for new therapies with novel mechanisms of action and broad therapeutic potential. The purine salvage pathway in P. falciparum is a validated drug target and the import of purines into the parasite, mediated by Equilibrative Nucleoside Transporters ( Pf ENTs), is a critical step in this pathway. Despite emerging as drug targets, the genetic diversity of Pf ENTs remains poorly characterized. Understanding this diversity is crucial for optimizing a potential purine-based drug development. Here, we report the genetic diversity in Pf ENT1 and Pf ENT4 in P. falciparum field isolates from geographical regions worldwide. Illumina-generated sequences and Variant Call Format (VCF) files for over 7,000 isolates were retrieved from the European Nucleotide Archive and Malaria Genomic Epidemiology Network data repositories respectively. VCF files were filtered to detect single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) in Pf ENT1 and Pf ENT4. Additionally, Illumina-generated sequences were analyzed to identify copy number variations (CNVs) in these genes. In both Pf ENT1 and Pf ENT4, only SNPs were identified as no InDels or CNVs were found. The frequency of missense variants in Pf ENT1 and Pf ENT4 was 4.5 % and 6.2 % respectively. Three common genetic variants, F36S, V129I, and L133F, were identified in Pf ENT1, with minor allele frequencies (MAF) of 4.0 %, 4.1 %, and 3.0 %, respectively. Additionally, four common genetic variants, I188F, N209I, A260T, and S383A, were found in Pf ENT4, with MAF of 2.2 %, 1.6 %, 1.5 %, and 38.9 %, respectively. In both genes, most genetic variants substituted non-polar amino acid residues with similar residues. The allele frequencies of the SNPs identified are similar ( p > 0.05) in rural and urban areas. In conclusion, this study revealed that Pf ENT1 and Pf ENT4 are highly conserved with low genetic diversity, emphasizing their importance and suitability as drug targets.