Structural Basis of the Promiscuous Inhibitor Susceptibility of Escherichia coli LpxC

Abstract

The LpxC enzyme in the lipid A biosynthetic pathway is one of the most promising and clinically unexploited antibiotic targets for treatment of multidrug-resistant Gram-negative infections. Progress in medicinal chemistry has led to the discovery of potent LpxC inhibitors with a variety of chemical scaffolds and distinct antibiotic profiles. The vast majority of these compounds, including the nanomolar inhibitors L-161,240 and BB-78485, are highly effective in suppressing the activity of E. coli LpxC (EcLpxC), but not divergent orthologs such as P. aeruginosa LpxC (PaLpxC) in vitro. The molecular basis for such promiscuous inhibition of EcLpxC has remained poorly understood. Here, we report the crystal structure of EcLpxC bound to L-161,240, providing the first molecular insight into L-161,240 inhibition. Additionally, structural analysis of the EcLpxC/L-161,240 complex together with the EcLpxC/BB-78485 complex reveals an unexpected backbone flipping of the Insert I βa-βb loop in EcLpxC in comparison with previously reported crystal structures of EcLpxC complexes with L-threonyl-hydroxamate-based broad-spectrum inhibitors. Such a conformational switch, which has only been observed in EcLpxC, but not in divergent orthologs such as PaLpxC, results in expansion of the active site of EcLpxC, enabling it to accommodate LpxC inhibitors with a variety of head groups, including compounds containing single (R- or S- enantiomers) or double substitutions at the neighboring Cα atom of the hydroxamate warhead group. These results highlight the importance of understanding inherent conformational plasticity of target proteins in lead optimization.

Crystal structure of A. aeolicus LpxC with bound product suggests alternate deacetylation mechanism

“…Analysis by Lee et al . suggested that Eco_LpxC exhibits significantly greater plasticity in the active site with greater potency for a variety of ligands in contrast to the relative potency against the LpxC enzyme from other pathogens . Ultimately, the consistency between Aae_LpxC structures suggested imposing constraints for modeling the reaction in this study was reasonable.…”

The Inhibition of Lipid A Biosynthesis—The Antidote Against Superbugs?

“…In 2002, British Biotech, now Vernalis, developed the sulfonamide BB-78485, which proved very active in vitro against E. coli (MIC = 1 μg mL −1 ) and having inhibitory potency against the E. coli LpxC enzyme in the nanomolar range (K i = 160 nm). [52] Importantly, comparison of the two E. coli structures with the corresponding one of the P. aeruginosa LpxC enzyme in complex with BB-78485 (PDB entry 2VES, [53] 1.9Å) revealed significant differences in the plasticity in the substrate-covering loop of the two enzymes that might account for their distinct inhibitory activity against P. aeruginosa versus E. coli. This lack of activity proved to be an issue, and the molecular bases that explain this poor efficacy in this pathogen were finally identified with the resolution of their enzyme/inhibitors complexes.…”

“…[54][55][56][57] The most relevant examples are: i) the 4-(phenylethynyl)benzamide derivatives LPC-004 and CHIR-090, [58,59] the 4-(phenylbuta-1,3-diyn-1-yl)benzamide derivatives LPC-009 and LPC-011, [60] all of which were developed by Chiron and the University of Washington; ii) the difluoromethyl derivative LPC-058 developed by C. -J. Lee et al; [61] iii) the pyridine methylsulfone LpxC-4 (PF-5081090), [62,63] which was discovered by Pfizer; and iv) the cyclopropane derivative ACHN-975 [56] developed by Achaogen (Figure 5). Diverse crystal structures of the LpxC enzyme from different species, including P. aeruginosa, [59,63,[65][66][67][68][69] Aquifex aeolicus, [67,[70][71][72][73][74][75] Yersinia enterocolitica [76] and E. coli, [52,77] have been solved, either as an apo-form or in complex mainly with inhibitors and these structures provide a good understanding at the molecular level of the differences in the inhibitory potency observed experimentally. The identification of these lead compounds was the result of the synthesis of a very large number of compounds in which mainly diverse moieties that mimic the fatty acid chain of the substrate were explored.…”

Structure, inhibition, and regulation of essential lipid A enzymes

“…Notably, it is inactive against P. aeruginosa . Such a lack of activity against P. aeruginosa LpxC appeared to be caused by the rigidity of the P. aeruginosa enzyme, whereas the E. coli enzyme is conformationally dynamic and can undergo a conformational switch that expands the active site to accommodate inhibitors with various head groups, including compounds with opposite stereo centers at the Cα position next to the hydroxamate group [46] ( Fig. 3E ).…”