A Yeast-Based Drug Discovery Platform To Identify Plasmodium falciparum Type II NADH Dehydrogenase Inhibitors

Abstract

Conventional methods utilizing in vitro protein activity assay or in vivo parasite survival to screen for malaria inhibitors suffer from high experimental background and/or inconvenience. Here we introduce a yeast-based system to facilitate chemical screen for specific protein or pathway inhibitors. The platform comprises several isogeneic Pichia strains that only differ in the target of interest, so that a compound which inhibits one strain but not the other is implicated in working specifically against the target. We used Plasmodium falciparum NDH2(PfNDH2), a type II NADH dehydrogenase, as a proof of principle to show how well this works. Three isogenic Pichia strains harboring respectively exogeneously introduced PfNDH2, its own complex I (a type I NADH dehydrogenase), and PfNDH2 with its own complex I were constructed. In a pilot screen of more than2000 compounds, we identified a highly specific inhibitor that acts on PfNDH2. This compound poorly inhibit the parasites at the asexual blood stage, however, is highly effective in repressing oocyst maturation in the mosquito stage. Our results demonstrate that the yeast cell based screen platform is feasible, efficient, economical and with very low background noise. Similar strategies could be extended to the functional screen for interacting molecules of other targets.

Plasmodium Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase

“…Various strategies can be employed to determine protein function, including single gene deletion, use of specific inhibitors, or the creation of recombinant systems in a model organism ( 9 , 25 , 26 ). Heterologous expression systems in nonpathogenic yeast have been applied to studies on eukaryotic proteins ( 27 ), including mitochondrial enzymes from P. falciparum ( 28 – 30 ), because of the following advantages: posttranslational modifications, expression in the appropriate organelles, fast and scalable growth, and easy genomic manipulation. Baker’s yeast, Saccharomyces cerevisiae , is one of the simplest eukaryotic model organisms and possesses three types of MDH (mitochondrial MDH1, cytosolic MDH2, and peroxisomal MDH3) ( 31 – 33 ), but not MQO.…”

An experimental target-based platform in yeast for screeningPlasmodium vivaxdeoxyhypusine synthase inhibitors

“…We have here established a S. cerevisiae surrogate genetics system for high-throughput screening to identify new inhibitors of P. vivax DHS. This approach was an advancement of previous yeast surrogate systems designed for target-selective inhibitor searches, circumventing challenges associated with in vitro growth of parasites [5,34,35]. Utilizing CRISPR-Cas9 homology-directed repair, we have incorporated new features including fluorescent markers and genetic integration of the orthologous DHS genes, greatly enhancing the platform's robustness and providing control over a wider range of target protein expression levels, using the weak regulatable MET3 promoter.…”

“…Target-based drug discovery campaigns typically rely on in vitro biochemical assays, which require protein purification and lack a cellular context [8,31,32]. Alternatively, designing systems with genetically modified S. cerevisiae strains can provide efficient platforms for target-based drug discovery within a eukaryotic cell [5][6][7][33][34][35].…”

The mystery of massive mitochondrial complexes: the apicomplexan respiratory chain

“…No known complex in myzozoans compensates for the proton-pumping ability of Complex I, and the effect of this potential loss on the proton-pumping capacity of the myzozoan mETC remains a question for future study. Humans do not have NDH2, and for this reason apicomplexan NDH2 has been the focus of drugdiscovery studies [23][24][25]. The events that led to the loss of Complex I in the myzozoan lineage are unknown, but one hypothesis points to a benefit in the reduction of superoxide generation [26].…”