Malaria remains a major public health issue. Infection with Plasmodium falciparum species parasites is most lethal. Increasing resistance to current antimalarial treatments makes new drug development imperative. Plasmodium parasites are obligate intracellular purine auxotrophs. Purine salvage pathways are therefore good targets for antimalarial drug development. An equilibrative nucleoside transporter, PfENT1, is the primary pathway for purine uptake by P. falciparum. However, there has been evidence of a secondary pathway that can import AMP as a purine source. This pathway remains to be characterized.
In Aim #1 I will characterize the AMP purine transport into P. falciparum through radiolabel substrate uptake inhibition experiments with purines and potential inhibitors. I will test whether AMP transport is mediated through a sodium-coupled or proton- coupled process. Results from these experiments will provide novel insight into P. falciparum biology. Previous work in the lab using a high throughput screen identified PfENT1 inhibitors. The hits block the transporter and kill malaria parasites in culture. However, these initial hits are not potent enough to be drugs.
In Aim #2, I will participate in the hit-to-lead medicinal chemistry process to optimize these PfENT1 inhibitor hits and determine the structure-activity relationships for these compounds. I will evaluate their potency in parasite cytotoxicity assays and their human cell toxicity using human hepatoma HepG2 cytotoxicity assays. This process will allow us to determine what chemical characteristics provide drug hits with the potential to be converted into drug candidates.
In Aim #3, I will determine whether naturally occurring non-synonymous single nucleotide polymorphisms (SNP) identified in genome sequencing of P. falciparum field isolates affect efficacy of the PfENT1 inhibitors that we are developing as potential antimalarial drugs. It is possible that the efficacy of PfENT1 inhibitors can be affected by the presence of SNPs in PfENT1. We have identified 13 non- synonymous SNPs in the more than 100 sequences in the PlasmoDB database. I will express PfENT1-SNPs in Saccharomyces cerevisiae by using a PfENT1-pCM189 yeast expression vector. I will determine whether the SNPs affect purine import and PfENT1 inhibitor efficacy. Available PfENT1-SNP expressing parasite isolates will also be tested using parasite cytotoxicity assays with inhibitors. These experiments will help us determine if PfENT1 SNPs could cause resistance to our inhibitors. These experiments will advance the drug development process for the PfENT1 inhibitors and hopefully lead to the development of successful antimalarial drugs. Participation in this project will provide me with the opportunity to learn about membrane transport physiology, parasite biology and the early stage drug development process. It will provide me with an excellent foundation for a career in biomedical research.
Malaria is a major public health problem and requires increased efforts to establish new targets for drug development due to emerging drug resistance. Plasmodium, the parasite that causes malaria, is purine auxotrophic and can be killed by targeting purine salvage pathways. This proposal aims to characterize and target malaria purine uptake pathways in order to generate novel antimalarial drugs. !
