Evaluation of the metS and murB Loci for Antibiotic Discovery Using Targeted Antisense RNA Expression Analysis in Bacillus anthracis

The biowarfare-relevant bacterial pathogen Bacillus anthracis contains two paralogs each of the metS and murB genes, which encode the important antibiotic target functions methionyl-tRNA synthetase and UDP-N-acetylenolpyruvoylglucosamine reductase, respectively. Empirical screens were conducted to detect and characterize gene fragments of each of these four genes that could cause growth reduction of B. anthracis when inducibly expressed from a plasmid-borne promoter. Numerous such gene fragments that were overwhelmingly in the antisense orientation were identified for the metS1 and murB2 alleles, while no such orientation bias was seen for the metS2 and murB1 alleles. Gene replacement mutagenesis was used to confirm the essentiality of the metS1 and murB2 alleles, and the nonessentiality of the metS2 and murB1 alleles, for vegetative growth. Induced transcription of RNA from metS1 and murB2 antisense-oriented gene fragments resulted in specific reduction of mRNA of their cognate genes. Attenuation of MetS1 enzyme expression hypersensitized B. anthracis cells to a MetS-specific antimicrobial compound but not to other antibiotics that affect cell wall assembly, fatty acid biosynthesis, protein translation, or DNA replication. Antisense-dependent reduction of MurB2 enzyme expression caused hypersensitivity to beta-lactam antibiotics, a synergistic response that has also been noted for the MurA-specific antibiotic fosfomycin. These experiments form the basis of mode-of-action detection assays that can be used in the discovery of novel MetS- or MurB-specific antibiotic drugs that are effective against B. anthracis or other gram-positive bacterial pathogens

Regulation of mprF by Antisense RNA Restores Daptomycin Susceptibility to Daptomycin-Resistant Isolates of Staphylococcus aureus▿

Mutations in mprF have been shown to result in reduced susceptibility to daptomycin and other cationic antibacterials. An mprF antisense-inducible plasmid was constructed and used to demonstrate that depletion of mprF can reestablish susceptibility to daptomycin. Inducing antisense to mprF also resulted in increased susceptibility to vancomycin and gentamicin but, paradoxically, decreased susceptibility to oxacillin. These results suggest that mprF mutations that reduce susceptibility to cationic antibacterials result in a gain-of-function phenotype

Comparison of the Essential Cellular Functions of the Two murA Genes of Bacillus anthracis▿

Targeted antisense and gene replacement mutagenesis experiments demonstrate that only the murA1 gene and not the murA2 gene is required for the normal cellular growth of Bacillus anthracis. Antisense-based modulation of murA1 gene expression hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of B. anthracis to this drug

Structure-Based Design of New Dihydrofolate Reductase Antibacterial Agents: 7‑(Benzimidazol-1-yl)-2,4-diaminoquinazolines

A new series of dihydrofolate reductase (DHFR) inhibitors, the 7-(benzimidazol-1-yl)-2,4-diaminoquinazolines, were designed and optimized for antibacterial potency and enzyme selectivity. The most potent inhibitors in this series contained a five-membered heterocycle at the 2-position of the benzimidazole, leading to highly potent and selective compounds that exploit the differences in the size of a binding pocket adjacent to the NADPH cofactor between the bacterial and human DHFR enzymes. Typical of these compounds is 7-((2-thiazol-2-yl)­benzimidazol-1-yl)-2,4 diaminoquinazoline, which is a potent inhibitor of <i>S. aureus</i> DHFR (<i>K</i>_i = 0.002 nM) with 46700-fold selectivity over human DHFR. This compound also has high antibacterial potency on Gram-positive bacteria with an MIC versus wild type <i>S. aureus</i> of 0.0125 μg/mL and a MIC versus trimethoprim-resistant <i>S. aureus</i> of 0.25 μg/mL. In vivo efficacy versus a <i>S. aureus</i> septicemia was demonstrated, highlighting the potential of this new series

Discovery of FabH/FabF Inhibitors from Natural Products

Condensing enzymes are essential in type II fatty acid synthesis and are promising targets for antibacterial drug discovery. Recently, a new approach using a xylose-inducible plasmid to express antisense RNA in Staphylococcus aureus has been described; however, the actual mechanism was not delineated. In this paper, the mechanism of decreased target protein production by expression of antisense RNA was investigated using Northern blotting. This revealed that the antisense RNA acts posttranscriptionally by targeting mRNA, leading to 5′ mRNA degradation. Using this technology, a two-plate assay was developed in order to identify FabF/FabH target-specific cell-permeable inhibitors by screening of natural product extracts. Over 250,000 natural product fermentation broths were screened and then confirmed in biochemical assays, yielding a hit rate of 0.1%. All known natural product FabH and FabF inhibitors, including cerulenin, thiolactomycin, thiotetromycin, and Tü3010, were discovered using this whole-cell mechanism-based screening approach. Phomallenic acids, which are new inhibitors of FabF, were also discovered. These new inhibitors exhibited target selectivity in the gel elongation assay and in the whole-cell-based two-plate assay. Phomallenic acid C showed good antibacterial activity, about 20-fold better than that of thiolactomycin and cerulenin, against S. aureus. It exhibited a spectrum of antibacterial activity against clinically important pathogens including methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Haemophilus influenzae

Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

<div><p>Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.</p> </div

Optimization of inhibitor scaffolds.

<p>For the fragment hit and inhibitor candidates <b>C1</b>, <b>C2</b>, <b>C3</b> and <b>C4,</b> identical cutaway views of solvent accessible surface representations of the active-site pockets of <i>E. faecalis</i> GyrB from the crystal structures of complexes of the inhibitors with the 24 kDa N-terminal fragment of GyrB from <i>E. faecalis</i> GyrB are shown. The bound inhibitors are drawn with green bonds, the conserved ATP-binding aspartate is drawn with blue bonds and the structural water molecule that plays a key role in substrate binding in GyrB and ParE is shown as a red sphere. Potential hydrogen-bonds between the inhibitors, aspartate and water molecule are depicted as dotted lines. Optimization of the pyrrolopyrimidine scaffold led to inhibitors like <b>C1</b> with good enzyme potency but only moderate Gram-negative antibacterial activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084409#B16" target="_blank">16</a>]. Expansion of the bicyclic pyrrolopyrimidine scaffold to a tricyclic pyrimidoindole scaffold <b>(C2)</b> fills an interior lipophilic pocket and offers superior optimization vectors to improve enzyme potency. Subsequent elaboration of the tricyclic scaffold with a fluorine atom at R₆ and an aminomethyl moiety at R₈ dramatically improved inhibitor potency and ligand efficiency. The 6-fluoro-<i>N-</i>methyl-9<i>H</i>-pyrimido[4,5-<i>b</i>]indol-8-amine scaffold quantitatively fills the lipophilic interior sub-pockets of the GyrB/ParE active-sites and adds a new hydrogen-bond. <b>C3</b> and <b>C4</b> demonstrate sub-nanomolar enzyme potency versus GyrB and ParE enzymes from a broad range of Gram-positive and Gram-negative pathogens; inhibition constants (<i>K</i>_i values) are shown for a representative enzyme panel that includes the full length GyrB and ParE enzymes from <i>E. faecalis</i>, Francisella tularensis, and <i>E. coli</i>. </p

Reduction in bioburden after 24 hrs in a neutropenic mouse thigh infection model.

<p>The MIC values for <b>C3</b>, <b>C4</b> and levofloxacin against the <i>E. coli</i> strain used in the study are 0.13, 0.13 and 0.03 μg/mL, respectively. </p