Antimicrobial agents targeting early-stage isoprenoid biosynthesis: design, synthesis and evaluation of acetylphosphonate inhibitors of DXP synthase

The global threat of antibiotic resistant bacteria necessitates renewed efforts at designing antibiotics to tackle this healthcare challenge. Battling or overcoming this hazard will require the united efforts of chemists and microbiologists to discover new targets for antibacterial agents and design new molecular scaffolds to inhibit these unique targets. In addition to finding these new targets, scientists need to better understand the complicated, endogenous defenses of bacteria, including the up-regulation of promiscuous multi-drug resistant (MDR) efflux pumps. One potential new target for antibacterial development is early-stage isoprenoid biosynthesis via the methylerythritol phosphate (MEP) pathway essential to bacteria, parasites and some plants but absent in humans which instead utilize the orthogonal mevalonate pathway. The first catalytic step in the MEP pathway involves the condensation of pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) in a thiamin diphosphate (ThDP)-dependent manner, to generate 1-deoxy-D-xylulose 5-phosphate (DXP) and carbon dioxide. This initial step is catalyzed by the unique biosynthetic enzyme, DXP synthase, and the product represents a metabolic branch point as DXP is the precursor for isoprenoid, thiamin and pyridoxal biosynthesis in bacteria. The importance of these three pathways in bacterial growth and division highlights DXP synthase as an attractive target for antibacterial development. DXP synthase is promiscuous in reference to acceptor substrates and possesses a unique domain arrangement and catalytic mechanism, which involves formation of a ternary complex, suggesting that selective inhibition should be achievable by using unnatural bisubstrate analogs which mimic potential donor and acceptor substrates. Towards this goal, we successfully designed, synthesized and evaluated a series of acetylphosphonate inhibitors which interact with the ThDP cofactor to form a phosphonolactylthiamin diphosphate (PLThDP) intermediate, which cannot undergo decarboxylation to form product. Two compounds in this series, butylacetylphosphonate (BAP) and benzylacetylphosphonate (BnAP) were shown to be selective inhibitors of DXP synthase. Further, antimicrobial studies show that these compounds can weakly inhibit bacterial cell growth in complex media. Rescue experiments with downstream metabolites and target overexpression strains confirm that they restrict bacterial growth by a mechanism involving inhibition of DXP synthase. One potential contributing factor to their weak antimicrobial activity is their susceptibility to efflux by the E. coli MDR pump, AcrAB-TolC. We report an observed trend with the length of the alkyl chain of the acetylphosphonate and the potential for uptake and efflux towards defining the optimal properties for a future generation of potent antimicrobial agents targeting DXP synthase