Development of a continuous assay for high throughput screening to identify inhibitors of the purine salvage pathway in Plasmodium falciparum

Abstract

Malaria, an infectious disease caused by protozoan parasites from the genus Plasmodium, represents a serious global health threat. The continued emergence of drug resistant strains has severely decreased current antimalarial drug efficacy and led to a perpetual race for drug discovery. Most protozoan parasites, including Plasmodium spp., are unable to synthesize purines de novo and instead rely on an essential purine salvage pathway for acquisition of purines from the infected host. Because purines are essential for Plasmodium growth and survival, the enzymes of the purine salvage pathway represent promising targets for drug discovery. Target-based high-throughput screening (HTS) assays traditionally focus on a single target, which severely limits the screening power of this type of approach. To circumvent this limitation, we have reconstituted the purine salvage pathway from Plasmodium falciparum in an assay combining four drug targets. This assay was developed for HTS and optimized to detect partial inhibition of any of the four enzymes in the pathway. Inhibitors of several enzymes in the pathway were identified in a pilot screen, with several compounds exhibiting effective inhibition when provided in micromolar amounts.

Keywords

• Purine salvage pathway
• Multiple targets
• High-throughput screening assay
• Pathway targeting