Mutagenesis of dihydrofolate reductase from Plasmodium falciparum: analysis in Saccharomyces cerevisiae of triple mutant alleles resistant to pyrimethamine or WR99210.

Inhibitors of dihydrofolate reductase (DHFR) have been a mainstay of chemotherapy of falciparum malaria for >50 years. Unfortunately, point mutations in DHFR are the major cause of resistance to drugs of this class and mutations have rapidly diminished the clinical effectiveness of these drugs. We designed a simple yeast-based system to produce and analyze point mutations in the Plasmodium falciparum DHFR domain of the DHFR-thymidylate synthase gene that confers resistance to pyrimethamine (PM), the major antifolate currently used in malaria treatment, or to WR99210, an experimental antifolate. We used PCR mutagenesis, screened >1000 DHFR alleles that encoded functional enzymes and studied approximately 100 that were more resistant than a naturally occurring resistant allele (N51I and S108N). The IC(50) values for both drugs were determined for a subset of 44 alleles that carried only a single new mutation. Mutations that increased resistance to PM 10-100 fold (to >10(-4) M) were identified in three regions of the DHFR domain - around amino acids 50, 188 and 213. In contrast, mutations that caused WR-resistance were far less common and only conferred approximately 10-fold resistance (to approximately 10(-7) M). Even more interesting, only the mutations at 188 increased resistance to WR and mutations in the 213 and other regions either had no effect or actually increased sensitivity to WR. This collateral hypersensitivity raises the possibility that opposing selection for resistance/sensitivity to PM and WR might be used to slow selection of populations of P. falciparum resistant to antifolate treatment.