Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay

Abstract

A cellular thermal shift assay (CETSA) protocol identifies and resolves antimalarial drug targets in P. falciparum. Defining drug targets in malaria Different classes of malaria drugs have been used for decades, even though the mechanisms of action have never been elucidated. To identify antimalarial drug targets, Dziekan et al. developed a protocol combining mass spectrometry and cellular thermal shift assay. The assay uses parasite lysate and intact infected red blood cells and was able to positively identify two known drug targets. They then investigated targets for quinine and mefloquine, revealing purine nucleoside phosphorylase as the top hit. Their results not only identify the likely target of these common drugs but also showcase an assay that could be widely used in antimalarial drug research. Mechanisms of action (MoAs) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need for a better resolved understanding of their MoAs and associated resistance mechanisms. In the present work, we implemented the cellular thermal shift assay coupled with mass spectrometry (MS-CETSA) for drug target identification in Plasmodium falciparum, the main causative agent of human malaria. We validated the efficacy of this approach for pyrimethamine, a folic acid antagonist, and E64d, a broad-spectrum cysteine proteinase inhibitor. Subsequently, we applied MS-CETSA to quinine and mefloquine, two important antimalarial drugs with poorly characterized MoAs. Combining studies in the P. falciparum parasite lysate and intact infected red blood cells, we found P. falciparum purine nucleoside phosphorylase (PfPNP) as a common binding target for these two quinoline drugs. Biophysical and structural studies with a recombinant protein further established that both compounds bind within the enzyme’s active site. Quinine binds to PfPNP at low nanomolar affinity, suggesting a substantial contribution to its therapeutic effect. Overall, we demonstrated that implementation of MS-CETSA for P. falciparum constitutes a promising strategy to elucidate the MoAs of existing and candidate antimalarial drugs.