Stretching Peptides to Generate Small Molecule ß-Strand Mimics.

Advances in the modulation of protein-protein interactions (PPIs) enable both characterization of PPI networks that govern diseases and design of therapeutics and probes. The shallow protein surfaces that dominate PPIs are challenging to target using standard methods, and approaches for accessing extended backbone structures are limited. Here, we incorporate a rigid, linear, diyne brace between side chains at the i to i+2 positions to generate a family of low-molecular-weight, extended-backbone peptide macrocycles. NMR and density functional theory studies show that these stretched peptides adopt stable, rigid conformations in solution and can be tuned to explore extended peptide conformational space. The diyne brace is formed in excellent conversions (>95%) and amenable to high-throughput synthesis. The minimalist structure-inducing tripeptide core (<300 Da) is amenable to further synthetic elaboration. Diyne-braced inhibitors of bacterial type 1 signal peptidase demonstrate the utility of the technique.

Initial Analysis of the Arylomycin D Antibiotics.

The arylomycins are a class of natural product antibiotics that inhibit bacterial type I signal peptidase and are under development as therapeutics. Four classes of arylomycins are known, arylomycins A-D. Previously, we reported the synthesis and analysis of representatives of the A, B, and C classes and showed that their spectrum of activity has the potential to be much broader than originally assumed. Along with a comparison of the mechanism of acquired and innate resistance, this led us to suggest that the arylomycins are latent antibiotics, antibiotics that once possessed broad-spectrum activity, but which upon examination today, have only narrow spectrum activity due to prior selection for resistance in the course of the competition with other microorganisms that drove their evolution in the first place. Interestingly, actinocarbasin, the only identified member of the arylomycin D class, has been reported to have activity against MRSA. To confirm and understand this activity, several actinocarbasin derivatives were synthesized. We demonstrate that the previously reported structure of actinocarbasin is incorrect, identify what is likely the correct scaffold, confirm that scaffold has activity against MRSA, and determine the origin of this activity.

Inhibition of Protein Secretion in Escherichia coli and Sub-MIC Effects of Arylomycin Antibiotics.

At sufficient concentrations, antibiotics effectively eradicate many bacterial infections. However, during therapy, bacteria are unavoidably exposed to lower antibiotic concentrations, and sub-MIC exposure can result in a wide variety of other effects, including the induction of virulence, which can complicate therapy, or horizontal gene transfer (HGT), which can accelerate the spread of resistance genes. Bacterial type I signal peptidase (SPase) is an essential protein that acts at the final step of the general secretory pathway. This pathway is required for the secretion of many proteins, including many required for virulence, and the arylomycins are a class of natural product antibiotics that target SPase. Here, we investigated the consequences of exposing Escherichia coli cultures to sub-MIC levels of an arylomycin. Using multidimensional protein identification technology mass spectrometry, we found that arylomycin treatment inhibits the proper extracytoplasmic localization of many proteins, both those that appear to be SPase substrates and several that do not. The identified proteins are involved in a broad range of extracytoplasmic processes and include a number of virulence factors. The effects of arylomycin on several processes required for virulence were then individually examined, and we found that, at even sub-MIC levels, the arylomycins potently inhibit flagellation, motility, biofilm formation, and the dissemination of antibiotic resistance via HGT. Thus, we conclude that the arylomycins represent promising novel therapeutics with the potential to eradicate infections while simultaneously reducing virulence and the dissemination of resistance.

Optimization of a ß-Lactam Scaffold for Antibacterial Activity via the Inhibition of Bacterial Type I Signal Peptidase.

ß-Lactam antibiotics, one of the most important class of human therapeutics, act via the inhibition of penicillin-binding proteins (PBPs). The unparalleled success in their development has inspired efforts to develop them as inhibitors of other targets. Bacterial type I signal peptidase is evolutionarily related to the PBPs, but the stereochemistry of its substrates and its catalytic mechanism suggest that ß-lactams with the 5S stereochemistry, as opposed to the 5R stereochemistry of the traditional ß-lactams, would be required for inhibition. We report the synthesis and evaluation of a variety of 5S penem derivatives and identify several with promising activity against both a Gram-positive and a Gram-negative bacterial pathogen. To our knowledge these are the first 5S ß-lactams to possess significant antibacterial activity and the first ß-lactams imparted with antibacterial activity via optimization of the inhibition of a target other than a PBP. Along with the privileged status of their scaffold and the promise of bacterial signal peptidase I (SPase) as a target, this activity makes these compounds promising leads for development as novel therapeutics.

Not just an antibiotic target: Exploring the role of type I signal peptidase in bacterial virulence.

The looming antibiotic crisis has prompted the development of new strategies towards fighting infection. Traditional antibiotics target bacterial processes essential for viability, whereas proposed antivirulence approaches rely on the inhibition of factors that are required only for the initiation and propagation of infection within a host. Although antivirulence compounds have yet to prove their efficacy in the clinic, bacterial signal peptidase I (SPase) represents an attractive target in that SPase inhibitors exhibit broad-spectrum antibiotic activity, but even at sub-MIC doses also impair the secretion of essential virulence factors. The potential consequences of SPase inhibition on bacterial virulence have not been thoroughly examined, and are explored within this review. In addition, we review growing evidence that SPase has relevant biological functions outside of mediating secretion, and discuss how the inhibition of these functions may be clinically significant.