Relevance of Higher-Order Epistasis in Drug Resistance.

We studied five chemically distinct but related 1,3,5-triazine antifolates with regard to their effects on growth of a set of mutants in dihydrofolate reductase. The mutants comprise a combinatorially complete data set of all 16 possible combinations of four amino acid replacements associated with resistance to pyrimethamine in the malaria parasite Plasmodium falciparum. Pyrimethamine was a mainstay medication for malaria for many years, and it is still in use in intermittent treatment during pregnancy or as a partner drug in artemisinin combination therapy. Our goal was to investigate the extent to which the alleles yield similar adaptive topographies and patterns of epistasis across chemically related drugs. We find that the adaptive topographies are indeed similar with the same or closely related alleles being fixed in computer simulations of stepwise evolution. For all but one of the drugs the topography features at least one suboptimal fitness peak. Our data are consistent with earlier results indicating that third order and higher epistatic interactions appear to contribute only modestly to the overall adaptive topography, and they are largely conserved. In regard to drug development, our data suggest that higher-order interactions are likely to be of little value as an advisory tool in the choice of lead compounds.