Abstract
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 15N-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.
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Acknowledgements
This research was supported by the US National Institutes of Health (NIH) Center for Quantitative Biology at Princeton University (P50GM071508). Additional support came from the Beckman Foundation, the US National Science Foundation (NSF) Dynamic Data Driven Applications Systems grant CNS-0540181, the American Heart Association grant 0635188N, NSF Career Award MCB-0643859 and the NIH grant AI078063 (to J.D.R.).
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Y.K.K. and J.D.R. designed experiments, analyzed data and wrote the paper. W.L. developed the LC-MS/MS method. E.M. wrote the computer code. N.K. and A.B. contributed to biochemical assays of FP-γ-GS activity. A.B. edited the paper.
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Kwon, Y., Lu, W., Melamud, E. et al. A domino effect in antifolate drug action in Escherichia coli. Nat Chem Biol 4, 602–608 (2008). https://doi.org/10.1038/nchembio.108
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DOI: https://doi.org/10.1038/nchembio.108
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