The 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.
Using 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 (Pfdhfr51I,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).
We have generated a number of yeast strains that can be successfully harnessed for the rapid and selective identification of urgently needed anti-parasitic agents.
Parasites kill millions of people every year and leave countless others with chronic debilitating disease. These diseases, which include malaria and sleeping sickness, mainly affect people in developing countries. For this reason, few drugs have been developed to treat them. To make matters worse, many parasites are developing resistance to the drugs that are available. Thus, there is an urgent need to develop new drugs, but this is hampered by the fact that most parasites are difficult or impossible to grow in the laboratory. To address this, we have engineered baker's yeast to be dependent on the function of enzymes from either parasites or humans. In all, our engineered yeast constructs encompass six parasites (causing malaria, schistosomiasis, leishmaniasis, sleeping sickness, and Chagas disease) and three different enzymes that are known or potential drug targets. Further, we have increased yeast's sensitivity to drugs by deleting the gene for its major drug efflux pump. Because yeast is robust and easy to grow in the laboratory, we can use a robot to screen for drugs that will kill yeast dependent on a parasite enzyme, but not touch yeast dependent on the equivalent human enzyme.