David J. Haydon

An Inhibitor of FtsZ with Potent and Selective Anti-Staphylococcal Activity

FtsZ is an essential bacterial guanosine triphosphatase and homolog of mammalian β-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.

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.

DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: synthesis and antibacterial activity.

The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.

An Improved Small-Molecule Inhibitor of FtsZ with Superior In Vitro Potency, Drug-Like Properties, and In Vivo Efficacy

ABSTRACT 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.

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

ABSTRACT 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%.

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.