Since 1990 mortality from Plasmodium falciparum malaria has increased, due primarily to the spread of drug-resistant parasites. Although antifolates are affordable and still clinically effective, their use has been compromised by the evolution of drug resistance. Resistance results from a small number of amino acid replacements in two enzymes for folate synthesis: dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). An understanding of the likely order in which these mutations were selected, as well as the resistance phenotypes of the intermediates and the biophysical basis of the resistance, would be important in prolonging the useful therapetic lifetime of antifolates and in developing novel and more effective antifolates. We have developed and verified a method for inferring the likely sequence of events in the evolution of drug resistance. The method also reveals the molecular mechanisms of resistance. We construct all possible combinations of relevant mutations, and assay each allele for its effect on the drug resistance phenotype of the organism and for its biochemical and biophysical properties. These data reveal the probability of each individual step in all possible genealogies of drug resistance, and hence the overall probability of each evolutionary pathway. Our previous work in another experimental system indicates that most of the probability comes from a small number of evolutionary pathways. Our experimental approach is ideally suited for dissecting the evolutionary pathways and biophysical basis'of the multiple antifolate mutations in DHFR. We therefore propose to construct all possible combinations of clinically important P. falciparum DHFR mutations. The resistance phenotype of each mutant combination will be assayed using the expression system in S. cerevisiae, and the biochemical and biophysical properties of each protein determined. Based on these data, a systems-level model will be developed that relates increased drug resistance to the biochemical and biophysical properties of the evolutionary intermediates. For future work, we will also take steps to develop an analogous DHPS system. This research has the potential to improve methods of resistance surveillance in the field, to provide basic information required for improved epidemiological modeling and drug deployment protocols, and to contribute fundamental insights1- into the rational design of novel antifolate drugs. LAY LANGUAGE SUMMARY: Drug-resistant malaria is an ever increasing problem. This year about 1.5 million African children will die from the disease. This research project will uncover why certain mutations in a particular gene contribute to resistance to a key affordable class of antimalaria drugs, the antifolates. The research may prolong the effectiveness of present antifolates, and help in the development of new ones.