Rational design of a new antibiotic class for drug-resistant infections.

The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including β-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of β-lactamases, the primary resistance mechanism associated with β-lactam therapy in Gram-negative bacteria.

Aldehyde-mediated bioconjugation via in situ generated ylides.

A technically simple approach for rapid, high-yielding and site-selective bioconjugation has been developed for both in vitro and cellular applications. This method involves the generation of maleimido-phosphonium ylides via 4-nitrophenol catalysis under physiological conditions followed by their Wittig reactions with aldehyde-appended biomolecules.

Titrating Levels of TolC in E. coli: A Sensitive Approach to Quantifying Efflux.

The susceptibility of small molecules to Gram-negative bacterial efflux is typically evaluated using an antibacterial activity-based efflux ratio, which is computed as the ratio of the antibacterial activity for a wild-type strain and its isogenic efflux mutant (typically lacking genes encoding major efflux pumps). The magnitude of the ratio is often used as an efflux index. However, early in drug discovery, hits with suboptimal physicochemical properties often lack whole cell inhibition against wild-type strains, which makes efflux ratios indeterminable.

Role of the Klebsiella pneumoniae TolC porin in antibiotic efflux.

The major Gram-negative gated efflux channel TolC has been extensively characterized in Escherichia coli but there is minimal information about Klebsiella pneumoniae TolC. Using an arabinose-inducible plasmid-based expression system, we show that the K. pneumoniae TolC complements the efflux defect in an E.

Evaluating LC-MS/MS To Measure Accumulation of Compounds within Bacteria.

A general method for determining bacterial uptake of compounds independent of antibacterial activity would be a valuable tool in antibacterial drug discovery. LC-MS/MS assays have been described, but it has not been shown whether the data can be used directly to inform medicinal chemistry. We describe the evaluation of an LC-MS/MS assay measuring association of compounds with bacteria, using a set of over a hundred compounds (inhibitors of NAD-dependent DNA ligase, LigA) for which in vitro potency and antibacterial activity had been determined.

Antibacterial Drug Discovery: Some Assembly Required.

Our limited understanding of the molecular basis for compound entry into and efflux out of Gram-negative bacteria is now recognized as a key bottleneck for the rational discovery of novel antibacterial compounds. Traditional, large-scale biochemical or target-agnostic phenotypic antibacterial screening efforts have, as a result, not been very fruitful. A main driver of this knowledge gap has been the historical lack of predictive cellular assays, tools, and models that provide structure-activity relationships to inform optimization of compound accumulation.

Acinetobacter baumannii OmpA Is a Selective Antibiotic Permeant Porin.

OmpA is a conserved, abundantly expressed outer membrane porin in Acinetobacter baumannii whose presumed role in antibiotic permeation has not been clearly demonstrated. In this report, we use a titratable heterologous expression system to express OmpA in isolation and demonstrate selective passage of small molecule antibiotics through OmpA. ETX2514, a recently discovered broad-spectrum β-lactamase inhibitor, in combination with sulbactam, is currently in clinical testing for the treatment of drug-resistant A.

Whole-Cell-Based Assay To Evaluate Structure Permeation Relationships for Carbapenem Passage through the Pseudomonas aeruginosa Porin OprD.

The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Discovery of novel classes of antibiotics with activity against these pathogens has been impeded by a fundamental lack of understanding of the molecular drivers underlying small molecule uptake. Although it is well-known that outer membrane porins represent the main route of entry for small, hydrophilic molecules across the Gram-negative cell envelope, the structure-permeation relationship for porin passage has yet to be defined.

Direct measurement of efflux in Pseudomonas aeruginosa using an environment-sensitive fluorescent dye.

Resistance-Nodulation-Division (RND) family pumps AcrB and MexB are the major efflux routes in Escherichia coli and Pseudomonas aeruginosa respectively. Fluorescent environment-sensitive dyes provide a means to study efflux pump function in live bacterial cells in real-time. Recently, we demonstrated the utility of this approach using the dye Nile Red to quantify AcrB-mediated efflux and measured the ability of antibiotics and other efflux pump substrates to compete with efflux of Nile Red, independent of antibacterial activity.

A fluorescent microplate assay quantifies bacterial efflux and demonstrates two distinct compound binding sites in AcrB.

A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli.

Sucrose metabolism contributes to in vivo fitness of Streptococcus pneumoniae.

We characterized two sucrose-metabolizing systems -sus and scr- and describe their roles in the physiology and virulence of Streptococcus pneumoniae in murine models of carriage and pneumonia. The sus and scr systems are regulated by LacI family repressors SusR and ScrR respectively. SusR regulates an adjacent ABC transporter (susT1/susT2/susX) and sucrose-6-phosphate (S-6-P) hydrolase (susH).

Catabolite control protein A (CcpA) contributes to virulence and regulation of sugar metabolism in Streptococcus pneumoniae.

We characterized the role of catabolite control protein A (ccpA) in the physiology and virulence of Streptococcus pneumoniae. S. pneumoniae has a large percentage of its genome devoted to sugar uptake and metabolism, and therefore, regulation of these processes is likely to be crucial for fitness in the nasopharynx and may play a role during invasive disease.

Colicins, spermine and cephalosporins: a competitive interaction with the OmpF eyelet.

The L3 loop is an important feature of the OmpF porin structure, contributing to both channel size and electrostatic properties. Colicins A and N, spermine, and antibiotics that use OmpF to penetrate the cell, were used to investigate the structure-function relationships of L3. Spermine was found to protect efficiently cells expressing wild-type OmpF from colicin action.

A biological role for prokaryotic ClC chloride channels.

An unexpected finding emerging from large-scale genome analyses is that prokaryotes express ion channels belonging to molecular families long studied in neurons. Bacteria and archaea are now known to carry genes for potassium channels of the voltage-gated, inward rectifier and calcium-activated classes, ClC-type chloride channels, an ionotropic glutamate receptor and a sodium channel. For two potassium channels and a chloride channel, these homologues have provided a means to direct structure determination.