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

Elizabeth Bilsland, Pınar Pir, Alex Gutteridge, Alexander Johns, Ross D. King, Stephen G. Oliver, Timothy G. Geary (Editor)

Research output: Contribution to journalArticlepeer-review

30 Citations (SciVal)
139 Downloads (Pure)

Abstract

Background
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.

Methodology/Principal Findings
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).

Conclusions/Significance
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.
Original languageEnglish
Article numbere1320
JournalPLoS Neglected Tropical Diseases
Volume5
Issue number10
DOIs
Publication statusPublished - 04 Oct 2011

Keywords

  • Antiprotozoal Agents/isolation & purification
  • Drug Evaluation, Preclinical/methods
  • High-Throughput Screening Assays/methods
  • Humans
  • Organisms, Genetically Modified
  • Protozoan Proteins/biosynthesis
  • Pyrimethamine/pharmacology
  • Recombinant Proteins/biosynthesis
  • Saccharomyces cerevisiae/drug effects
  • Tetrahydrofolate Dehydrogenase/biosynthesis

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