Potent DNA gyrase inhibitors bind asymmetrically to their target using symmetrical bifurcated halogen bonds

Anja Kolarič,Nikola Minovski,Thomas Germe,Nicolas P. Burton,Marko Anderluh,Anthony Maxwell,Martina Hrast,Judit Vörös,David M. Lawson,Clare E. M. Stevenson

doi:10.1038/s41467-020-20405-8

Anja Kolarič, Nikola Minovski + Show 8 more

Open Access

https://doi.org/10.1038/s41467-020-20405-8

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Abstract

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase–DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.

Highlights

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now
By using cleavage assays with E. coli gyrase, we have proven that our compounds stabilize single-stranded cleavage complexes, as expected for NBTIs
A genuine breakthrough in understanding the mechanism of the NBTI mode of action was made possible by obtaining the crystal structure of one of our inhibitors (4) in a ternary complex with gyrase and a doubly nicked DNA fragment

Summary

Introduction

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. A Protein Data Bank (PDB)[2] query with the same search terms gives 190 different crystal structures and a very recent cryoelectron microscopic structure of the complete Escherichia coli DNA gyrase in a ternary complex with DNA and a small molecule GyrA inhibitor gepotidacin[3]. The reason for this popularity is clear: DNA gyrase is an essential bacterial type II topoisomerase that is involved in the maintenance of the correct spatial DNA topology in bacteria[4]. The NBTIs form a gyrase–DNA–inhibitor ternary complex (as demonstrated by Staphylococcus aureus DNA gyrase)[10] and have a somewhat similar intercalating mechanism of action to fluoroquinolones with a single inhibitor molecule bound centrally between the two scissile DNA bonds and in a pocket between the two GyrA subunits, as demonstrated for gepotidacin[10,11]

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