Functional Expression of Parasite Drug Targets and Their Human Orthologs in Yeast

Abstract

BackgroundThe exacting nutritional requirements and complicated life cycles of parasites mean that they are not always amenable to high-throughput drug screening using automated procedures. Therefore, we have engineered the yeast Saccharomyces cerevisiae to act as a surrogate for expressing anti-parasitic targets from a range of biomedically important pathogens, to facilitate the rapid identification of new therapeutic agents.Methodology/Principal FindingsUsing pyrimethamine/dihydrofolate reductase (DHFR) as a model parasite drug/drug target system, we explore the potential of engineered yeast strains (expressing DHFR enzymes from Plasmodium falciparum, P. vivax, Homo sapiens, Schistosoma mansoni, Leishmania major, Trypanosoma brucei and T. cruzi) to exhibit appropriate differential sensitivity to pyrimethamine. Here, we demonstrate that yeast strains (lacking the major drug efflux pump, Pdr5p) expressing yeast (ScDFR1), human (HsDHFR), Schistosoma (SmDHFR), and Trypanosoma (TbDHFR and TcDHFR) DHFRs are insensitive to pyrimethamine treatment, whereas yeast strains producing Plasmodium (PfDHFR and PvDHFR) DHFRs are hypersensitive. Reassuringly, yeast strains expressing field-verified, drug-resistant mutants of P. falciparum DHFR (Pfdhfr 51I,59R,108N) are completely insensitive to pyrimethamine, further validating our approach to drug screening. We further show the versatility of the approach by replacing yeast essential genes with other potential drug targets, namely phosphoglycerate kinases (PGKs) and N-myristoyl transferases (NMTs).Conclusions/SignificanceWe have generated a number of yeast strains that can be successfully harnessed for the rapid and selective identification of urgently needed anti-parasitic agents.

The utility of yeast as a tool for cell-based, target-directed high-throughput screening

“…Using data from previous screening campaigns to remove non-specific hits, 39 compounds were selected for secondary selectivity and dose-dependency assays; this identified four compounds as potent and highly selective. One of these compounds induced high cAMP levels in a mammalian cell-based assay and the same compound induced cortisol production (associated with PDE11 inactivation) in these cells (Ceyhan et al 2012). Collectively these studies present the largest body of work in the literature describing the use of yeast for cell-based, target-directed HTS.…”

Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

“…Using yeast as a tool for drug screening against parasites is a strategy that has been successfully employed [5], [21], [75]. This system allows the identification of drugs acting specifically on the parasite enzyme since their effect on transfected yeast mutants growing in permissive and nonpermissive media can be compared (for a recent review, see [76]).…”

Pyrimidine metabolism in schistosomes: A comparison with other parasites and the search for potential chemotherapeutic targets

“…The enzyme from of P. lophurae differed from the host enzyme in greater molecular weight, pH optimum, substrate, cofactor specificity, stimulation by salts (Platzer, 1974b), and higher sensitivity to pyrimethamine inhibition (Platzer, 1974b; Bilsland et al, 2011). The schistosomal and filarial dihydrofolate reductases, on the other hand, closely resemble the enzyme from rat liver.…”

“…There is less of a difference between the apparent K NADPH of the parasite and rat liver enzymes. Both schistosomal and filarial dihydrofolate are less sensitive or equisensitive to inhibition by folate analogues than the mammalian enzyme (Jaffe, 1971; Jaffe et al, 1972; Bilsland et al, 2011). This suggests that it is unlikely that these compounds could be selectively toxic as antischistosomal or antifilarial agents.…”