Abstract

Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA (‘poisoning’) in an analogous fashion to fluoroquinolones, but without the requirement for the water–metal–ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase–DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases.

Highlights

  • Antibiotics are a cornerstone of modern medicine, underpinning modern clinical and ambulatory care

  • A DNA-cleavage assay performed with purified Escherichia coli DNA gyrase shows that the IPY t1 induces levels of double-stranded cleavage comparable to those obtained with ciprofloxacin (Figure 1)

  • We have characterized a new class of nonquinolone bacterial topoisomerase inhibitors, the IPYs, and show that they efficiently inhibit the supercoiling activity of purified DNA gyrase due to their ability to stabilize a cleavage complex

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Summary

Introduction

Antibiotics are a cornerstone of modern medicine, underpinning modern clinical and ambulatory care. The ability to prevent and treat infections has led to a huge drop in mortality and morbidity, and indirectly allowed the development of sophisticated surgical procedures. Few discoveries can claim to have had such an all-encompassing and far-reaching effect on human well being. The levels of antimicrobial resistance (AMR) worldwide are increasing [1,2] while recent decades have seen a paucity of new antibacterial compounds reaching the clinic [3] or approved for use [4]. The current perceived low return on investment in antibacterials does not encourage significant investment by industry. The discovery and optimization of new antibacterials is intrinsically challenging and clinical trials face challenges as well [5]

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