pc190723 and benzamide

Bacterial resistance to antibiotics makes previously manageable infections again disabling and lethal, highlighting the need for new antibacterial strategies. In this regard, inhibition of the bacterial division process by targeting key protein FtsZ has been recognized as an attractive approach for discovering new antibiotics. Binding of small molecules to the cleft between the N-terminal guanosine triphosphate (GTP)-binding and the C-terminal subdomains allosterically impairs the FtsZ function, eventually inhibiting bacterial division. Nonetheless, the lack of appropriate chemical tools to develop a binding screen against this site has hampered the discovery of FtsZ antibacterial inhibitors. Herein, we describe the first competitive binding assay to identify FtsZ allosteric ligands interacting with the interdomain cleft, based on the use of specific high-affinity fluorescent probes. This novel assay, together with phenotypic profiling and X-ray crystallographic insights, enables the identification and characterization of FtsZ inhibitors of bacterial division aiming at the discovery of more effective antibacterials.

Topics: Allosteric Site; Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Benzamides; Crystallography, X-Ray; Cytoskeletal Proteins; Fluorescence Polarization; Fluorescent Dyes; Ligands; Microbial Sensitivity Tests; Protein Binding; Pyridines; Small Molecule Libraries; Staphylococcus aureus; Structure-Activity Relationship

Antibiotic resistance among clinically significant bacterial pathogens is becoming a prevalent threat to public health, and new antibacterial agents with novel mechanisms of action hence are in an urgent need. Utilizing computational docking method and structure-based optimization strategy, we rationally designed and synthesized two series of isoxazol-3-yl- and isoxazol-5-yl-containing benzamide derivatives that targeted the bacterial cell division protein FtsZ. Evaluation of their activity against a panel of Gram-positive and -negative pathogens revealed that compounds B14 and B16 that possessed the isoxazol-5-yl group showed strong antibacterial activity against various testing strains, including methicillin-resistant Staphylococcus aureus and penicillin-resistant S. aureus. Further molecular biological studies and docking analyses proved that the compound functioned as an effective inhibitor to alter the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which finally terminated the cell division and caused cell death. Taken together, these results could suggest a promising chemotype for development of new FtsZ-targeting bactericidal agent.

Topics: Anti-Bacterial Agents; Bacillus pumilus; Bacillus subtilis; Bacterial Proteins; Benzamides; Cell Survival; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Drug Design; HeLa Cells; Humans; Isoxazoles; Microbial Sensitivity Tests; Molecular Structure; Staphylococcus aureus; Structure-Activity Relationship

As an essential protein for bacterial cell division, the tubulin-like FtsZ protein has been selected as a target for development of next generation antimicrobials. PC190723 is a fluoride-containing benzamide compound developed as a FtsZ inhibitor that selectively inhibits growth of multidrug resistant Gram-positive bacteria.. Our aim was to investigate the mechanism of resistance to PC109723 conferred by over-expression of a gene, rfiA, in an environmental bacterium Arthrobacter A3.. The investigations included analysis of the effect of PC109723 on wild-type Arthrobacter A3 and a recombinant strain over-expressing rfiA, in vivo localization of RfiA, in vitro measurements of fluorine release from PC109723 by membrane extracts from the over-expression strain combined with mass spectrophotometric analysis of reaction products, and modelling of RfiA structure.. We describe a novel protein, RfiA, from Arthrobacter A3 that confers PC190723 resistance. RfiA is a PAP2 domain-containing polytopic transmembrane protein that can modify the fluoridated benzamide ring that is critical for high affinity binding of PC190723 with FtsZ.. RfiA-mediated modification of PC190723 is the first reported instance of resistance to this antibiotic involving a change to its structure. We predict that adoption of PC190723 or related benzamides as antimicrobials in clinical practice will lead to the acquisition by resistant pathogens of a gene encoding this subfamily of proteins.

Topics: Arthrobacter; Bacterial Proteins; Benzamides; Cytoskeletal Proteins; Drug Resistance, Bacterial; Membrane Proteins; Microbial Sensitivity Tests; Phylogeny; Pyridines; Thiazoles

The clinical development of FtsZ-targeting benzamide compounds like PC190723 has been limited by poor drug-like and pharmacokinetic properties. Development of prodrugs of PC190723 (e.g., TXY541) resulted in enhanced pharmaceutical properties, which, in turn, led to improved intravenous efficacy as well as the first demonstration of oral efficacy in vivo against both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Despite being efficacious in vivo, TXY541 still suffered from suboptimal pharmacokinetics and the requirement of high efficacious doses. We describe here the design of a new prodrug (TXA709) in which the Cl group on the pyridyl ring has been replaced with a CF3 functionality that is resistant to metabolic attack. As a result of this enhanced metabolic stability, the product of the TXA709 prodrug (TXA707) is associated with improved pharmacokinetic properties (a 6.5-fold-longer half-life and a 3-fold-greater oral bioavailability) and superior in vivo antistaphylococcal efficacy relative to PC190723. We validate FtsZ as the antibacterial target of TXA707 and demonstrate that the compound retains potent bactericidal activity against S. aureus strains resistant to the current standard-of-care drugs vancomycin, daptomycin, and linezolid. These collective properties, coupled with minimal observed toxicity to mammalian cells, establish the prodrug TXA709 as an antistaphylococcal agent worthy of clinical development.

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Benzamides; Cells, Cultured; Cytoskeletal Proteins; Daptomycin; Dogs; Half-Life; Humans; Linezolid; Methicillin; Methicillin Resistance; Methicillin-Resistant Staphylococcus aureus; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Prodrugs; Pyridines; Rats; Staphylococcal Infections; Thiazoles; Vancomycin

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.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Benzamides; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Drug Design; Microbial Sensitivity Tests; Molecular Structure; Oxazoles; Staphylococcus aureus; Structure-Activity Relationship