Abstract
Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.
Highlights
The ever-present and rapidly growing threat of antibiotic-resistant bacteria, coupled with a significant void in antibiotic drug discovery, underscores the need for the continuing identification of new bacterial targets for the development of therapies with a novel mode of action
Crystal structures of enolase bound with SF2312 or KSF were generated through co-crystallization of the recombinant E. coli enolase with an excess amount of inhibitor and Mg2+ to ensure the stability of the protein-ligand complex
The combination of molecular dynamics (MD) simulations, crystal structures, biochemical and kinetic characterization, and antibacterial activity analysis has allowed us to elucidate the basis of E. coli enolase inhibition observed with SF2312 and its carbonyl substituted congener (KSF)
Summary
The ever-present and rapidly growing threat of antibiotic-resistant bacteria, coupled with a significant void in antibiotic drug discovery, underscores the need for the continuing identification of new bacterial targets for the development of therapies with a novel mode of action. A metalloenzyme involved in carbon metabolism, is essential to bacterial glycolysis and the generation of metabolites used in several bacterial cell processes Despite their critical nature, there has been little effort directed towards developing antibiotics targeting the glycolytic enzymes. While the individual isoforms are involved in numerous non-glycolytic biological processes, the expression of multiple human isoforms primarily provides redundancy for their role in glycolysis[4] The importance of this redundancy is highlighted by certain lineages of glioblastoma where the gene encoding ENO1 is collaterally deleted along with tumor suppressor genes during a translocation event. We identified a series of antimicrobial tropolones that target the E. coli enolase, supporting the hypothesis that inhibition of enolase can be an effective strategy for the development of new antibacterial agents[6] Further support for this approach was found with the naturally occurring antibiotic SF2312. The structural studies, corroborated by biochemical, molecular dynamics simulations and microbiological data indicate that SF2312 is a potent inhibitor of E. coli enolase, providing a paradigm for further optimization of its antibacterial properties as a combinatorial therapy with phosphonic acid antibiotics such as fosfomycin
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