title: Functional expression of parasite drug targets and their human orthologs in yeast.
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.
  
  
  RESULTS
  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 ((Sc)DFR1), human ((Hs)DHFR), Schistosoma ((Sm)DHFR), and Trypanosoma ((Tb)DHFR and (Tc)DHFR) DHFRs are insensitive to pyrimethamine treatment, whereas yeast strains producing Plasmodium ((Pf)DHFR and (Pv)DHFR) DHFRs are hypersensitive. Reassuringly, yeast strains expressing field-verified, drug-resistant mutants of P. falciparum DHFR ((Pf)dhfr(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
  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.
authors: Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, United Kingdom.
authors: Elizabeth
authors: E
authors: Bilsland
authors: Pınar
authors: P
authors: Pir
authors: Alex
authors: A
authors: Gutteridge
authors: Alexander
authors: A
authors: Johns
authors: Ross D
authors: RD
authors: King
authors: Stephen G
authors: SG
authors: Oliver
mesh_terms: D000981:Antiprotozoal Agents / Q000302:isolation & purification* / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D030781:Organisms, Genetically Modified; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000981:Antiprotozoal Agents; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D011739:Pyrimethamine