#23: Investigation of Phosphomannomutase as an Antimalarial Drug Target

Abstract

Abstract Background Malaria continues to pose an enormous economic and global health threat, killing over 200,000 people annually, primarily children under the age of 5. With the constant barrier of antimalarial resistance and the rise of delayed clearance by artemisinin, it is especially important to identify drug/target pairs that rapidly kill parasites. We study targetable metabolic pathways in the malaria parasite, Plasmodium falciparum, to guide such future drug development against this disease. In recent years, we have discovered that a large family of hydrolases, the Haloacid Dehalogenase (HAD) Superfamily of proteins, are implicated in regulating a variety of P. falciparum metabolic pathways, which can lead to dramatic changes in central carbon metabolism and drug resistance. We now turn our attention to a related HAD protein, the putative phosphomannomutase in these parasites, HAD5, responsible for the interconversion of mannose-6-phosphate and mannose-1-phosphate. This is an essential process for all stages of the parasite, and thus has potential as a broad antimalarial target. We examined the role of HAD5 in these parasites, and its potential to be chemically inhibited. Methods Recombinant protein was generated and purified for enzymatic assays to determine HAD5 activity and test inhibitor potency against HAD5 compared to recombinant human orthologs, PMM1 and PMM2. In parallel, CRISPR/Cas9 was used to generate inducible knockdown parasite strains to demonstrate this gene’s essentiality and its role in parasite biology. Parasite growth was measured by flow cytometry and light microscopy. Immunofluorescence analysis (IFA) was used to track the parasite development on a molecular scale. Results Inhibition of HAD5 was achieved in biochemical assays, with an IC50 of 68µM in our most potent compound, representing roughly 10-fold increased potency against the parasite protein compared to human orthologs. In culture, knockdown of HAD5 leads to interrupted egress from and reinvasion into red blood cells, culminating in parasite death. In IFA-visualized parasites, reinvasion-facilitating proteins were no longer anchored to parasite surfaces, accounting for the inhibition of the parasite life cycle. Conclusion In the search for new antimalarial targets, identifying proteins that are essential across multiple parasite life-stages while being distinct from human orthologs is necessary to block parasite transmission, cure symptomatic infection, and minimize off-target effects. HAD5 is an essential protein in malaria parasites that is expressed throughout the parasite’s life cycle, and can be specifically targeted by inhibitors, giving it promise as a future drug target.