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Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3., (© 2024. The Author(s).)

Many systemic cancer chemotherapies comprise a combination of drugs, yet all clinically used antibody-drug conjugates (ADCs) contain a single-drug payload. These combination regimens improve treatment outcomes by producing synergistic anticancer effects and slowing the development of drug-resistant cell populations. In an attempt to replicate these regimens and improve the efficacy of targeted therapy, the field of ADCs has moved towards developing techniques that allow for multiple unique payloads to be attached to a single antibody molecule with high homogeneity. However, the methods for generating such constructs-homogeneous multi-payload ADCs-are both numerous and complex owing to the plethora of reactive functional groups that make up the surface of an antibody. Here, by summarizing and comparing the methods of both single- and multi-payload ADC generation and their key preclinical and clinical results, we provide a timely overview of this relatively new area of research. The methods discussed range from branched linker installation to the incorporation of unnatural amino acids, with a generalized comparison tool of the most promising modification strategies also provided. Finally, the successes and challenges of this rapidly growing field are critically evaluated, and from this, future areas of research and development are proposed., (© 2024. Springer Nature Limited.)

Design of the next-generation of therapeutics, biosensors, and molecular tools for basic research requires that we bring protein activity under control. Each protein has unique properties, and therefore, it is critical to tailor the current techniques to develop new regulatory methods and regulate new proteins of interest (POIs). This perspective gives an overview of the widely used stimuli and synthetic and natural methods for conditional regulation of proteins., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Fusion proteins play an essential role in the biosciences but suffer from several key limitations, including the requirement for N-to-C terminal ligation, incompatibility of constituent domains, incorrect folding, and loss of biological activity. This perspective focuses on chemical and enzymatic approaches for the post-translational generation of well-defined protein-protein conjugates, which overcome some of the limitations faced by traditional fusion techniques. Methods discussed range from chemical modification of nucleophilic canonical amino acid residues to incorporation of unnatural amino acid residues and a range of enzymatic methods, including sortase-mediated ligation. Through summarizing the progress in this rapidly growing field, the key successes and challenges associated with using chemical and enzymatic approaches are highlighted and areas requiring further development are discussed.

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody-drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol ( CPO-PFP ), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2 × cys-GFP , containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.