Characterization of Caulobacter crescentus FtsZ protein using dynamic light scattering.

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.

Structural and functional characterization of mycobactericidal ubiquitin-derived peptides in model and bacterial membranes.

The mycobactericidal properties of macrophages include the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal ubiquitin-derived peptides (Ub-peptides). To improve our understanding of interactions of ubiquitin-derived peptides with mycobacteria, we further characterized the structure and function of bactericidal Ub-peptide Ub2. We found that Ub2 adopts a ß-sheet conformation in the context of sodium dodecyl sulfate micelles and phospholipid (1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) vesicles that was dependent upon the primary sequence of the peptide. Point mutations in Ub2 that reduced the net charge of the peptide decreased Ub2 bactericidal activity. We investigated Ub-peptide function in the context of model membranes and intact bacteria. Differential scanning calorimetry analysis demonstrated that Ub2 inserts into and perturbs model phospholipid vesicles. In addition, we demonstrate that Ub2 disrupts the integrity of the mycobacterial membrane, equilibrates the transmembrane potential, and is localized within both the mycobacterial membrane and cytoplasm of treated bacteria. Finally, we identified additional bactericidal Ub-peptides and characterized their activity and structure. This study provides new insight into the mycobactericidal mechanisms of Ub-peptides.

DCAP: a broad-spectrum antibiotic that targets the cytoplasmic membrane of bacteria.

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.

A Cell-Based Screen Identifies HDAC Inhibitors as Activators of RIG-I Signaling.

Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1,200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Combination treatment of tumor cell lines with Entinostat increased RIG-I induced cell death in a mammary carcinoma cell line that is resistant to either Entinostat or RIG-I agonist alone. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations.

Identification and Characterization of Small-Molecule IRF3-Dependent Immune Activators for Pharmaceutical Development.

We sought to develop a small-molecule activator of interferon regulatory factor 3 (IRF3), an essential innate immune transcription factor, which could potentially be used therapeutically in multiple disease settings. Using a high-throughput screen, we identified small-molecule entities that activate a type I interferon response, with minimal off-target NFκB activation. We identified 399 compounds at a hit rate of 0.24% from singlicate primary screening. Secondary screening included the primary hits and additional compounds with similar chemical structures obtained from other library sources and resulted in 142 candidate compounds. The hit compounds were sorted and ranked to identify compound groups with activity in both human and mouse backgrounds to facilitate animal model engagement for translational development. Chemical modifications within two groups of small molecules produced leads with improved activity over original hits. Furthermore, these leads demonstrated activity in ex vivo cytokine release assays from human blood- and mouse bone marrow-derived macrophages. Dependence on IRF3 was demonstrated using bone marrow-derived macrophages from IRF3-deficient mice, which were not responsive to the molecules. To identify the upstream pathway leading to IRF3 activation, we used a library of CRISPR knockout cell lines to test the key innate immune adaptor and receptor molecules. These studies indicated a surprising toll-interleukin-1 receptor-domain-containing-adapter-inducing interferon-ß-dependent but TLR3/4-independent mechanism of IRF3 activation.

Chemical-biological studies of subcellular organization in bacteria.

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.

Oligochlorophens are potent inhibitors of Bacillus anthracis.

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 x 10(-10) per generation, which is approximately 10-fold lower than that of commercial antibiotics used to treat this human pathogen.

Targeting quinolone- and aminocoumarin-resistant bacteria with new gyramide analogs that inhibit DNA gyrase.

Bacterial DNA gyrase is an essential type II topoisomerase that enables cells to overcome topological barriers encountered during replication, transcription, recombination, and repair. This enzyme is ubiquitous in bacteria and represents an important clinical target for antibacterial therapy. In this paper we report the characterization of three exciting new gyramide analogs-from a library of 183 derivatives-that are potent inhibitors of DNA gyrase and are active against clinical strains of gram-negative bacteria (Escherichia coli, Shigella flexneri, and Salmonella enterica; 3 of 10 wild-type strains tested) and gram-positive bacteria (Bacillus spp., Enterococcus spp., Staphylococcus spp., and Streptococcus spp.; all 9 of the wild-type strains tested). E. coli strains resistant to the DNA gyrase inhibitors ciprofloxacin and novobiocin display very little cross-resistance to these new gyramides. In vitro studies demonstrate that the new analogs are potent inhibitors of the DNA supercoiling activity of DNA gyrase (IC50s of 47-170 nM) but do not alter the enzyme's ATPase activity. Although mutations that confer bacterial cells resistant to these new gyramides map to the genes encoding the subunits of the DNA gyrase (gyrA and gyrB genes), overexpression of GyrA, GyrB, or GyrA and GyrB together does not suppress the inhibitory effect of the gyramides. These observations support the hypothesis that the gyramides inhibit DNA gyrase using a mechanism that is unique from other known inhibitors.

Diphenylether-Modified 1,2-Diamines with Improved Drug Properties for Development against Mycobacterium tuberculosis.

New treatments for tuberculosis infection are critical to combat the emergence of multidrug- and extensively drug-resistant Mycobacterium tuberculosis (Mtb). We report the characterization of a diphenylether-modified adamantyl 1,2-diamine that we refer to as TBL-140, which has a minimal inhibitory concentration (MIC99) of 1.2 µg/mL. TBL-140 is effective against drug-resistant Mtb and nonreplicating bacteria. In addition, TBL-140 eliminates expansion of Mtb in cell culture infection assays at its MIC. To define the mechanism of action of this compound, we performed a spontaneous mutant screen and biochemical assays. We determined that TBL-140 treatment affects the proton motive force (PMF) by perturbing the transmembrane potential (ΔΨ), consistent with a target in the electron transport chain (ETC). As a result, treated bacteria have reduced intracellular ATP levels. We show that TBL-140 exhibits greater metabolic stability than SQ109, a structurally similar compound in clinical trials for treatment of MDR-TB infections. Combined, these results suggest that TBL-140 should be investigated further to assess its potential as an improved therapeutic lead against Mtb.

Gyramides prevent bacterial growth by inhibiting DNA gyrase and altering chromosome topology.

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 abnormally localized, condensed chromosomes 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. 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.

Inhibitors of bacterial tubulin target bacterial membranes in vivo.

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.

The Synthesis and Antimicrobial Activity of Heterocyclic Derivatives of Totarol.

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.

N-Benzyl-3-sulfonamidopyrrolidines are a New Class of Bacterial DNA Gyrase Inhibitors.

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.