Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase

Abstract

Objectives To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches. Methods Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863. Results Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive. Conclusions Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.

Activity of Tricyclic Pyrrolopyrimidine Gyrase B Inhibitor against Mycobacterium abscessus

“…Screening series of advanced TB actives against M. abscessus identified several compounds with in vivo activity, including inhibitors of RNA polymerase (7), ATP synthase (8), Leucyl-tRNA synthetase (9, 10), DNA gyrase (11) and DNA clamp DnaN (12). Expanding on this strategy, we asked whether the recently identified novel class of tricyclic pyrrolopyrimidines (TPPs (13)), targeting DNA gyrase in Mycobacterium tuberculosis and various other bacteria (14, 15) is active against M. abscessus .…”

“…Similar to SPR719, TPPs were shown to bind and inhibit the ATPase domain of the gyrase B subunit in M. tuberculosis (14). To determine whether this novel class of inhibitors is active against M. abscessus, the minimum inhibitory concentration (MIC) of a representative TPP compound (TPP8, compound #8 in (15) and Fig.…”

“…synthetase (9,10), DNA gyrase (11) and DNA clamp DnaN (12). Expanding on this strategy, we asked whether the recently identified novel class of tricyclic pyrrolopyrimidines (TPPs (13)), targeting DNA gyrase in Mycobacterium tuberculosis and various other bacteria (14,15) is active against M. abscessus.…”

“…Benzimidazoles target the ATPase domain of the DNA gyrase complex, located on its B subunits and required to drive the catalytic cycle (20), distinct from the fluoroquinolone binding site. Similar to SPR719, TPPs were shown to bind and inhibit the ATPase domain of the gyrase B subunit in M. tuberculosis (14). To determine whether this novel class of inhibitors is active against M. abscessus, the minimum inhibitory concentration (MIC) of a representative TPP compound (TPP8, compound #8 in (15) and Fig.…”

In Vitro Resistance against DNA Gyrase Inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus

“…1 ) belongs to a novel class of DNA gyrase inhibitor with an on-target mechanism of action distinct from the fluoroquinolones and not shared by currently marketed antibiotics. Targeting the ATPase domain of Gyrase B, the drug acts as competitive ATP binding inhibitor ( 6 – 9 ). This synthetic inhibitor is active against Mycobacterium tuberculosis and non-tuberculous mycobacteria, including M. avium and M. abscessus ( 6 , 10 , 11 ).…”