[Show abstract][Hide abstract] ABSTRACT: Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors-the gyramides-are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have an abnormally localized, condensed chromosome that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation, which is different from the mechanism of the fluoroquinolones and aminocoumarin antibiotics. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division.
ACS Chemical Biology 04/2014; 9(6). DOI:10.1021/cb500154m · 5.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: FtsZ is a homolog of eukaryotic tubulin that is widely conserved among bacteria and coordinates the assembly of the cell division machinery. FtsZ plays a central role in cell replication and is a target of interest for antibiotic development. Several FtsZ inhibitors have been reported. We characterized the mechanism of these compounds in bacteria and found that many of them disrupt the localization of membrane-associated proteins, including FtsZ, by reducing the transmembrane potential or perturbing membrane permeability. We tested whether the reported phenotypes of a broad collection of FtsZ inhibitors disrupt the transmembrane potential in Bacillus subtilis strain 168. Using a combination of flow cytometry and microscopy, we found that zantrin Z1, cinnamaldehyde, totarol, sanguinarine, and viriditoxin decreased the B. subtilis transmembrane potential or perturbed membrane permeability, and influenced the localization of the membrane-associated, division protein MinD. These studies demonstrate that small molecules that disrupt membrane function in bacterial cells produce phenotypes that are similar to the inhibition of proteins associated with membranes in vivo, including bacterial cytoskeleton homologs, such as FtsZ. The results provide a new dimension for consideration in the design and testing of inhibitors of bacterial targets that are membrane-associated and provide additional insight into the structural characteristics of antibiotics that disrupt the membrane.
Medicinal Chemistry Communication 03/2013; 4(1):112-119. DOI:10.1039/C2MD20127E · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The synthesis and antimicrobial activity heterocyclic analogs of the diterpenoid totarol are described. An advanced synthetic intermediate with a ketone on the A-ring is used to attach fused heterocycles and a carbon-to-nitrogen atom replacement is made on the B-ring by de novo synthesis. A-ring analogs with an indole attached exhibit, for the first time, enhanced antimicrobial activity relative to the parent natural product. Preliminary experiments demonstrate that the indole analogs do not target the bacterial cell division protein FtsZ as had been hypothesized for totarol.
[Show abstract][Hide abstract] ABSTRACT: Persistent infections are frequently caused by dormant and biofilm-associated bacteria, which often display characteristically slow growth. Antibiotics that require rapid cell growth may be ineffective against these organisms and thus fail to prevent reoccurring infections. In contrast to growth-based antimicrobial agents, membrane-targeting drugs effectively kill slow-growing bacteria. Herein we introduce 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), a potent broad-spectrum antibiotic that reduces the transmembrane potential of Gram-positive and Gram-negative bacteria and causes mislocalization of essential membrane-associated proteins, including MinD and FtsA. Importantly, DCAP kills nutrient-deprived microbes and sterilizes bacterial biofilms. DCAP is lethal against bacterial cells, has no effect on red blood cell membranes, and only decreases the viability of mammalian cells after ≥6 h. We conclude that membrane-active compounds are a promising solution for treating persistent infections. DCAP expands the limited number of compounds in this class of therapeutic small molecules and provides new opportunities for the development of potent broad-spectrum antimicrobial agents.
Journal of the American Chemical Society 06/2012; 134(28):11322-5. DOI:10.1021/ja302542j · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering (DLS) spectroscopy as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP, and FtsZ monomers. Measurements of the diffusion coefficient of the polymers demonstrate that their length is remarkably stable until the free GTP is consumed. We estimated the mean size of the FtsZ polymers within this interval of stable length to be between 9 and 18 monomers. The rates of FtsZ polymerization and depolymerization are likely influenced by the concentration of GDP, as the repeated addition of GTP to FtsZ increased the rate of polymerization and slowed down depolymerization. Increasing the FtsZ concentration did not change the size of FtsZ polymers; however, it increased the rate of the depolymerization reaction by depleting free GTP. Using transmission electron microscopy we observed that FtsZ forms linear polymers in solutions which rapidly convert to large bundles upon contact with surfaces at time scales as short as several seconds. Finally, the best studied small molecule that binds to FtsZ, PC190723, had no stabilizing effect on Caulobacter crescentus FtsZ filaments in vitro, which complements previous studies with Escherichia coli FtsZ and confirms that this class of small molecules binds Gram-negative FtsZ weakly.
[Show abstract][Hide abstract] ABSTRACT: The subcellular organization of biological molecules is a critical determinant of many bacterial processes, including growth, replication of the genome, and division, yet the details of many mechanisms that control intracellular organization remain unknown. Decoding this information will impact the field of bacterial physiology and can provide insight into eukaryotic biology, including related processes in mitochondria and chloroplasts. Small molecule probes provide unique advantages in studying these mechanisms and manipulating the organization of biomolecules in live bacterial cells. In this review, we describe small molecules that are available for investigating subcellular organization in bacteria, specifically targeting FtsZ, MreB, peptidoglycan, and lipid bilayers. We discuss how these probes have been used to study microbiological questions and conclude by providing suggestions about important areas in which chemical-biological approaches will have a revolutionary impact on the study of bacterial physiology.
[Show abstract][Hide abstract] ABSTRACT: This paper characterizes N-benzyl-3-sulfonamidopyrrolidines (gyramides) as DNA gyrase inhibitors. Gyramide A was previously shown to exhibit antimicrobial activity that suggested it inhibited bacterial cell division. In this study, we conducted target identification studies and identified DNA gyrase as the primary target of gyramide A. The gyramide A resistance-determining region in DNA gyrase is adjacent to the DNA cleavage gate and is a new site for inhibitor design. We studied the antibiotic effects of gyramides A-C in combination with the Gram-negative efflux pump inhibitor MC-207,110 (60 μM). The gyramides had a minimum inhibitory concentration of 10-40 μM against Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, and Streptococcus pneumoniae; the compounds were ineffective against Enterococcus faecalis. The IC(50) of gyramides A-C against E. coli DNA gyrase was 0.7- 3.3 μM. The N-benzyl-3-sulfonamidopyrrolidines described in this manuscript represent a starting point for development of antibiotics that bind a new site in DNA gyrase.
[Show abstract][Hide abstract] ABSTRACT: Bacterial cytoskeletal proteins are an emerging set of targets for antibiotic development. This paper describes oligochlorophen
analogs based on the monomer 4-chloro-2,6-dimethylphenol as antimicrobial agents against Bacillus anthracis. The most potent analogs have a MIC of 160 to 320 nM against B. anthracis and may target the cytoskeletal protein FtsZ. B. anthracis develops resistance to the oligochlorophens at a rate of 4.34 × 10−10 per generation, which is ∼10-fold lower than that of commercial antibiotics used to treat this human pathogen.