Mass spectrometry technologies for measurement of cellular metabolism are opening new avenues to explore drug activity. Trimethoprim is an antibiotic that inhibits bacterial dihydrofolate reductase (DHFR). Kinetic flux profiling with 15 N-labeled ammonia in Escherichia coli reveals that trimethoprim leads to blockade not only of DHFR but also of another critical enzyme of folate metabolism: folylpoly-γ-glutamate synthetase (FP-γ-GS). Inhibition of FP-γ-GS is not directly due to trimethoprim. Instead, it arises from accumulation of DHFR's substrate dihydrofolate, which we show is a potent FP-γ-GS inhibitor. Thus, owing to the inherent connectivity of the metabolic network, falling DHFR activity leads to falling FP-γ-GS activity in a domino-like cascade. This cascade results in complex folate dynamics, and its incorporation in a computational model of folate metabolism recapitulates the dynamics observed experimentally. These results highlight the potential for quantitative analysis of cellular metabolism to reveal mechanisms of drug action.Enzyme inhibition is among the best established mechanisms of drug action 1 . Nevertheless, even for enzyme inhibitors, the mechanisms of action are often incompletely understood 2 . For example, drugs known to be inhibitors of one enzyme may further bind to unidentified enzymes or indirectly interact with other pathways. In part, this limited understanding is due to the scope and complexity of cellular chemical reactions as well as the associated difficulty of tracking many reactions at once. The recent development of technologies that can measure many cellular metabolites 3-5 and metabolic fluxes 6-8 in parallel may allow for more complete elucidation of actions of enzyme inhibitors. Here we apply LC-MS/MS to explore the effects of the DHFR inhibitor trimethoprim (1) in Escherichia coli by tracking both metabolite concentrations and fluxes throughout the folate pathway.Folates are cofactors that accept or donate one-carbon units for the biosynthesis of essential metabolites including purines, thymine (2), methionine (3) and glycine (4). Folate metabolism is the target of many therapeutics, including antibiotics (trimethoprim and sulfa drugs) 9-11 and anticancer agents (methotrexate, 5, and pemetrexed, 6) 12 . Folates are synthesized from GTP (7), p-aminobenzoic acid (pABA, 8) and glutamates (9) and can exist in three different oxidation/reduction states: PteGlu n (pteroylglutamate, or folate,
Despite the therapeutic importance of antifolates, the links between their direct antimetabolite activity and downstream consequences remain incompletely understood. Here we employ metabolomics to examine the complete metabolic effects of the antibiotic trimethoprim in E. coli. In rich media, trimethoprim treatment causes thymineless death. In minimal media, in contrast, trimethoprim addition results in rapid stoppage of cell growth and stable cell stasis. We show that initial impairment of cell growth is due to rapid depletion of glycine and associated activation of the stringent response. Long-term stasis is due to purine insufficiency. Thus, E. coli has dual systems for surviving folate depletion and avoiding thymineless death: a short-term response based on sensing of amino acids and a long-term response based on sensing of nucleotides.
Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using Ϫ75°C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly 13 C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena. (J Am Soc Mass Spectrom 2007, 18, 898 -909)
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