Unrecognized cyclic structure in bacterial folate biosynthesis pathway plays a key role in trimethoprim-sulfamethoxazole synergism.

Trimethoprim-sulfamethoxazole is a highly synergistic antimicrobial drug combination in that their combined activity is far greater than the sum of their individual activities. It was previously understood that this drug combination is synergistic because these two drugs inhibit sequential steps of bacterial folate biosynthesis pathway.

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Apr 20, 2018
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Trimethoprim-sulfamethoxazole is a highly synergistic antimicrobial drug combination in that their combined activity is far greater than the sum of their individual activities. It was previously understood that this drug combination is synergistic because these two drugs inhibit sequential steps of bacterial folate biosynthesis pathway.

We are molecular microbiologists who target bacterial metabolism to develop novel strategies to treat bacterial infections. If any drug combination can inhibit sequential steps in a linear biosynthetic pathway to produce synergistic activity like trimethoprim-sulfamethoxazole, it would be very easy to find novel synergistic antimicrobial combinations. One day, we discussed the rationale for this project with a graduate student whose expertise is mathematical modeling of biochemical systems. He told us that some of the antimicrobial drug interactions are predictable by mathematical modeling and showed us a paper 1. The paper performed mathematical analysis of two inhibitors that act on different sites within a metabolic pathway. Based on the analysis, when the two inhibitors target sequential steps of a linear metabolic pathway, they should not show synergistic activity. Then, why does the trimethoprim-sulfamethoxazole combination show strong synergistic activity? This was the motivation of our recent study published in Nature Communications.

We took a very careful look at the bacterial folate biosynthesis pathway and noticed that there is an unrecognized cyclic pathway structure connecting the downstream portion of the pathway to the upstream portion of the pathway via purine biosynthesis pathway (Figure).  We used genetic and biochemical approaches to demonstrate the pathway is not only connected on a pathway map, but also the pathway is functionally connected. Intriguingly, targets of trimethoprim and sulfamethoxazole are both within this cyclic structure of the pathway. To investigate if the cyclic structure of the pathway is essential for the synergy, we used another antimicrobial compound that targets a site within the folate biosynthesis pathway but located outside of the cyclic pathway structure.  When this compound was combined with trimethoprim or sulfamethoxazole, the combinations showed some extent of positive interaction but substantially less than the strong synergistic activity of the trimethoprim and sulfamethoxazole combination.

Why is the cyclic pathway structure essential for trimethoprim-sulfamethoxazole synergism? When two inhibitors act on sequential steps in a linear biosynthetic pathway, an inhibitor that targets upstream of the pathway potentiates the activity of the other inhibitor by limiting the metabolic flux toward the downstream of the pathway. In the case of trimethoprim-sulfamethoxazole, sulfamethoxazole targets the upstream and potentiates trimethoprim. However, this mono-potentiation is not sufficient to produce strongly synergistic activity. Our study revealed that the cyclic pathway structure allows trimethoprim to potentiate sulfamethoxazole by limiting the metabolic flux toward the folate precursor biosynthesis pathway. We propose cyclic mutual potentiation of the two drugs results in amplified depletion of folate that produces a strong synergism. We believe this finding tells us new ways to look for other drug combinations that will have similarly potent synergistic activity.

Bacterial folate pathway and targets of anti-folate compounds are shown on the left. Graphical representations of checkerboard assay are shown on the right. Synergy was assessed by calculating FICI. FICIm, minimum value of FICI in the tested combinations is shown. GTP, guanosine-5’-triphosphate. IMP, inosine 5'-monophosphate. DHPPP, dihydropterin pyrophosphate. PABA p-aminobenzoic acid. SMX, sulfamethoxazole. TMP, trimethoprim. MAC1739792,3,3-dichloro-1-(3-nitrophenyl) prop-2-en-1-one.

Written by Yusuke Minato and Anthony Baughn.

  1. Harvey, R. J. Interaction of two inhibitors which act on different enzymes of a metabolic pathway. J Theor Biol 74, 411-437 (1978).
  2. Zlitni, S., Ferruccio, L. F. & Brown, E. D. Metabolic suppression identifies new antibacterial inhibitors under nutrient limitation. Nat Chem Biol 9, 796-804, doi:10.1038/nchembio.1361 (2013).
Go to the profile of Yusuke Minato

Yusuke Minato

Research Associate, University of Minnesota Medical School

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