Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase.

1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate. DXP is at a metabolic branch point in bacteria, feeding into the methylerythritol phosphate pathway to indispensable isoprenoids and acting as a precursor for biosynthesis of essential cofactors in central metabolism, pyridoxal phosphate and ThDP, the latter of which is also required for DXP synthase catalysis. DXP synthase follows a unique random sequential mechanism and possesses an unusually large active site. These features have guided the design of sterically demanding alkylacetylphosphonates (alkylAPs) toward the development of selective DXP synthase inhibitors. alkylAPs studied here display selective, low µM inhibitory activity against DXP synthase. They are weak inhibitors of bacterial growth in standard nutrient rich conditions. However, bacteria are significantly sensitized to most alkylAPs in defined minimal growth medium, with minimal inhibitory concentrations (MICs) ranging from low µM to low mM and influenced by alkyl-chain length. The longest analog (C8) displays the weakest antimicrobial activity and is a substrate for efflux via AcrAB-TolC. The dependence of inhibitor potency on growth environment emphasizes the need for antimicrobial screening conditions that are relevant to the in vivo microbial microenvironment during infection. DXP synthase expression and thiamin supplementation studies offer support for DXP synthase as an intracellular target for some alkylAPs and reveal both the challenges and intriguing aspects of these approaches to study target engagement.

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

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.

Selective inhibition of E. coli 1-deoxy-D-xylulose-5-phosphate synthase by acetylphosphonates().

DXP synthase catalyzes the formation of 1-deoxy-D-xylulose 5-phosphate, an essential precursor in pathogen isoprenoid biosynthesis. The selective inhibition of this ThDP-dependent transformation is a challenging goal in the development of isoprenoid biosynthesis inhibitors. Potent, selective inhibitors could lead to new anti-infective agents. Here, we demonstrate selective inhibition of E. coli DXP synthase by butylacetylphosphonate.

Oligo(ethylene oxide) self-assembled monolayers with self-limiting packing densities for the inhibition of nonspecific protein adsorption.

We have created a molecule that forms self-assembled monolayers (SAMs) on Au, possessing the characteristics for inhibition of nonspecific protein adsorption, i.e., uniformly distributed, loosely packed, conformationally mobile, hydrated ethylene oxide (EO) chains of near optimal packing densities. SAMs of the bipodal molecule CH(3)O(CH(2)CH(2)O)(5)CH(2)CON(CH(2)CH(2)CH(2)SCOCH(3))(2) [N,N-(bis-3'-thioacetylpropyl)-3,6,9,12,15,18-hexaoxanonadecanamide (BTHA)] on polycrystalline Au are described. Spectroscopic ellipsometry (SE) and reflection-absorption infrared spectroscopy data indicate that BTHA SAM thickness and EO chain disorder closely match that of partially formed monothio-(EO)(5-6)CH(3) SAMs when they exhibit maximum inhibition of protein adsorption. However, in contrast to the monothio-(EO)(5-6)CH(3) SAMs, the BTHA SAM thickness and EO chain disorder remain constant in the presence of unbound molecules because of the structurally imposed upper limit of one EO chain per two Au occupancy sites. SE data indicate high resistance to protein adsorption for bovine serum albumin, fibrinogen, and a mixture of the two, suggesting uniform EO surface coverage on a length scale at least equal to the smallest dimension of these proteins.