Analysis in yeast of antimalaria drugs that target the dihydrofolate reductase of Plasmodium falciparum.

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.

Temperature-sensitive mutants of yeast exhibiting a rapid inhibition of protein synthesis.

Certain temperature-sensitive (ts(-)) mutants of yeast which cannot be corrected by nutritional supplementation exhibited a rapid cessation of protein synthesis after a shift to the restrictive temperature. Genetic and biochemical tests permitted a division of these mutants into four classes. This division was based upon genetic complementation patterns among the mutants and an investigation of glucose incorporation into macromolecules and polyribosome content in the mutants after a shift to the restrictive temperature. A study of these parameters in the parent strain (ts(+)) in the presence of certain well-characterized inhibitors allowed a tentative identification of the biochemical defects in each of the four classes. The properties of the mutants in class IA were consistent with the hypothesis that they result from a defect in the initiation of polypeptide chains or in ribonucleic acid synthesis; mutants in class IB from a defect in the elongation of polypeptide chains; mutants in class IIA from a defect in energy metabolism; and mutants in class IIB from a lesion affecting membrane function.