Interactions between Gepotidacin and Escherichia coli Gyrase and Topoisomerase IV: Genetic and Biochemical Evidence for Well-Balanced Dual-Targeting

Abstract

Antimicrobial resistance is a global threat to human health. Therefore, efforts have been made to develop new antibacterial agents that address this critical medical issue. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial in clinical development. Recently, phase III clinical trials for gepotidacin treatment of uncomplicated urinary tract infections caused by uropathogens, including Escherichia coli, were stopped for demonstrated efficacy. Because of the clinical promise of gepotidacin, it is important to understand how the compound interacts with its cellular targets, gyrase and topoisomerase IV, from E. coli. Consequently, we determined how gyrase and topoisomerase IV mutations in amino acid residues that are involved in gepotidacin interactions affect the susceptibility of E. coli cells to the compound and characterized the effects of gepotidacin on the activities of purified wild-type and mutant gyrase and topoisomerase IV. Gepotidacin displayed well-balanced dual-targeting of gyrase and topoisomerase IV in E. coli cells, which was reflected in a similar inhibition of the catalytic activities of these enzymes by the compound. Gepotidacin induced gyrase/topoisomerase IV-mediated single-stranded, but not double-stranded, DNA breaks. Mutations in GyrA and ParC amino acid residues that interact with gepotidacin altered the activity of the compound against the enzymes and, when present in both gyrase and topoisomerase IV, reduced the antibacterial activity of gepotidacin against this mutant strain. Our studies provide insights regarding the well-balanced dual-targeting of gyrase and topoisomerase IV by gepotidacin in E. coli.

Gyrase and Topoisomerase IV: Recycling Old Targets for New Antibacterials to Combat Fluoroquinolone Resistance

“…The NBTI LHS sits in a pocket in the DNA on the 2-fold axis of the complex, midway between the two DNA cleavage sites, and the RHS sits in a pocket on the 2-fold axis between the two GyrA subunits. ,,, One of the most important interactions between gyrase/topoisomerase IV and the NBTI is with an aspartic acid residue (Asp82 in E. coli GyrA), which forms a hydrogen bond with the basic nitrogen. NBTIs do not interact directly with the serine or acidic residues that are critical for fluoroquinolone binding. ,,, Even though the binding sites of fluoroquinolones and NBTIs do not overlap, it appears that both drugs cannot bind to the active site of gyrase/topoisomerase IV simultaneously. ,, …”

“…Because of the recent emergence of NBTIs as an antibacterial class with clinical potential, relatively little is known about resistance. However, mutations in gyrase/topoisomerase IV that decrease the sensitivity of laboratory strains and clinical isolates to this drug class have been reported. ,,− A prominent mutation occurs at the aspartic acid residue that has been shown to interact with NBTIs in structural studies. ,, The response of NBTIs to fluoroquinolone resistance mutations varies across compounds and species. Whereas moderate to high levels of resistance are observed in some drug-species combinations, in others, such as MGIs and M. tuberculosis gyrase, compounds are more potent and efficacious against enzymes that carry a fluoroquinolone resistance mutation. ,,,,, …”

“…As a result of their binding modality, NBTIs inhibit overall catalytic activity of gyrase and topoisomerase IV and act as topoisomerase poisons. ,,,− However, in contrast to fluoroquinolones, NBTIs enhance primarily single-stranded (as opposed to double-stranded) DNA breaks. ,,,,, Furthermore, in most cases, the binding of NBTIs actually suppresses the ability of gyrase/topoisomerase IV to generate double-stranded DNA breaks. ,,,, The mechanistic basis for the induction of single-stranded and suppression of double-stranded DNA breaks by NBTIs is not fully understood. However, it is believed that following cleavage of one DNA strand, the NBTI induces sufficient distortion in the active site of bacterial type II topoisomerases that it prevents these enzymes from cleaving the second strand. ,,, …”

“…As discussed above, a major drawback to fluoroquinolones is their unbalanced targeting of gyrase and topoisomerase IV, which has decreased the effectiveness of this drug class due to the development of clinical resistance. ,,, This drawback may be overcome by some members of the NBTI class. For example, the triazaacenaphthylene gepotidacin displays well-balanced, dual targeting against both type II enzymes in E. coli . ,, Whereas there is no loss of susceptibility to gepotidacin in strains that contain a resistance mutation in either gyrase or topoisomerase IV, strains that contain a mutation in both type II topoisomerases were found to be more than 100-fold less sensitive to the triazaacenaphthylene. , Thus, in at least some species, it appears that gepotidacin (and potentially other NBTIs) may be able to kill bacteria equally well through either gyrase or topoisomerase IV. It is assumed that this well-balanced dual-targeting of gyrase and topoisomerase IV will diminish the emergence of resistance and increase the clinical lifespan of gepotidacin and related compounds.…”

“… 13 , 18 , 72 , 218 Even though the binding sites of fluoroquinolones and NBTIs do not overlap, it appears that both drugs cannot bind to the active site of gyrase/topoisomerase IV simultaneously. 18 , 207 , 219 …”