Structural and Functional Studies of Bacterial Enolase, a Potential Target Against Gram-Negative Pathogens.

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation, RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activity of α-, β- and γ- substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring plays an important role in the biological activity of the investigated compounds. Both α- and β- substituted phenyl derivatives of tropolone were the most active with MIC values in the range of 11-14 µg/ml. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring β-thujaplicin and α- and β- substituted phenyl derivatives of tropolones with IC50 values in range of 8 to 11 µM. Ligand binding parameters were assessed by ITC and DSC techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.