Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate

Abstract

The unique methylerythritol phosphate pathway for isoprenoid biosynthesis is essential in most bacterial pathogens. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes a distinct thiamin diphosphate (ThDP)-dependent reaction to form DXP from D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate and represents a potential anti-infective drug target. We have previously demonstrated that the unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhibits selective inhibition of Escherichia coli DXP synthase over mammalian ThDP-dependent enzymes. Here, we report the selective inhibition by BAP against recombinant DXP synthase homologs from Mycobacterium tuberculosis, Yersinia pestis and Salmonella enterica. We also demonstrate antimicrobial activity of BAP against both Gram-negative and Gram-positive strains (including E. coli, S. enterica and Bacillus anthracis), and several clinically isolated pathogens. Our results suggest a mechanism of action involving inhibition of DXP synthase and show that BAP acts synergistically with established antimicrobial agents, highlighting a potential strategy to combat emerging resistance in bacterial pathogens.

Figure 1

DXP synthase catalyzes the first step in IDP/DMADP biosynthesis and represents a branch point in bacterial metabolism.

Figure 2

Proposed mechanism of selective inhibition of DXP synthase by butylacetylphosphonate.

Figure 3

Butylacetylphosphonate (BAP) inhibition of E. coli MG1655 growth is rescued by 1-deoxy-D-xylulose (DX), thiamin (Th) and E. coli DXP synthase (Dxs) overexpression. A) Fractional growth at 12.5 hours in the presence and absence of BAP and DX. Mean and standard error of four replicates are shown. ^*p = 0.022 (unpaired t test comparing DX treated and untreated cultures at 0.66 mM BAP). B) Fractional growth at 16 hours in the presence and absence of BAP and thiamin. Mean and standard error of four replicates is shown. ^***p = 0.0000258 (unpaired t test comparing thiamin treated and untreated cultures at 165 μM BAP). C) Growth curve over 16 hours, in the absence or presence of BAP and thiamin. D) Fractional growth at 16 hours determined for E. coli BL21 cells only, cells transformed with empty pET37b vector, vector expressing recombinant E. coli DXP synthase protein (dxs-pET37b), or vector expressing the catalytically inactive ⁽¹⁵⁾ E370A variant (E370A dxs-pET37b). Mean and standard error of four replicates is shown.