Peptide stapling is a strategy for constraining short peptides typically in an alpha-helical conformation. Stapling is carried out by covalently linking the side-chains of two amino acids, thereby forming a peptide macrocycle. There is an expanding repertoire of stapling techniques based on different macrocyclisation chemistries. In this tutorial review, we categorise and analyse key examples of peptide stapling in terms of their synthesis and applicability to biological systems.

A divergent synthetic strategy for generating helical p53 peptides bearing functionalised staple linkages, allowing for efficient optimisation of cellular activity.

Since the advent of click chemistry in 2001, the 1,4-disubstituted triazole has become an increasingly common motif in chemical sensors. Although these click-derived triazoles are generally used as a convenient method of ligation, their prevalence in chemosensors can be attributed to their ability to bind both cations and anions. In this critical review, we present an overview of the wide range of chemosensors that contain click-derived triazoles, with a particular focus on those cases where the triazole plays a functional, rather than merely a structural, role. Examples are categorised based on method of detection and key structural features, providing a complete picture of the current state of click-based chemosensors, as well as potential future directions for sensor design. (140 references).

Compartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae, based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments afford protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.

Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.

We report a double-click macrocyclization approach for the design of constrained peptide inhibitors having non-helical or extended conformations. Our targets are the tankyrase proteins (TNKS), poly(ADP-ribose) polymerases (PARP) that regulate Wnt signaling by targeting Axin for degradation. TNKS are deregulated in many different cancer types, and inhibition of TNKS therefore represents an attractive therapeutic strategy. However, clinical development of TNKS-specific PARP catalytic inhibitors is challenging due to off-target effects and cellular toxicity. We instead targeted the substrate-recognition domain of TNKS, as it is unique among PARP family members. We employed a two-component strategy, allowing peptide and linker to be separately engineered and then assembled in a combinatorial fashion via click chemistry. Using the consensus substrate-peptide sequence as a starting point, we optimized the length and rigidity of the linker and its position along the peptide. Optimization was further guided by high-resolution crystal structures of two of the macrocyclized peptides in complex with TNKS. This approach led to macrocyclized peptides with submicromolar affinities for TNKS and high proteolytic stability that are able to disrupt the interaction between TNKS and Axin substrate and to inhibit Wnt signaling in a dose-dependent manner. The peptides therefore represent a promising starting point for a new class of substrate-competitive inhibitors of TNKS with potential for suppressing Wnt signaling in cancer. Moreover, by demonstrating the application of the double-click macrocyclization approach to non-helical, extended, or irregularly structured peptides, we greatly extend its potential and scope, especially given the frequency with which such motifs mediate protein–protein interactions.

Peptide cyclization is a useful strategy for the stabilization of short flexible peptides into well-defined bioactive conformations, thereby enhancing their ability to interact with proteins and other important biomolecules. We present an optimized procedure for the stabilization of linear diazido peptides in an α-helical conformation upon reaction with dialkynyl linkers under Cu(I) catalysis. As this procedure generates side chain-cyclized peptides bearing a bis-triazole linkage, it is referred to as 'double-click' stapling. Double-click stapling can enhance the binding affinity, proteolytic stability and cellular activity of a peptide inhibitor. A distinguishing feature of double-click stapling is the efficiency with which peptides bearing different staple linkages can be synthesized, thus allowing for modular control over peptide bioactivity. This protocol describes the double-click reaction between a 1,3-dialkynylbenzene linker and peptides that contain azidoornithine. Subsequent peptide purification and confirmation steps are also described. The entire double-click stapling protocol can be completed in ∼48 h, including two overnight lyophilization steps.

The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10–25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.