Förster Resonance Energy Transfer Assay for Investigating the Reactivity of Thioesters in Biochemistry and Native Chemical Ligation.

Thioesters are considered to be "energy-rich" functional groups that are susceptible to attack by thiolate and amine nucleophiles while remaining hydrolytically stable at neutral pH, which enables thioester chemistry to take place in an aqueous medium. Thus, the inherent reactivity of thioesters enables their fundamental roles in biology and unique applications in chemical synthesis. Here, we investigate the reactivity of thioesters that mimic acyl-coenzyme A (CoA) species and S-acylcysteine modifications as well as aryl thioesters applied in chemical protein synthesis by native chemical ligation (NCL). We developed a fluorogenic assay format for the direct and continuous investigation of the rate of reaction between thioesters and nucleophiles (hydroxide, thiolate, and amines) under various conditions and were able to recapitulate previously reported reactivity of thioesters. Further, chromatography-based analyses of acetyl- and succinyl-CoA mimics revealed striking differences in their ability to acylate lysine side chains, providing insight into nonenzymatic protein acylation. Finally, we investigated key aspects of native chemical ligation reaction conditions. Our data revealed a profound effect of the tris-(2-carboxyethyl)phosphine (TCEP) commonly used in systems where thiol-thioester exchange occurs, including a potentially harmful hydrolysis side reaction. These data provide insight into the potential optimization of native chemical ligation chemistry.

In Vivo Neutralization of Myotoxin II, a Phospholipase A2 Homologue from Bothrops asper Venom, Using Peptides Discovered via Phage Display Technology.

Many snake venom toxins cause local tissue damage in prey and victims, which constitutes an important pathology that is challenging to treat with existing antivenoms. One of the notorious toxins that causes such effects is myotoxin II present in the venom of the Central and Northern South American viper, Bothrops asper. This Lys49 PLA2 homologue is devoid of enzymatic activity and causes myotoxicity by disrupting the cell membranes of muscle tissue. To improve envenoming therapy, novel approaches are needed, warranting the discovery and development of inhibitors that target key toxins that are currently difficult to neutralize. Here, we report the identification of a new peptide (JB006), discovered using phage display technology, that is capable of binding to and neutralizing the toxic effects of myotoxin II in vitro and in vivo. Through computational modeling, we further identify hypothetical binding interactions between the toxin and the peptide to enable further development of inhibitors that can neutralize myotoxin II.

On-Resin Peptide Cyclization Using the 3-Amino-4-(Methylamino)Benzoic Acid MeDbz Linker.

Cyclic peptides are becoming increasingly important in drug discovery due to their specific binding properties, larger surface area compared to small molecules, and their ready and modular synthetic accessibility. In this protocol, we describe an on-resin, cleavage-inducing cyclization methodology for the synthesis of cyclic thiodepsipeptides and cyclic homodetic peptides using the 3-amino-4-(methylamino)benzoic acid (MeDbz) linker. We further describe three post-cyclization one-pot procedures, which include desulfurization, disulfide bond formation, and S-alkylation of cysteine residues.

Rearrangement of Thiodepsipeptides by S → N Acyl Shift Delivers Homodetic Autoinducing Peptides.

Group behavior in many bacteria relies on chemically induced communication called quorum sensing (QS), which plays important roles in the regulation of colonization, biofilm formation, and virulence. In Gram-positive bacteria, QS is often mediated by cyclic ribosomally synthesized and posttranslationally modified peptides (RiPPs). In staphylococci, for example, most of these so-called autoinducing peptides (AIPs) contain a conserved thiolactone functionality, which has also been predicted to constitute a structural feature of AIPs from other genera. Here, we show that pentameric AIPs from Lactiplantibacillus plantarum, Clostridium perfringens, and Listeria monocytogenes that were previously presumed to be thiolactone-containing structures readily rearrange to become homodetic cyclopeptides. This finding has implications for the developing understanding of cross-species and potential cross-genus communication of bacteria and may help guide the discovery of peptide ligands to perturb their function.

Peptide Inhibitors of the α-Cobratoxin-Nicotinic Acetylcholine Receptor Interaction.

Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure-function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.

Effect of Co-inhabiting Coagulase Negative Staphylococci on S. aureus agr Quorum Sensing, Host Factor Binding, and Biofilm Formation.

Staphylococcus aureus is a commensal colonizer of both humans and animals, but also an opportunistic pathogen responsible for a multitude of diseases. In recent years, colonization of pigs by methicillin resistant S. aureus has become a problem with increasing numbers of humans being infected by livestock strains. In S. aureus colonization and virulence factor expression is controlled by the agr quorum sensing system, which responds to and is activated by self-generated, autoinducing peptides (AIPs). AIPs are also produced by coagulase negative staphylococci (CoNS) commonly found as commensals in both humans and animals, and interestingly, some of these inhibit S. aureus agr activity. Here, we have addressed if cross-communication occurs between S. aureus and CoNS strains isolated from pig nares, and if so, how properties such as host factor binding and biofilm formation are affected. From 25 pig nasal swabs we obtained 54 staphylococcal CoNS isolates belonging to 8 different species. Of these, none were able to induce S. aureus agr as monitored by reporter gene fusions to agr regulated genes but a number of agr-inhibiting species were identified including Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus arlettae, Staphylococcus lentus, and Staphylococcus chromogenes. After establishing that the inhibitory activity was mediated via AgrC, the receptor of AIPs, we synthesized selective AIPs to explore their effect on adhesion of S. aureus to fibronectin, a host factor involved in S. aureus colonization. Here, we found that the CoNS AIPs did not affect adhesion of S. aureus except for strain 8325-4. When individual CoNS strains were co-cultured together with S. aureus we observed variable degrees of biofilm formation which did not correlate with agr interactions. Our results show that multiple CoNS species can be isolated from pig nares and that the majority of these produce AIPs that inhibit S. aureus agr. Further they show that the consequences of the interactions between CoNS and S. aureus are complex and highly strain dependent.

Identification of autoinducing thiodepsipeptides from staphylococci enabled by native chemical ligation.

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non-S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus.

Direct Peptide Cyclization and One-Pot Modification Using the MeDbz Linker.

The one-pot synthesis and modification of cyclic peptides through a self-cleaving on-resin protocol is described. We apply Dawson's MeDbz linker to achieve direct intramolecular peptide cyclization by thioesterification followed by S → N acyl shift. This native chemical ligation approach requires no activating additive and allows direct modification of the crude cyclic peptides in one-pot. The strategy was applied to synthesize 5 cyclic peptide natural products of varying ring size. Finally, one-pot modifications include desulfurization, fluorophore conjugation, and intramolecular disulfide formation.

Corrigendum: Cross-Talk between Staphylococcus aureus and Other Staphylococcal Species via the agr Quorum Sensing System.

[This corrects the article on p. 1733 in vol. 7, PMID: 27877157.].

Structure-Activity Relationship Study Based on Autoinducing Peptide (AIP) from Dog Pathogen S. schleiferi.

Herein, an effective protocol for solid-phase synthesis of peptide thiolactones by concomitant ring closure and cleavage from the solid support is reported. The strategy was applied for mapping the importance of the structural features in S. schleiferi AIP (5) by performing an alanine scan and truncation of this natural compound. This furnished some of the most potent inhibitors of accessory gene regulator (agr)-I in the human pathogen S. aureus reported to date.