ANALYSIS IN YEAST OF ANTIMALARIA DRUGS THAT TARGET THE DIHYDROFOLATE-REDUCTASE OF PLASMODIUM-FALCIPARUM

Pyrimethamine and cycloguanil are competitive inhibitors of the Plasmo dium enzyme dihydrofolate reductase (DHFR). They have been effective t reatments for malaria, but rapid selection of populations of the paras ite resistant to these drugs has compromised their effectiveness. Para sites resistant to either drug usually have point mutations in the dhf r gene, but the frequency of these mutations is unknown. To study drug resistance more effectively, we transferred the DHFR domain of the dh fr-thymidylate synthase gene from a drug-sensitive line of P. falcipar um to a strain of the budding yeast, Saccharomyces cerevisiae, that la cks endogenous DHFR activity. Expression of the P. falciparum dhfr is controlled by the yeast dhfr 5' and 3' regulatory regions and the hete rologous enzyme provided all of the functions of the yeast dhfr gene. These yeast were susceptible to pyrimethamine and cycloguanil at low c oncentrations 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 a nd cycloguanil (IC50 > 10(-6) M); resistance of the yeast depended on the dhfr allele they expressed. The experimental drug WR99210 efficien tly killed all three yeast strains (IC50 about 10(-8) M) but the pyr(R ) strains showed collateral hypersensitivity to drug. The yeast transf ormants 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. S uch compounds would be excellent candidates for development of treatme nts with a longer life in clinical practice. (C) 1997 Elsevier Science B.V.