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.
Highlights
Parasitic diseases such as malaria, schistosomiasis, leishmaniasis, sleeping sickness, and Chagas disease affect millions of people every year, leading to severe morbidity and death
Defining the drug targets We have defined our candidate drug targets based on the following criteria: (i) the target should be an enzyme that is essential in yeast; (ii) the target should be essential, or predicted to be essential, in most parasites; (iii) it may be present in human (a ‘humanized’ yeast strain will be used as a control in the screens); (iv) there should be a low similarity between human and parasite proteins; (v) the target should be one suggested by the TDR targets database
Complementation We have constructed a series of four plasmids containing the coding sequences for human dihydrofolate reductase (DHFR), phosphoglycerate kinases (PGKs), N-myristoyl transferases (NMTs), and FPS and transformed these into diploid yeast strains that are heterozygous deletion mutants for the gene encoding the corresponding essential enzyme, namely: DFR1 (ScDHFR), PGK1 (ScPGK), NMT1 (ScNMT) or ERG20 (ScFPS)
Summary
Parasitic diseases such as malaria, schistosomiasis, leishmaniasis, sleeping sickness, and Chagas disease affect millions of people every year, leading to severe morbidity and death. For example malaria, caused by parasites of the genus Plasmodium, kills 1–3 million people every year (http://www.who.int/mediacentre/factsheets/fs094/ en/index.html). 207 million people (http://www.who.int/mediacentre/factsheets/fs115/en/ index.html) [2], and causing the death of greater than 300,000 individuals per annum [3]. Sleeping sickness (or African trypanosomiasis), caused by Trypanosoma brucei, infects about 300,000 people each year leading to about 40,000 deaths [5,6]. Leishmaniasis, caused by Leishmania spp. is endemic in 88 countries affecting 12 million people (http://www.who.int/leishmaniasis/en/). It is traditionally treated with antimony compounds, but resistance to this class of drugs is increasing and very few novel drugs are under development. 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
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