Insights into the Mechanism of Inhibition of Novel Bacterial Topoisomerase Inhibitors from Characterization of Resistant Mutants of Staphylococcus aureus

Abstract

The type II topoisomerases DNA gyrase and topoisomerase IV are clinically validated bacterial targets that catalyze the modulation of DNA topology that is vital to DNA replication, repair, and decatenation. Increasing resistance to fluoroquinolones, which trap the topoisomerase-DNA complex, has led to significant efforts in the discovery of novel inhibitors of these targets. AZ6142 is a member of the class of novel bacterial topoisomerase inhibitors (NBTIs) that utilizes a distinct mechanism to trap the protein-DNA complex. AZ6142 has very potent activity against Gram-positive organisms, including Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. In this study, we determined the frequencies of resistance to AZ6142 and other representative NBTI compounds in S. aureus and S. pneumoniae. The frequencies of selection of resistant mutants at 4؋ the MIC were 1.7 ؋ 10 ؊8 for S. aureus and <5.5 ؋ 10 ؊10 for S. pneumoniae. To improve our understanding of the NBTI mechanism of inhibition, the resistant S. aureus mutants were characterized and 20 unique substitutions in the topoisomerase subunits were identified. Many of these substitutions were located outside the NBTI binding pocket and impact the susceptibility of AZ6142, resulting in a 4-to 32-fold elevation in the MIC over the wild-type parent strain. Data on cross-resistance with other NBTIs and fluoroquinolones enabled the differentiation of scaffold-specific changes from compound-specific variations. Our results suggest that AZ6142 inhibits both type II topoisomerases in S. aureus but that DNA gyrase is the primary target. Further, the genotype of the resistant mutants suggests that domain conformations and DNA interactions may uniquely impact NBTIs compared to fluoroquinolones.

Gepotidacin (GSK2140944) In Vitro Activity against Gram-Positive and Gram-Negative Bacteria

“…Molecular characterization to determine the mechanism of the reduced susceptibility of these isolates was not performed. Previously, Lahiri et al showed for S. aureus that after initial bacterial killing in time-kill kinetics, a partial rebound of growth occurred when testing a representative NBTI compound (6). They also noted variations in the frequency of spontaneous resistance when testing S. aureus against representative NBTIs and found that the mutations that occurred were in the gyrase subunit.…”

In Vitro Activity of Gepotidacin (GSK2140944) against Neisseria gonorrhoeae

“…Gepotidacin and other mechanistically related (but structurally different) NBTIs have a mode of action which differs from fluoroquinolones in that they bind to the DNA-protein complex at a location that is away from the cleavage site and do not form a cleavage complex (12,13). Since gepotidacin and NBTIs bind at a different location than fluoroquinolones, they have demonstrated in vitro activity against strains resistant to other antibacterial classes, including fluoroquinolones (7,8).…”

Discovery of Small‐Molecule Antibiotics against a Unique tRNA‐Mediated Regulation of Transcription in Gram‐Positive Bacteria

“…New approaches to combat antibiotic resistance include targeting the synthesis of aminoacyl‐tRNA synthetases (aaRS) and charging of tRNA, DNA replication, RNA transcription, the action of topoisomerases and riboswitches that carry out essential bacterial cell functions . Riboswitches are suitable targets for intervention as they have highly selected metabolite receptors located within the 5′‐untranslated region (5′UTR) of messenger RNAs (mRNA) controlling transcription or translation and are validated targets for intervention .…”

“…New approaches to combat antibiotic resistance include targeting the synthesis of aminoacyl-tRNA synthetases (aaRS) and chargingo ft RNA, DNA replication,R NA transcription,t he action of topoisomerases and riboswitches that carry out essentialb acterial cell functions. [10][11][12][13][14][15] Riboswitches are suitable targets for intervention as they have highly selected metabolite receptors located within the 5'-untranslated region (5'UTR) of messenger RNAs (mRNA) controlling transcription or translation [16] and are validated targets for intervention. [17,18] For example,a na nalogue of an atural metabolite could control expression of as ingle essential gene by outcompeting the metabolite or alteringt he RNA conformation and disrupting normalexpression patterns.…”