title: Analysis in yeast of antimalaria drugs that target the dihydrofolate reductase of Plasmodium falciparum.
abstract: Pyrimethamine and cycloguanil are competitive inhibitors of the Plasmodium enzyme dihydrofolate reductase (DHFR). They have been effective treatments for malaria, but rapid selection of populations of the parasite resistant to these drugs has compromised their effectiveness. Parasites resistant to either drug usually have point mutations in the dhfr gene, but the frequency of these mutations is unknown. To study drug resistance more effectively, we transferred the DHFR domain of the dhfr-thymidylate synthase gene from a drug-sensitive line of P. falciparum to a strain of the budding yeast, Saccharomyces cerevisiae, that lacks endogenous DHFR activity. Expression of the P. falciparum dhfr is controlled by the yeast dhfr 5' and 3' regulatory regions and the heterologous enzyme provided all of the functions of the yeast dhfr gene. These yeast were susceptible to pyrimethamine and cycloguanil at low concentrations that inhibit P. falciparum (IC50 about 10(-8) and 10(-7) M, respectively). Yeast expressing constructs with dhfr alleles from pyrimethamine-resistant strains were resistant to both pyrimethamine and cycloguanil (IC50 > 10(-6) M); resistance of the yeast depended on the dhfr allele they expressed. The experimental drug WR99210 efficiently killed all three yeast strains (IC50 about 10(-8) M) but the pyrR strains showed collateral hypersensitivity to drug. The yeast transformants carrying the drug-sensitive allele can now be screened quickly and quantitatively to identify new drugs or combinations of drugs and determine which drugs select resistant parasites least efficiently. Such compounds would be excellent candidates for development of treatments with a longer life in clinical practice.
authors.affiliation: Department of Genetics, University of Washington, Seattle 98195-7360, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Wooden
authors.forename: L H
authors.initials: LH
authors.lastname: Hartwell
authors.forename: B
authors.initials: B
authors.lastname: Vasquez
authors.forename: C H
authors.initials: CH
authors.lastname: Sibley
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D002738:Chloroquine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004351:Drug Resistance; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D017125:Genes, Protozoan; D010616:Phenanthrenes / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects* / Q000235:genetics; D014227:Triazines / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D005493:Folic Acid Antagonists; D010616:Phenanthrenes; D011994:Recombinant Proteins; D014227:Triazines; C006201:BRL 6231; D002738:Chloroquine; D013762:Tetrahydrofolate Dehydrogenase; C023768:halofantrine; D011739:Pyrimethamine