Discovery and structure-activity relationships of a novel oxazolidinone class of bacterial type II topoisomerase inhibitors.

There is an increasingly urgent and unmet medical need for novel antibiotic drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. Novel bacterial type II topoisomerase inhibitors (NBTIs) are of high interest due to limited cross-resistance with fluoroquinolones, however analogues with Gram-negative activity often suffer from hERG channel inhibition. A novel series of bicyclic-oxazolidinone inhibitors of bacterial type II topoisomerase were identified which display potent broad-spectrum anti-bacterial activity, including against MDR strains, along with an encouraging in vitro safety profile. In vivo proof of concept was achieved in a A. baumannii mouse thigh infection model.

Design, synthesis and antibacterial properties of pyrimido[4,5-b]indol-8-amine inhibitors of DNA gyrase.

According to the World Health Organization (WHO), approximately 1.7 million deaths per year are caused by tuberculosis infections. Furthermore, it has been predicted that, by 2050, antibacterial resistance will be the cause of approximately 10 million deaths annually if the issue is not tackled. As a result, novel approaches to treating broad-spectrum bacterial infections are of vital importance. During the course of our wider efforts to discover unique methods of targeting multidrug-resistant (MDR) pathogens, we identified a novel series of amide-linked pyrimido[4,5-b]indol-8-amine inhibitors of bacterial type II topoisomerases. Compounds from the series were highly potent against gram-positive bacteria and mycobacteria, with excellent potency being retained against a panel of relevant Mycobacterium tuberculosis drug-resistant clinical isolates.

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

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 Gram-positive 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 MIC90 values were 4 and 8 µg/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 <10-8 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 [IC50], >100 µM). 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.

Discovery and structure-activity relationships of a novel isothiazolone class of bacterial type II topoisomerase inhibitors.

There is an urgent and unmet medical need for new antibacterial drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. During the course of our wider efforts to discover and exploit novel mechanism of action antibacterials, we have identified a novel series of isothiazolone based inhibitors of bacterial type II topoisomerase. Compounds from the class displayed excellent activity against both Gram-positive and Gram-negative bacteria with encouraging activity against a panel of MDR clinical Escherichia coli isolates when compared to ciprofloxacin. Representative compounds also displayed a promising in vitro safety profile.

In vitro biological evaluation of novel broad-spectrum isothiazolone inhibitors of bacterial type II topoisomerases.

OBJECTIVES: To evaluate the in vitro biological properties of a novel class of isothiazolone inhibitors of the bacterial type II topoisomerases. METHODS: Inhibition of DNA gyrase and topoisomerase IV activity was assessed using DNA supercoiling and decatenation assays. MIC and MBC were determined according to CLSI guidelines. Antibacterial combinations were assessed using a two-dimensional chequerboard MIC method. Spontaneous frequency of resistance was measured at various multiples of the MIC. Resistant mutants were generated by serial passage at subinhibitory concentrations of antibacterials and genetic mutations were determined through whole genome sequencing. Mammalian cytotoxicity was evaluated using the HepG2 cell line. RESULTS: Representative isothiazolone compound REDX04957 and its enantiomers (REDX05967 and REDX05990) showed broad-spectrum bactericidal activity against the ESKAPE organisms, with the exception of Enterococcus spp., as well as against a variety of other human bacterial pathogens. Compounds retained activity against quinolone-resistant strains harbouring GyrA S83L and D87G mutations (MIC ≤4 mg/L). Compounds inhibited the supercoiling activity of wild-type DNA gyrase and the decatenation function of topoisomerase IV. Frequency of resistance of REDX04957 at 4× MIC was <9.1 × 10(-9). Against a panel of recent MDR isolates, REDX05967 demonstrated activity against Acinetobacter baumannii with MIC50 and MIC90 of 16 and 64 mg/L, respectively. Compounds showed a lack of cytotoxicity against HepG2 cells at 128 mg/L. CONCLUSIONS: Isothiazolone compounds show potent activity against Gram-positive and -negative pathogens with a dual targeting mechanism-of-action and a low potential for resistance development, meriting their continued investigation as broad-spectrum antibacterial agents.

Efficacy of a Novel Tricyclic Topoisomerase Inhibitor in a Murine Model of Neisseria gonorrhoeae Infection.

There is an urgent need for new antibiotics to treat multidrug-resistant Neisseria gonorrhoeae In this report, the microbiology, in vivo pharmacokinetics, and efficacy of REDX05931, a representative novel tricyclic topoisomerase inhibitor, were evaluated. REDX05931 demonstrated high oral bioavailability in mice and reduced N. gonorrhoeae infection after a single dose in a mouse model of gonorrhea. These data support the potential of this series of small molecules as a new treatment for drug-resistant gonorrheal infections.

Biological profiling of novel tricyclic inhibitors of bacterial DNA gyrase and topoisomerase IV.

OBJECTIVES: The objective of this study was to characterize the in vitro and in vivo biological properties of a novel series of small-molecule bacterial type IIA topoisomerase inhibitors. METHODS: Bacterial susceptibility testing was performed by broth microdilution. Resistance frequencies were determined by plating bacteria onto agar containing test compound and enumerating mutants. Bacteria were passaged using subinhibitory concentrations of antibacterials to generate resistance. Target enzyme inhibition was determined by exposure to antibacterials and DNA; topoisomers were visualized by gel electrophoresis. Oral and intravenous pharmacokinetic profiles were determined in mice. In vivo efficacy was determined using a mouse model of septicaemia and thigh infection with MSSA and MRSA, respectively. RESULTS: Representative compounds REDX04139, REDX05604 and REDX05931 demonstrated in vitro potency against a range of Gram-positive and fastidious Gram-negative pathogens. Clinical isolate testing revealed REDX04139 and REDX05931 had MIC90 values of 0.25 and 0.5 mg/L, respectively, for MRSA and MIC90 values of 2 mg/L for streptococci. REDX04139 was bactericidal in vitro against Staphylococcus aureus at 8× MIC over 6 h. Pharmacokinetic profiling of REDX04139 and REDX05604 in mice revealed low clearance and excellent bioavailability (≥71%). REDX04139 provided 100% survival against S. aureus in a mouse septicaemia model, while REDX05604 reduced bacterial load by up to 3.7 log units in the MRSA mouse thigh infection model. CONCLUSIONS: Redx Pharma has discovered a novel series of topoisomerase inhibitors that are being further developed for drug-resistant bacteria.

Discovery and in vivo evaluation of alcohol-containing benzothiazoles as potent dual-targeting bacterial DNA supercoiling inhibitors.

A series of dual-targeting, alcohol-containing benzothiazoles has been identified with superior antibacterial activity and drug-like properties. Early lead benzothiazoles containing carboxylic acid moieties showed efficacy in a well-established in vivo model, but inferior drug-like properties demanded modifications of functionality capable of demonstrating superior efficacy. Eliminating the acid group in favor of hydrophilic alcohol moieties at C(5), as well as incorporating solubilizing groups at the C(7) position of the core ring provided potent, broad-spectrum Gram-positive antibacterial activity, lower protein binding, and markedly improved efficacy in vivo.

Design, synthesis and structure-activity relationships of substituted oxazole-benzamide antibacterial inhibitors of FtsZ.

The design, synthesis and structure-activity relationships of a series of oxazole-benzamide inhibitors of the essential bacterial cell division protein FtsZ are described. Compounds had potent anti-staphylococcal activity and inhibited the cytokinesis of the clinically-significant bacterial pathogen Staphylococcus aureus. Selected analogues possessing a 5-halo oxazole also inhibited a strain of S. aureus harbouring the glycine-to-alanine amino acid substitution at residue 196 of FtsZ which conferred resistance to previously reported inhibitors in the series. Substitutions to the pseudo-benzylic carbon of the scaffold improved the pharmacokinetic properties by increasing metabolic stability and provided a mechanism for creating pro-drugs. Combining multiple substitutions based on the findings reported in this study has provided small-molecule inhibitors of FtsZ with enhanced in vitro and in vivo antibacterial efficacy.

Design, synthesis and biological evaluation of α-substituted isonipecotic acid benzothiazole analogues as potent bacterial type II topoisomerase inhibitors.

The discovery and optimisation of a new class of benzothiazole small molecules that inhibit bacterial DNA gyrase and topoisomerase IV are described. Antibacterial properties have been demonstrated by activity against DNA gyrase ATPase and potent activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes and Haemophilus influenzae. Further refinements to the scaffold designed to enhance drug-likeness included analogues bearing an α-substituent to the carboxylic acid group, resulting in excellent solubility and favourable pharmacokinetic properties.

Biological evaluation of benzothiazole ethyl urea inhibitors of bacterial type II topoisomerases.

The type II topoisomerases DNA gyrase (GyrA/GyrB) and topoisomerase IV (ParC/ParE) are well-validated targets for antibacterial drug discovery. Because of their structural and functional homology, these enzymes are amenable to dual targeting by a single ligand. In this study, two novel benzothiazole ethyl urea-based small molecules, designated compound A and compound B, were evaluated for their biochemical, antibacterial, and pharmacokinetic properties. The two compounds inhibited the ATPase activity of GyrB and ParE with 50% inhibitory concentrations of <0.1 µg/ml. Prevention of DNA supercoiling by DNA gyrase was also observed. Both compounds potently inhibited the growth of a range of bacterial organisms, including staphylococci, streptococci, enterococci, Clostridium difficile, and selected Gram-negative respiratory pathogens. MIC90s against clinical isolates ranged from 0.015 µg/ml for Streptococcus pneumoniae to 0.25 µg/ml for Staphylococcus aureus. No cross-resistance with common drug resistance phenotypes was observed. In addition, no synergistic or antagonistic interactions between compound A or compound B and other antibiotics, including the topoisomerase inhibitors novobiocin and levofloxacin, were detected in checkerboard experiments. The frequencies of spontaneous resistance for S. aureus were <2.3 × 10(-10) with compound A and <5.8 × 10(-11) with compound B at concentrations equivalent to 8× the MICs. These values indicate a multitargeting mechanism of action. The pharmacokinetic properties of both compounds were profiled in rats. Following intravenous administration, compound B showed approximately 3-fold improvement over compound A in terms of both clearance and the area under the concentration-time curve. The measured oral bioavailability of compound B was 47.7%.

An improved small-molecule inhibitor of FtsZ with superior in vitro potency, drug-like properties, and in vivo efficacy.

The bacterial cell division protein FtsZ is an attractive target for small-molecule antibacterial drug discovery. Derivatives of 3-methoxybenzamide, including compound PC190723, have been reported to be potent and selective antistaphylococcal agents which exert their effects through the disruption of intracellular FtsZ function. Here, we report the further optimization of 3-methoxybenzamide derivatives towards a drug candidate. The in vitro and in vivo characterization of a more advanced lead compound, designated compound 1, is described. Compound 1 was potently antibacterial, with an average MIC of 0.12 µg/ml against all staphylococcal species, including methicillin- and multidrug-resistant Staphylococcus aureus and Staphylococcus epidermidis. Compound 1 inhibited an S. aureus strain carrying the G196A mutation in FtsZ, which confers resistance to PC190723. Like PC190723, compound 1 acted on whole bacterial cells by blocking cytokinesis. No interactions between compound 1 and a diverse panel of antibiotics were measured in checkerboard experiments. Compound 1 displayed suitable in vitro pharmaceutical properties and a favorable in vivo pharmacokinetic profile following intravenous and oral administration, with a calculated bioavailability of 82.0% in mice. Compound 1 demonstrated efficacy in a murine model of systemic S. aureus infection and caused a significant decrease in the bacterial load in the thigh infection model. A greater reduction in the number of S. aureus cells recovered from infected thighs, equivalent to 3.68 log units, than in those recovered from controls was achieved using a succinate prodrug of compound 1, which was designated compound 2. In summary, optimized derivatives of 3-methoxybenzamide may yield a first-in-class FtsZ inhibitor for the treatment of antibiotic-resistant staphylococcal infections.

Characterization of three novel mechanosensitive channel activities in Escherichia coli.

Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS. Deletion of both proteins severely compromises survival of hypoosmotic shock. However, like many bacteria, E. coli cells possess other MscS-type genes (kefA, ybdG, ybiO, yjeP and ynaI). Two homologs, MscK (kefA) and YbdG, have been characterized as mechanosensitive channels that play minor roles in maintaining cell integrity. Additional channel openings are occasionally observed in patches derived from mutants lacking MscS, MscK and MscL. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we complete the identification of the remaining E. coli mechanosensitive channels YnaI, YbiO and YjeP. The latter is the major component of the previously described MscM activity (~300 pS), while YnaI (~100 pS) and YbiO (~1000 pS) were previously unknown. Expression of native YbiO is NaCl-specific and RpoS-dependent. A Δ7 strain was created with all seven E. coli mechanosensitive channel genes deleted. High level expression of YnaI, YbiO or YjeP proteins from a multicopy plasmid in the Δ7 strain (MJFGH) leads to substantial protection against hypoosmotic shock. Purified homologs exhibit high molecular masses that are consistent with heptameric assemblies. This work reveals novel mechanosensitive channels and discusses the regulation of their expression in the context of possible additional functions.

Creating an antibacterial with in vivo efficacy: synthesis and characterization of potent inhibitors of the bacterial cell division protein FtsZ with improved pharmaceutical properties.

3-Methoxybenzamide (1) is a weak inhibitor of the essential bacterial cell division protein FtsZ. Alkyl derivatives of 1 are potent antistaphylococcal compounds with suboptimal drug-like properties. Exploration of the structure-activity relationships of analogues of these inhibitors led to the identification of potent antistaphylococcal compounds with improved pharmaceutical properties.

Antibacterial alkoxybenzamide inhibitors of the essential bacterial cell division protein FtsZ.

3-Methoxybenzamide is a weak inhibitor of the essential bacterial cell division protein FtsZ. Exploration of the structure-activity relationships of 3-methoxybenzamide analogues led to the identification of potent anti-staphylococcal compounds.

An inhibitor of FtsZ with potent and selective anti-staphylococcal activity.

FtsZ is an essential bacterial guanosine triphosphatase and homolog of mammalian beta-tubulin that polymerizes and assembles into a ring to initiate cell division. We have created a class of small synthetic antibacterials, exemplified by PC190723, which inhibits FtsZ and prevents cell division. PC190723 has potent and selective in vitro bactericidal activity against staphylococci, including methicillin- and multi-drug-resistant Staphylococcus aureus. The putative inhibitor-binding site of PC190723 was mapped to a region of FtsZ that is analogous to the Taxol-binding site of tubulin. PC190723 was efficacious in an in vivo model of infection, curing mice infected with a lethal dose of S. aureus. The data validate FtsZ as a target for antibacterial intervention and identify PC190723 as suitable for optimization into a new anti-staphylococcal therapy.

Novel inhibitors of bacterial cytokinesis identified by a cell-based antibiotic screening assay.

The continuous emergence of antibiotic resistance demands that novel classes of antibiotics continue to be developed. The division machinery of bacteria is an attractive target because it comprises seven or more essential proteins that are conserved almost throughout the bacteria but are absent from humans. We describe the development of a cell-based assay for inhibitors of cell division and its use to isolate a new inhibitor of FtsZ protein, a key player in the division machinery. Biochemical, cytological, and genetic data are presented that demonstrate that FtsZ is the specific target for the compound. We also describe the effects of more potent analogues of the original hit compound that act on important pathogens, again at the level of cell division. The assay and the compounds have the potential to provide novel antibiotics with no pool of pre-existing resistance. They have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery.

Transcriptional responses of intestinal epithelial cells to infection with Vibrio cholerae.

Vibrio cholerae is a noninvasive enteric bacterium that causes the severe diarrheal disease cholera. Candidate cholera vaccines have been engineered by deleting genes encoding known virulence factors in V. cholerae; however, many of these attenuated strains were still reactogenic in human volunteers. In this study, DNA arrays were utilized to monitor the transcriptional responses of human intestinal epithelial cells (T84) to eight strains of V. cholerae, including attenuated, toxigenic, and environmental isolates. cDNA probes generated from host RNA samples were hybridized against low- and high-density gene arrays. V. cholerae induced the transcription of a variety of host genes and repressed the expression of a lower number of genes. Expression patterns were confirmed for certain genes by reverse transcriptase PCR and enzyme-linked immunosorbent assays. A core subset of genes was found to be differentially regulated in all experiments. These genes included genes involved in innate mucosal immunity, intracellular signaling, and cellular proliferation. Reactogenic vaccine strains induced greater expression of genes for certain proinflammatory cytokines than nonreactogenic strains. Wild-type and attenuated derivatives induced and repressed many genes in common, although there were differences in the transcription profiles. These results indicate that the types of host genes modulated by attenuated V. cholerae, and the extent of their induction, may mediate the symptoms seen with reactogenic cholera vaccine strains.

A role for mechanosensitive channels in survival of stationary phase: regulation of channel expression by RpoS.

The mechanosensitive (MS) channels MscS and MscL are essential for the survival of hypoosmotic shock by Escherichia coli cells. We demonstrate that MscS and MscL are induced by osmotic stress and by entry into stationary phase. Reduced levels of MS proteins and reduced expression of mscL- and mscS-LacZ fusions in an rpoS mutant strain suggested that the RNA polymerase holoenzyme containing sigmaS is responsible, at least in part, for regulating production of MS channel proteins. Consistent with the model that the effect of sigmaS is direct, the MscS and MscL promoters both use RNA polymerase containing sigmaS in vitro. Conversely, clpP or rssB mutations, which cause enhanced levels of sigmaS, show increased MS channel protein synthesis. RpoS null mutants are sensitive to hypoosmotic shock upon entry into stationary phase. These data suggest that MscS and MscL are components of the RpoS regulon and play an important role in ensuring structural integrity in stationary phase bacteria.