NBTI 5463 Is a Novel Bacterial Type II Topoisomerase Inhibitor with Activity against Gram-Negative Bacteria and In Vivo Efficacy

Abstract

, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections. Gram-negative pathogens have become an increased focus for antibiotic development with the continued erosion of the efficacy of current therapies (1). Current options to treat Gramnegative infections are becoming alarmingly limited due to the organisms' abilities to evade existing antibiotic classes by employing a broad array of resistance mechanisms (2). Multidrug-resistant (MDR) Gram-negative bacteria represent important nosocomial pathogens and are responsible for a significant proportion of infections in patients in hospital and intensive care unit (ICU) settings (3). It is clear that additional agents effective against Gram-negative organisms, in particular Pseudomonas aeruginosa, are needed (4).The bacterial topoisomerases have proven to be very effective targets for the fluoroquinolone class of antibiotics (5, 6). Bacterial type II topoisomerases are enzymes that mediate transient double-strand DNA breaks and participate in DNA replication and decatenation reactions (7). DNA gyrase can introduce negative supercoils and controls the level of supercoiling in the bacterial chromosomal DNA (8, 9). Topoisomerase IV is most efficient in decatenating activity, and participates in daughter chromosome separation (10, 11). DNA gyrase is a heterotetramer composed of two copies of each of two protein subunits, GyrA and GyrB (12). Topoisomerase IV is similarly a tetramer of two homodimeric subunits, designated ParC and ParE (13). The fluoroquinolone antibiotics inhibit DNA replication by forming complexes of the drug with DNA bound to the topoisomerase enzyme. This complex acts as a poison for DNA replication, blocking the progression of the replication fork and subsequently inducing the formation of double-strand breaks in the chromosome (14). Despite clinical success, the utility of the fluoroquinolones has eroded over time with use, due primarily to point mutations in the two target enzymes, bacterial gyrase and topoisomerase IV, as well as drug efflux pump mechanisms (15).In this report, we describe the properties of a novel bacterial type II topoisomerase inhibitor (NBTI), NBTI 5463. Mechanistic studies with NBTIs have revealed that members of this class are similar to the fluoroquinolones in tha...

Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor

“…As previously reported, these compounds inhibit the two topoisomerase targets in the bacterial cell, gyrase and topoisomerase IV, in a manner distinct from that of the fluoroquinolone class, with inhibition not resulting in a cleavage complex (19,20). The NBTI compounds were also found to maintain MIC activity against strains with topoisomerase mutations that impair fluoroquinolone target inhibition (18). In the initial report, we identified first-step resistance mutations in Pseudomonas aeruginosa exclusively in the nfxB regulator gene controlling the expression of the MexCD, OprJ efflux pump system (21).…”

“…There have been several reports of novel, nonquinolone compounds that interact with bacterial type II topoisomerases gyrase and topoisomerase IV (TopoIV); however, these are almost exclusively active primarily against Grampositive pathogens, although one class of tricyclic compounds has broader activity (10)(11)(12)(13)(14)(15)(16)(17). We have recently reported on a chemical class of topoisomerase inhibitors with activity against several Gram-negative multidrug resistant pathogens; these compounds have been termed novel bacterial type II topoisomerase inhibitors (NBTI) (11,18). As previously reported, these compounds inhibit the two topoisomerase targets in the bacterial cell, gyrase and topoisomerase IV, in a manner distinct from that of the fluoroquinolone class, with inhibition not resulting in a cleavage complex (19,20).…”

Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

“…When there is success in attaining cellular bioactivity, close attention must be paid to overcoming intrinsic resistance mechanisms such as efflux and permeability. Such efforts have shown promise in recent years with the development of hydroxamate LpxC inhibitors (84)(85)(86), novel bacterial type II topoisomerase inhibitors (NBTIs) (87,88), tricyclic GyrB/ParE (TriBE) inhibitors (89), and pyrrolocytosine ribosome inhibitors (90). Although these classes of compounds have not yet reached clinical utility, they demonstrate progress and inspire confidence that novel Gram-negative antibiotics can be discovered.…”

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development

“…(Ehmann and Lahiri 2014) A follower of the same series, gepotidacin 45 (Mayer and Janin 2014), is in several clinical phase II trials investigating the treatment of respiratory tract infections caused by S. pneumoniae, acute bacterial skin and skin structure infections caused by S. aureus and uncomplicated urogenital gonorrhea (Paris 2015). Additionally, researchers at AstraZeneca reported further NTBIs showing potent activity against Gram-positive bacteria such as AZD9742 46 (Reck et al 2012) and NTBI 5463 47 (Dougherty et al 2014). The latter presents a 4-aminocyclohexyl bridge between the two aromatic regions with promising activity against Gram-negative pathogens.…”