Optimization of physicochemical properties and safety profile of novel bacterial topoisomerase type II inhibitors (NBTIs) with activity against Pseudomonas aeruginosa

Self Cite

), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4-to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16-and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

Section: Discussionmentioning

confidence: 93%

“…The NBTI series of compounds, including NBTI 5463, was developed with the goal of improved Gram-negative pathogen coverage and minimizing the potential for hERG cardiac channel inhibition (30). Earlier compounds with chemical similarities to the NBTI series were focused primarily on Gram-positive antibacterial activities (10-16).…”

Section: Discussionmentioning

confidence: 99%

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

Nayar

Dougherty

Reck

et al. 2015

Self Cite

“…The addition of a fluorine atom in the southern group of REDX07623 appears to increase hERG affinity in comparison to that in its matched pair REX06276 with IC 50 s of 8.2 and Ͼ33 M, respectively. Introduction of more polar groups to reduce the logD of NBTI compounds has been shown to be associated with reduced hERG inhibition (22). REDX06181 displayed the lowest logD of the compounds tested and showed the most attenuated hERG inhibition with an IC 50 of Ͼ100 M. However, its antibacterial potency was reduced compared to that of other compounds with a higher logD, such as REDX07623.…”

Section: Discussionmentioning

confidence: 99%

“…A number of advanced NBTI molecules have been described in the literature, including NXL101 (16), AZD9742 (17), NBTI 5463 (18), and gepotidacin (19), which recently successfully completed phase II human clinical evaluation for the treatment of uncomplicated urogenital gonorrhea caused by Neisseria gonorrhoeae (ClinicalTrials registration number NCT02294682). The NBTI pharmacophore, however, has been associated with cardiovascular and other safety liabilities (17,(20)(21)(22)(23). Therefore, a key aim in the development of NBTIs is achieving broad antibacterial potency, including against challenging Gram-negative pathogens, while maintaining satisfactory safety margins.…”

mentioning

confidence: 99%

Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria

Charrier

Salisbury

Savage

et al. 2017

The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the in vitro properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of Escherichia coli DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Grampositive and Gram-negative pathogens, including biodefence microorganisms and Mycobacterium tuberculosis. No cross-resistance with fluoroquinolone-resistant Staphylococcus aureus and E. coli isolates was observed. Measured MIC 90 values were 4 and 8 g/ml against a panel of contemporary multidrug-resistant isolates of Acinetobacter baumannii and E. coli, respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically Ͻ10 Ϫ8 against E. coli and A. baumannii at concentrations equivalent to 4-fold the MIC. Compound-resistant E. coli mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary in vitro safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC 50 ], Ͼ100 M). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable in vitro safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents.

“…Responsible for slow nonspecific diffusion of solutes into the bacterium, it has been shown that OprF folds into two conformations, with less than 5% of the porin present in the openchannel conformer, severely limiting penetration of molecules into the bacterium (50). It is for this multitude of reasons that often agents which are active on most other Gram-negative bacteria are less effective on Pseudomonas aeruginosa (13,(51)(52)(53), and finding compounds that inhibit this organism can be difficult. Again, it is imperative to understand the roles efflux and permeability play in the intrinsic resistance of this organism when developing the next generation of antibiotics.…”

mentioning

confidence: 99%

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

Balibar

Grabowicz

2016

bGram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens. With emerging multidrug resistance and a limited number of treatment options, bacterial infections pose an ever growing threat to human health. Gram-negative bacteria are particularly recalcitrant to antimicrobial intervention due to their cell envelope structure. Possessing two membranes with different chemical properties (1) and containing an arsenal of efflux pumps (2), Gram-negative bacteria are capable of both excluding and expelling molecules, rendering them resistant to many drugs. Despite major Gram-negative pathogens being classified as urgent or serious threats by the CDC (3), no Gram-negative-active antibiotic with a new mechanism of action has been introduced in over 40 years. Of the 28 new antibiotics approved since 2000, only 18 are indicated for treatment of Gram-negative bacteria (4), and all of those are derived from the four legacy classes ␤-lactams, tetracyclines, macrolides, and fluoroquinolones, which were first introduced in 1942 (5), 1948 (6), 1952 (7), and 1967 (8), respectively. Novel derivatives of old antibiotics often have limited spectra of coverage and can only do so much to overcome existing mechanisms of resistance; therefore, introduction of novel classes of antibiotics is critical.There is no shortage of potential targets in the antibacterial space given that the essential gene set for several Gram-negative pathogens has been defined (9-12). However, despite genetically demonstrating target essentiality, cognate inhibitors with exquisite in vitro activity often have no measurable cellular activity (13,14). The reasons for this can be attributed to a lack of penetration of compounds into bacteria, efflux of molecules out of bacteria, insufficient inhibition of the target, metabolism within the cells, or a combination of the aforementioned factors. Understanding the reason for failure to translate enzymatic 50% inhibitory concentrations (IC 50 s) into cellular MICs is paramou...