Metabolomic investigation of trimethoprim's drug action

Antifolates play a major role in the treatment of various diseases such as cancer, autoimmune disorders, and bacterial infections. In this thesis, we explore the mechanism of action of antifolates using the antimicrobial drug trimethoprim, an inhibitor of bacterial dihydrofolate reductase (DHFR), a key enzyme in the folate pathway. We develop a method to detect the full spectrum of intracellular folates using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To determine absolute levels of selected folates in Escherichia coli (E. coli), we apply an isotope-ratio-based approach: intracellular folate concentrations are determined by growing cells in media containing uniformly labeled 13 C-glucose and spiking purified, unlabeled folate standards into folate extracts. Next, we apply kinetic flux profiling to measure folate fluxes, and combine flux and concentration measurements to investigate the effects of trimethoprim on folate metabolism. We find that trimethoprim leads to blockade not only of the target enzyme DHFR, but also of another critical enzyme of folate metabolism, folylpoly-gamma-glutamate synthetase (FP-gamma-GS). FP-gamma-GS activity is not inhibited directly by trimethoprim but by the accumulation of the DHFR-substrate dihydrofolate following DHFR-inhibition. We support these findings with in vitro assays and a computational model of folate metabolism. Finally, we examine the dynamics of the metabolome following trimethoprim treatment in exponentially growing E. coli. Trimethoprim is bactericidal in rich media but merely leads to bacteriostasis in minimal media. We find that in minimal media, trimethoprim treatment rapidly depletes intracellular glycine, which stops cell growth through activation of the stringent response. Purines are slowly depleted, inducing a cellular metabolic state resembling phosphate starvation, a natural stress which bacteria have evolved to survive. These results highlight the application of full-spectrum metabolomic and kinetic flux profiling technologies to holistically understand drug action.