Oligomeric protein interference validates druggability of aspartate interconversion in Plasmodium falciparum

Abstract

The appearance of multi‐drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross‐reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging—particularly in the most dangerous malaria parasite, Plasmodium falciparum . Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.

New directions in antimalarial target validation

“…Therefore, interference with a nonfunctional subunit might have a significant effect on the biological activity of these enzymes, as previously demonstrated [124,125]. A recent study was carried out targeting two key-enzymes of the aspartate metabolism pathway, aspartate aminotransferase (PfAspAT, Figure 4) and malate dehydrogenase (PfMDH, Figure 5) [122]. Multiple factors led to PfAspAT and PfMDH to be considered good candidates for the application of PIA based-inhibition for the first time: both enzymes present a low degree of conservation for the residues that form the oligomeric interfaces, unlike the residues that compose the active-site [122,124] and both possess their active sites formed across the interface between 2 monomers, and thus are dependent on their oligomeric state to be fully active [124,126].…”

“…OPRT; orotidine 5ʹ-phosphate decarboxylase, ODCase), aspartate metabolism (aspartate aminotransferase, AspAT and aspartate transcarbamoylase, ATC), vitamin B6 biosynthesis (pseudo enzyme, Pdx1.2) and malate metabolism (malate dehydrogenase, MDH) among other carbon metabolism pathways [9,121]. The oligomeric Protein Interference Assay (PIA), our recently proposed validation technique, takes advantage of oligomerization by disrupting oligomeric interfaces using mutant subunits of the target candidates and causing a significant reduction in specific enzyme activity [10,122] (Figure 3(b,c)). The biomechanics of the assembly of oligomer subunits and evolution of oligomeric interfaces make PIA remarkably interesting.…”

“…A recent study was carried out targeting two key-enzymes of the aspartate metabolism pathway, aspartate aminotransferase (PfAspAT, Figure 4) and malate dehydrogenase (PfMDH, Figure 5) [122]. Multiple factors led to PfAspAT and PfMDH to be considered good candidates for the application of PIA based-inhibition for the first time: both enzymes present a low degree of conservation for the residues that form the oligomeric interfaces, unlike the residues that compose the active-site [122,124] and both possess their active sites formed across the interface between 2 monomers, and thus are dependent on their oligomeric state to be fully active [124,126]. Subsequently, in vitro experiments have demonstrated that the introduction of mutations on the oligomeric surfaces can impair the function of these enzymes and the mutant forms of both enzymes can form oligomers with the wild-type forms, also impairing their function [122].…”

“…Multiple factors led to PfAspAT and PfMDH to be considered good candidates for the application of PIA based-inhibition for the first time: both enzymes present a low degree of conservation for the residues that form the oligomeric interfaces, unlike the residues that compose the active-site [122,124] and both possess their active sites formed across the interface between 2 monomers, and thus are dependent on their oligomeric state to be fully active [124,126]. Subsequently, in vitro experiments have demonstrated that the introduction of mutations on the oligomeric surfaces can impair the function of these enzymes and the mutant forms of both enzymes can form oligomers with the wild-type forms, also impairing their function [122]. In short, endogenous expression of mutant forms of both these enzymes in transfected parasites demonstrated that, although the introduction of AspAT and MDH mutants alone did not result in significant effect on parasite proliferation in bloodstage cultures, the transfection of parasites with both plasmids resulted in a significant reduction in parasite proliferation in aspartate-limited media (used to mimic physiological conditions) [122].…”

“…Subsequently, in vitro experiments have demonstrated that the introduction of mutations on the oligomeric surfaces can impair the function of these enzymes and the mutant forms of both enzymes can form oligomers with the wild-type forms, also impairing their function [122]. In short, endogenous expression of mutant forms of both these enzymes in transfected parasites demonstrated that, although the introduction of AspAT and MDH mutants alone did not result in significant effect on parasite proliferation in bloodstage cultures, the transfection of parasites with both plasmids resulted in a significant reduction in parasite proliferation in aspartate-limited media (used to mimic physiological conditions) [122]. Aspartate is known to be the least common of all the amino acids available within the human serum, with measurements suggesting a concentration of < 20 µM [127].…”