Antimicrobial peptides (AMPs), also known as host defense peptides, are short and generally positively charged peptides found in a wide variety of life forms from microorganisms to humans. Most AMPs have the ability to kill microbial pathogens directly, whereas others act indirectly by modulating the host defense systems. Against a background of rapidly increasing resistance development to conventional antibiotics all over the world, efforts to bring AMPs into clinical use are accelerating. Several AMPs are currently being evaluated in clinical trials as novel anti-infectives, but also as new pharmacological agents to modulate the immune response, promote wound healing, and prevent post-surgical adhesions. In this review, we provide an overview of the biological role, classification, and mode of action of AMPs, discuss the opportunities and challenges to develop these peptides for clinical applications, and review the innovative formulation strategies for application of AMPs.

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Antimicrobial Peptides: An Emerging Category of Therapeutic Agents

Over the past decade, peptide drug discovery has experienced a revival of interest and scientific momentum, as the pharmaceutical industry has come to appreciate the role that peptide therapeutics can play in addressing unmet medical needs and how this class of compounds can be an excellent complement or even preferable alternative to small molecule and biological therapeutics. In this Perspective, we give a concise description of the recent progress in peptide drug discovery in a holistic manner, highlighting enabling technological advances affecting nearly every aspect of this field: from lead discovery, to synthesis and optimization, to peptide drug delivery. An emphasis is placed on describing research efforts to overcome the inherent weaknesses of peptide drugs, in particular their poor pharmacokinetic properties, and how these efforts have been critical to the discovery, design, and subsequent development of novel therapeutics.

The Current State of Peptide Drug Discovery: Back to the Future?

Bioconjugation methodologies have proven to play a central enabling role in the recent development of biotherapeutics and chemical biology approaches. Recent endeavours in these fields shed light on unprecedented chemical challenges to attain bioselectivity, biocompatibility, and biostability required by modern applications. In this review the current developments in various techniques of selective bond forming reactions of proteins and peptides were highlighted. The utility of each endogenous amino acid-selective conjugation methodology in the fields of biology and protein science has been surveyed with emphasis on the most relevant among reported transformations; selectivity and practical use have been discussed.

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Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation

The native chemical ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro- and nanotechnology research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and physical chemistry studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O, S-, N, S-, or N, Se-acyl shift systems as thioester or selenoester surrogates and discusses the special molecular features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N, S-acyl migrations and selective dechalcogenation reactions.

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

Convergent Chemical Synthesis of Proteins by Ligation of Peptide Hydrazides

Protein chemical synthesis by native peptide ligation of unprotected peptide segments is an interesting complement and potential alternative to the use of living systems for producing proteins. Actually, tremendous efforts are focused on the design of one-pot strategies allowing the assembly of three peptide segments. The goal is to get rapid access to small proteins (less than 150 amino acid residues), while saving intermediate purification steps and obtaining the products in good yield. Such methods are gaining increasing significance for the study of protein function and appear as a potential option for producing various protein-based therapeutics currently under development. To date, proteins were mainly assembled by sequential native chemical ligation (NCL) or extended methodologies in the C-to-N direction (for recent achievements, see Refs. [8, 9]). NCL involves the chemoselective ligation of a Cterminal peptide thioester, usually an alkylthioester, with an N-terminal cysteine (Cys) peptide. The one-pot sequential Cto-N ligation of three peptide segments designed by Kent et al. is increasingly used for synthesizing proteins. Methods that enable the assembly of peptide segments in the reverse N-to-C direction are rare. 11] Fundamentally, the combination of N-to-C and C-to-N assembly techniques is at the basis of the convergent total synthesis of proteins. The general principle of the one-pot assembly of three peptide segments in the N-to-C direction is illustrated in Scheme 1. Ligation of peptide segments A-X and H-Cys-B-Y yields segment A-Cys-B-Y (Scheme 1, ligation 1). Group Y must ideally be inert during ligation 1 or at least be significantly less reactive than group X to avoid oligomerization or cyclization of segment B. Activation of group Y into Y* subsequently allows the ligation with the third segment H-Cys-C (Scheme 1, ligation 2). For designing a one-pot process working in the N-to-C direction, this activation must be carried out in situ after ligation 1 by using reagents compatible with ligation 2. Furthermore, the Y* group must enable an efficient ligation with the Cys segment C. To date only few one-pot strategies have been described that work in the N-to-C direction and enable the coupling of three peptide segments. 5, 12] Fundamentally, these methods, such as kinetically controlled ligation, rely on the differential reactivity of X and Y groups for peptide-bond formation. In other words, the purity of the target polypeptide is highly dependent on the C-terminal residues of A and B segments and more generally on the accessibility of the reactive ends. Clearly, a strategy in which Y is inert during the first ligation step would bypass these limitations and constitute a critical advance. Herein we show that the combination of NCL and SEA ligation (Scheme 1) permitted design of a solution to this important problem. Reaction of a peptide featuring a C-terminal bis(2-sulfanylethyl)amido group, called SEA hereafter (Scheme 1), with a Cys peptide results in the formation of a native peptide bond in water at pH 7. This reaction probably proceeds via Scheme 1. Total protein synthesis by one-pot assembly of three peptide segments in the N-to-C direction. The first step is a native chemical ligation between thioester segment A and Cys segment B, during which the cyclic disulfide SEA acts as a blocked thioester group (SEA = bis(2-sulfanylethyl)amido). Activation of SEA into SEA by reduction with a phosphine and addition of the third Cys segment C triggers the second ligation step.

A One‐Pot Three‐Segment Ligation Strategy for Protein Chemical Synthesis

The chemical synthesis of peptides or small proteins is often an important step in many research projects and has stimulated the development of numerous chemical methodologies. The aim of this review is to give a substantial overview of the solid phase methods developed for the production or purification of polypeptides. The solid phase peptide synthesis (SPPS) technique has facilitated considerably the access to short peptides (<50 amino acids). However, its limitations for producing large homogeneous peptides have stimulated the development of solid phase covalent or non-covalent capture purification methods. The power of the native chemical ligation (NCL) reaction for protein synthesis in aqueous solution has also been adapted to the solid phase by the combination of novel linker technologies, cysteine protection strategies and thioester or N,S-acyl shift thioester surrogate chemistries. This review details pioneering studies and the most recent publications related to the solid phase chemical synthesis of large peptides and proteins.

Solid phase protein chemical synthesis.

A statistical view of protein chemical synthesis using NCL and extended methodologies.

Although the majority of proteins used for biomedical research are produced using living systems such as bacteria, biological means for producing proteins can be advantageously complemented by protein semisynthesis or total chemical synthesis. The latter approach is particularly useful when the proteins to be produced are toxic for the expression system or show unusual features that cannot be easily programmed in living organisms. The aim of this review is to provide a wide overview of the use of chemical protein synthesis in medicinal chemistry with a special focus on the production of post-translationally modified proteins and backbone cyclized proteins.

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Ouafâa El Mahdi

Papers

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

A One‐Pot Three‐Segment Ligation Strategy for Protein Chemical Synthesis

Solid phase protein chemical synthesis.

A statistical view of protein chemical synthesis using NCL and extended methodologies.

Chemical Protein Synthesis in Medicinal Chemistry.