In Vitro Evaluation of Common Antimicrobial Solutions Used for Breast Implant Soaking and Breast Pocket Irrigation-Part 2: Efficacy Against Biofilm-Associated Bacteria.

BACKGROUND: Biofilm-associated bacteria have been observed in both breast implant revision and tissue expander-implant exchange surgeries. The utilization of antimicrobial solutions in breast surgery, especially those containing triple antibiotics (TAB) and/or 10% povidone-iodine (PI), may help reduce existing biofilm-associated bacteria, which is particularly important in a mature breast pocket that may contain residual bacteria from a previously colonized implant surface or, theoretically, bacteria that may arrive postoperatively through hematogenous spread. OBJECTIVES: A series of in vitro assessments was performed to evaluate the antimicrobial utility of TAB and PI, either alone or in combination, against preformed biofilm-associated bacteria. METHODS: Preformed biofilm-associated gram-positive and gram-negative bacterial strains were exposed to TAB and PI ± TAB for up to 30 minutes in a bacterial time-kill assay. Efficacy of various dilutions of PI and the effects of serum protein on PI efficacy were also investigated. RESULTS: TAB was ineffective at the timeframes tested when utilized alone; when utilized in conjunction with PI, significant log reduction of all biofilm-associated bacterial species tested was achieved when treated for at least 5 minutes. PI alone at a concentration of 25% or higher was also effective, although its efficacy was negatively affected by increasing serum protein concentration only for Staphylococcus epidermidis. CONCLUSIONS: Our data indicate that PI-containing solutions significantly reduce biofilm-associated bacteria, suggesting potential utility for breast pocket irrigation during revision or exchange surgeries. Care should be taken to minimize excessive dilution of PI to maintain efficacy.

Ribosomal Synthesis of Thioether-Bridged Bicyclic Peptides.

Many biologically active peptides found in nature exhibit a bicyclic structure wherein a head-to-tail cyclic backbone is further constrained by an intramolecular linkage connecting two side chains of the peptide. Accordingly, methods to access macrocyclic peptides sharing this overall topology could be of significant value toward the discovery of new functional entities and bioactive compounds. With this goal in mind, we recently developed a strategy for enabling the biosynthesis of thioether-bridged bicyclic peptides in living bacterial cells. This method involves a split intein-catalyzed head-to-tail cyclization of a ribosomally produced precursor peptide, combined with inter-sidechain cross-linking through a genetically encoded cysteine-reactive amino acid. This approach can be applied to direct the formation of structurally diverse bicyclic peptides with high efficiency and selectivity in living Escherichia coli cells and provides a platform for the generation of combinatorial libraries of genetically encoded bicyclic peptides for screening purposes.

Ribosomal Synthesis of Natural-Product-Like Bicyclic Peptides in Escherichia coli.

Methods to access natural-product-like macrocyclic peptides can disclose new opportunities for the exploration of this important structural class for chemical biology and drug discovery applications. Here, the scope and mechanism of a novel strategy for directing the biosynthesis of thioether-bridged bicyclic peptides in bacterial cells was investigated. This method entails split intein-catalyzed head-to-tail cyclization of a ribosomally produced precursor peptide, combined with inter-side-chain crosslinking through a genetically encoded cysteine-reactive amino acid. This strategy could be successfully applied to achieve formation of structurally diverse bicyclic peptides with high efficiency and selectivity in Escherichia coli. Insights into the sequence of reactions underlying the peptide bicyclization process were gained from time-course experiments. Finally, the potential utility of this methodology toward the discovery of macrocyclic peptides with enhanced functional properties was demonstrated through the isolation of a bicyclic peptide with sub-micromolar affinity for streptavidin.

Bioinspired strategy for the ribosomal synthesis of thioether-bridged macrocyclic peptides in bacteria.

Inspired by the biosynthetic logic of lanthipeptide natural products, a new methodology was developed to direct the ribosomal synthesis of macrocyclic peptides constrained by an intramolecular thioether bond. As a first step, a robust and versatile strategy was implemented to enable the cyclization of ribosomally derived peptide sequences via a chemoselective reaction between a genetically encoded cysteine and a cysteine-reactive unnatural amino acid (O-(2-bromoethyl)-tyrosine). Combination of this approach with intein-catalyzed protein splicing furnished an efficient route to achieve the spontaneous, post-translational formation of structurally diverse macrocyclic peptides in bacterial cells. The present peptide cyclization strategy was also found to be amenable to integration with split intein-mediated circular ligation, resulting in the intracellular synthesis of conformationally constrained peptides featuring a bicyclic architecture.

The designer leptin antagonist peptide Allo-aca compensates for short serum half-life with very tight binding to the receptor.

The leptin receptor antagonist peptide Allo-aca exhibits picomolar activities in various cellular systems and sub-mg/kg subcutaneous efficacies in animal models making it a prime drug candidate and target validation tool. Here we identified the biochemical basis for its remarkable in vivo activity. Allo-aca decomposed within 30 min in pooled human serum and was undetectable beyond the same time period from mouse plasma during pharmacokinetic measurements. The C max of 8.9 µg/mL at 5 min corresponds to approximately 22% injected peptide present in the circulation. The half-life was extended to over 2 h in bovine vitreous fluid and 10 h in human tears suggesting potential efficacy in ophthalmic diseases. The peptide retained picomolar anti-proliferation activity against a chronic myeloid leukemia cell line; addition of a C-terminal biotin label increased the IC50 value by approximately 200-fold. In surface plasmon resonance assays with the biotin-labeled peptide immobilized to a NeutrAvidin-coated chip, Allo-aca exhibited exceptionally tight binding to the binding domain of the human leptin receptor with ka = 5 × 10(5) M(-1) s(-1) and kdiss = 1.5 × 10(-4) s(-1) values. Peptides excel in terms of high activity and selectivity to their targets, and may activate or inactivate receptor functions considerably longer than molecular turnovers that take place in experimental animals.

Synthesis of side chain N,N'-diaminoalkylated derivatives of basic amino acids for application in solid-phase peptide synthesis.

Despite the enormous therapeutic potential, the clinical use of peptides has been limited by their poor bioavailability and low stability under physiological conditions. Hence, efforts have been undertaken to alter peptide structure in ways to improve their pharmacological properties. Inspired by the importance of basic amino acids in biological systems and the remarkable versatility displayed by lysine during the synthesis of complex peptide scaffolds, this chapter describes a simple procedure that enables rapid access to protected N,N'-diaminoalkylated basic amino acid building blocks suitable for standard solid-phase peptide synthesis. This procedure allows preparation of symmetrical, as well as unsymmetrical, dialkylated amino acid derivatives that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analog in which the lysine residue was substituted with its synthetic polyamino derivate. The substitution provided indolicidin analog with increase net positive charge, more ordered secondary structure in biological membranes mimicking conditions, and enhanced antibacterial activity without altering hemolytic activity. Taking into consideration the increasing interest for peptides with unusual structural features due to their improved biological properties, the described synthesis of polyfunctional amino acid building blocks is of particular practical value.

Solid-phase guanidinylation of peptidyl amines compatible with standard Fmoc-chemistry: formation of monosubstituted guanidines.

With the growing importance of peptides and peptidomimetics as potential therapeutic agents, a continuous synthetic interest has been shown for their modification to provide more stable and bioactive analogs. Among many approaches, peptide/peptidomimetic guanidinylation offers access to analogs possessing functionality with strong basic properties, capable of forming stable intermolecular H-bonds, charge pairing, and cation-π interactions. Therefore, guanidinium functional group is considered as an important pharmacophoric element. Although a number of methods for solid-phase guanidinylation reactions exist, only a few are fully compatible with standard Fmoc solid-phase peptide chemistry.In this chapter we summarize the solid-phase guanidinylation methods fully compatible with standard Fmoc-synthetic methodology. This includes use of direct guanidinylating reagents such as 1-H-pyrazole-1-carboxamidine and triflylguanidine, and guanidinylation with di-protected thiourea derivatives in combination with promoters such as Mukaiyama's reagent, N-iodosuccinimide, and N,N'-diisopropylcarbodiimide.

Direct access to side chain N,N'-diaminoalkylated derivatives of basic amino acids suitable for solid-phase peptide synthesis.

A simple and efficient one-pot procedure that enables rapid access to orthogonally protected N,N'-diaminoalkylated basic amino acid building blocks fully compatible with standard Boc and Fmoc solid-phase peptide synthesis is reported. Described synthetic approach includes double reductive alkylation of N (α)-protected diamino acids with N-protected amino aldehydes in the presence of sodium cyanoborohydride. This approach allows preparation of symmetrical, as well as unsymmetrical, basic amino acid derivatives with branched side-chains that can be further modified, enhancing their synthetic utility. The suitability of the synthesized branched basic amino acid building blocks for use in standard solid-phase peptide synthesis has been demonstrated by synthesis of an indolicidin analogue in which the lysine residue was substituted with the synthetic derivative N (α)-(9H-fluorenyl-9-methoxycarbonyl)-N (ß),N (ß) '-bis[2-(tert-butoxycarbonylamino)ethyl]-L-2,3-diaminopropionic acid. This substitution resulted in an analogue with more ordered secondary structure in 2,2,2-trifluoroethanol and enhanced antibacterial activity without altering hemolytic activity.

Discovery of novel inhibitors of a disintegrin and metalloprotease 17 (ADAM17) using glycosylated and non-glycosylated substrates.

A disintegrin and metalloprotease (ADAM) proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off-target toxicity. We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative to active site inhibitors. Potential exosites in ADAM structures have been reported, but no studies describing substrate features necessary for exosite interactions exist. Analysis of ADAM cognate substrates revealed that glycosylation is often present in the vicinity of the scissile bond. To study whether glycosylation plays a role in modulating ADAM activity, a tumor necrosis factor α (TNFα) substrate with and without a glycan moiety attached was synthesized and characterized. Glycosylation enhanced ADAM8 and -17 activities and decreased ADAM10 activity. Metalloprotease (MMP) activity was unaffected by TNFα substrate glycosylation. High throughput screening assays were developed using glycosylated and non-glycosylated substrate, and positional scanning was conducted. A novel chemotype of ADAM17-selective probes was discovered from the TPIMS library (Houghten, R. A., Pinilla, C., Giulianotti, M. A., Appel, J. R., Dooley, C. T., Nefzi, A., Ostresh, J. M., Yu, Y., Maggiora, G. M., Medina-Franco, J. L., Brunner, D., and Schneider, J. (2008) Strategies for the use of mixture-based synthetic combinatorial libraries. Scaffold ranking, direct testing in vivo, and enhanced deconvolution by computational methods. J. Comb. Chem. 10, 3-19; Pinilla, C., Appel, J. R., Borràs, E., and Houghten, R. A. (2003) Advances in the use of synthetic combinatorial chemistry. Mixture-based libraries. Nat. Med. 9, 118-122) that preferentially inhibited glycosylated substrate hydrolysis and spared ADAM10, MMP-8, and MMP-14. Kinetic studies revealed that ADAM17 inhibition occurred via a non-zinc-binding mechanism. Thus, modulation of proteolysis via glycosylation may be used for identifying novel, potentially exosite binding compounds. The newly described ADAM17 inhibitors represent research tools to investigate the role of ADAM17 in the progression of various diseases.

Glycation of a lysine-containing tetrapeptide by D-glucose and D-fructose--influence of different reaction conditions on the formation of Amadori/Heyns products.

The site specificity, extent, and nature of modification of the tetrapeptide, Leu-Ser-Lys-Leu (1), incubated with d-glucose or d-fructose in methanol, or in phosphate buffer of pH 5.7, 7.4, and 8.0 were investigated. The generated mono- and di-glycated Amadori (1-deoxy-d-fructosyl derivatives) and Heyns rearrangement products (N-alkylated glucosamine/mannosamine derivatives) were isolated and characterized by NMR and mass spectrometry. The results identified the epsilon-amino group of the Lys residue as the preferential glycation site in tetrapeptide 1. Under all conditions investigated, glucose afforded higher yields of glycation products than fructose. In the reactions carried out in buffer, glycation at pH 7.4 and 8.0 was much faster than at pH 5.7.