Antimalarial drug resistance is a problem that impedes global efforts in the control of malaria (Balikagala et al., 2021, WHO, 2024). Overcoming this resistance requires the development of new therapeutic agents with broad therapeutic potential and novel mechanism of action (Ippolito et al., 2021).

The purine transport system in Plasmodium falciparum has garnered a lot of interest for years as a drug target (Quashie et al., 2008, Frame et al., 2015a, Cheviet et al., 2019). P. falciparum cannot synthesize purines de novo and relies on salvaging them from host erythrocytes to support essential processes such as DNA and RNA synthesis (de Koning et al., 2005). Purine import into the parasite, which is mediated by Equilibrative Nucleoside Transporters (PfENTs), is a critical step in the purine salvage pathway. PfENT1 serves as the primary route for purine uptake in P. falciparum and is essential for the parasite’s intraerythrocytic survival. This transporter has independently been validated as a promising target for antimalarial drug development by multiple research groups (El Bissati et al., 2006, Quashie et al., 2008, Frame et al., 2015b, Zhang et al., 2018). PfENT4, like PfENT1, is localized to the parasite’s plasma membrane and is capable of purine uptake when expressed in Xenopus laevis oocytes (Frame et al., 2012), but to date, it has been much less studied.

Despite the purine transport system in P. falciparum emerging as a promising drug target, both as conduits of purine antimetabolites and as essential proteins in their own right, the genetic diversity of the PfENTs has not been investigated. Genetic diversity in ENTs could influence the efficacy of potential purine analogues such as may be developed as antimalarial drugs, as even small sequence changes can impact drug uptake without compromising the uptake of the original substrate (Mäser et al., 1999, Munday et al., 2015, Alghamdi et al., 2020). Consequently, the parasite may select for any pre-existing resistant variants in the population if these disable the binding of inhibitors or uptake of cytotoxic antimetabolites but retain transport of the natural substrate. An extensive polymorphic nature in PfENTs resulting from the presence of genetic variants may alter the structure and possibly the function of the proteins, giving rise to subgroups in a population which may eventually be, or more easily become, resistant to purine-based antimalarial drugs and promote (eventual) therapeutic failure.

Antimalarial drug resistance could result from the natural genetic diversity in the parasite population (Meyer et al., 2002). Naturally occurring genetic variants, such as single nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and copy number variations (CNVs) in drug targets can provide parasites with a fitness advantage and enhance their resistance to new drugs (Meyer et al., 2002, Ravenhall et al., 2019). SNPs, InDels, and CNVs in target proteins contribute to resistance against many antimalarials (Meyer et al., 2002, Ravenhall et al., 2019). Considering the high cost and labour-intensive nature of the drug development process, investigating the polymorphic nature of a drug target prior to the drug development process is essential in understanding whether genetic diversity may limit exploiting the drug target through the potential emergence and spread of resistance alleles. For instance, as observed for the P2/TbAT1 transporter of Trypanosoma brucei brucei which is critical for the uptake of several trypanocides including melarsoprol (Carter and Fairlamb, 1993, De Koning et al., 2000, de Koning and Jarvis, 2001), the resistance allele almost disappeared after withdrawal of the drug from the region (Kazibwe et al., 2009), although it persisted where the drug continued to be used (Graf et al., 2013). Thus, understanding the genetic diversity in a drug target early in the drug development process may aid in the substantiation and validation of the drug targets and aid improved drug design.

It is worth noting that a common strategy that most pathogens, including P. falciparum, use in evading their hosts’ immune responses is the accumulation of genetic variants within their genome (Terheggen et al., 2014, Ravenhall et al., 2016, Naung et al., 2022). The P. falciparum parasite exhibits enormous genetic diversity which contributes to the success of the parasite in overcoming antimalarial therapy, and this undermines malaria control interventions (Meyer et al., 2002, Neafsey et al., 2008, Ravenhall et al., 2016, Apinjoh et al., 2019). With the accumulation of such genetic variants within the genome of P. falciparum, chemotherapy may be ineffective against the complete repertoire of circulating genetic variants of the parasite and result in a “sieve effect” with higher efficacy against normal strains and low to no efficacy against rarer variant strains (Ravenhall et al., 2016, Gill and Sharma, 2022).

Previous studies reported the presence of genetic variants in the transmembrane segments of ENTs (Endres and Unadkat, 2005, Visser et al., 2007, Arendt and Ullman, 2010, Stewart et al., 2010, Munday et al., 2015), with some of these genetic variants affecting substrate affinity and/or inhibitor efficacy. With regards to the PfENTs, previous studies by Riegelhaupt et al. (2010) and Sosa et al. (2020) reported the presence of SNPs in PfENT1. Also, Carter et al. (2000) and Parker et al. (2000) reported quite contradictory findings in substrate affinity and specificity for PfENT1 although they both used the same expression system (Xenopus laevis oocytes). However, the amino acid sequences of PfENT1 used by Carter et al. (2000) and by Parker et al. (2000) differed in amino acid sequence at position 385, being phenylalanine or leucine, respectively.

Thus, to evaluate whether it is possible to successfully exploit the PfENTs in antimalarial drug development, it is necessary to catalogue circulating genetic variants including InDels and CNVs in PfENTs using isolates from different geographical locations across the world. With the availability of high-quality whole genome sequences of thousands of field isolates of P. falciparum through the Malaria Genomic Epidemiology Network (MalariaGEN) project, it has become feasible to incorporate genetic diversity studies into antimalarial drug development processes.

In addition to genetic diversity, the level of expression of the transporters is correlated to the rate of uptake and therefore to the efficacy of the drugs, but there is a paucity of data on the expression profile of PfENTs in field isolates. It has been demonstrated that PfENT1 and PfENT4 are both expressed in laboratory-adapted strains of the parasite (Frame et al., 2015a). However, previous studies reported variation in the expression of some genes in field isolates compared to laboratory-adapted parasites (Pinto et al., 1997, Daily et al., 2005, Tarr et al., 2018) and in T. b. brucei and T. equiperdum, the ENT transporter AT1 was found to be present and unmutated but not expressed in some resistant strains (Stewart et al., 2010), indicating that in at least some protozoan parasites the non-expression, rather than a genetic mutation of the coding sequence, can be the cause of drug resistance. A potentially confounding factor, however, is that gene expression profiles of parasites maintained in cultures for long periods of time may not reflect a true gene expression pattern as seen in parasite populations that are actively circulating in malaria-endemic areas (Kengne-Ouafo et al., 2023). Therefore, to target the PfENTs with purine analogues, it is crucial to investigate the gene expression profile of PfENT1 and PfENT4 in field isolates alongside their genetic diversity.