Titia

Nature Chemical Biology, Published online: 05 December 2023; doi:10.1038/s41589-023-01481-5

Qi et al. used phage-assisted evolution to optimize SlugCas9, a compact Cas9 nuclease, for NNG PAM recognition and developed a SlugCas9-NNG based adenine base editor for single AAV delivery.

Selective detection of disease-associated changes in the cellular glycocalyx is a foundation of modern targeted therapies. Detecting minor changes in the density and identity of glycans on the cell surface is a technological challenge exacerbated by lack of 1:1 correspondence between cellular DNA/RNA and glycan structures on cell surface. We demonstrate that multivalent displays of up to 300 lectins on DNA-barcoded M13 phage on a liquid lectin array (LiLA), detects subtle differences in composition and density of glycans on cells ex vivo and in immune cells or organs in animals. For example, constructs displaying 73 copies of diCBM40 lectin per 700x5 nm virion ({varphi}-CBM73) exhibit non-linear ON/OFF-like recognition of sialoglycans on the surface of normal and cancer cells. In contrast, a high-valency {varphi}-CBM290 display, or soluble diCBM40, exhibit canonical progressive scaling in binding with increased epitope density; these constructs cannot amplify the subtle differences detected by {varphi}-CBM73. Similarly, multivalent displays of diCBM40 and Siglec-7 detect differences in the glycocalyx between stem-like and non-stem populations in cancer cells that are not detected with soluble lectins. Multivalent display of lectins on M13 scaffold with protected DNA inside the phage offer non-destructive detection of minor differences in glycocalyx in cells in vitro and in vivo not feasible to currently available technologies.

Nature Chemical Biology, Published online: 20 November 2023; doi:10.1038/s41589-023-01484-2

Oxygen sensitivity hampers applications of metal-dependent CO2 reductases. Here, Oliveira et al. describe how an allosteric disulfide bond controls the activity of a CO2 reductase, preventing its physiological reduction during transient O2 exposure and allowing aerobic handling of the enzyme.

A library of disulfide linkers was established for Cys−Cys−(Cys) double/triple click stapling in only 1 min, bridging from 3 to 18 amino acid residues to deliver 18- to 48-membered locked peptides. The opposite unstapling process proceeded smoothly, making the reversible cross-linking reagents potentially suitable for peptide delivery.

Abstract

A disulfide click strategy is disclosed for stapling to enhance the metabolic stability and cellular permeability of therapeutic peptides. A 17-membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S-terminal peptides from 3 to 18 amino acid residues to provide 18- to 48-membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti-HCT-116 activity with a locked α-helical conformation (IC₅₀=6.81 μM vs. biological incompetence for acyclic linear peptides), which could be unstapled for rehabilitation of the native peptides under the assistance of tris(2-carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three-dimensional conformations, enabling their cellular uptake followed by release of the disulfides for peptide delivery.

Cyclic peptides combine a number of favorable properties that make them attractive for drug development. Today, more than 40 therapeutics based on cyclic peptides are in use, and new, powerful technologies for their development suggest that this number could grow rapidly. In this review, we feature cyclic peptide drugs developed in the past, discuss the properties of this molecule class, describe technological advances, and discuss current challenges and opportunities for developing the next generation of cyclic peptide therapeutics.

Abstract

Cyclic peptides are fascinating molecules abundantly found in nature and exploited as molecular format for drug development as well as other applications, ranging from research tools to food additives. Advances in peptide technologies made over many years through improved methods for synthesis and drug development have resulted in a steady stream of new drugs, with an average of around one cyclic peptide drug approved per year. Powerful technologies for screening random peptide libraries, and de novo generating ligands, have enabled the development of cyclic peptide drugs independent of naturally derived molecules and now offer virtually unlimited development opportunities. In this review, we feature therapeutically relevant cyclic peptides derived from nature and discuss the unique properties of cyclic peptides, the enormous technological advances in peptide ligand development in recent years, and current challenges and opportunities for developing cyclic peptides that address unmet medical needs.

An inspiring collaboration between pharmaceutical industry and academia enabled discovery, structure elucidation, and biosynthetic pathway analysis of streptnatamide A, a new cyclic peptide isolated from secondary metabolites of a marine invertebrate-associated Streptomyces sp. This study highlights the application of mass spectrometry tools that enables the rapid unambiguous structure elucidation of this complex molecule.

Abstract

Cyclic peptides have been excellent source of drug leads. With the advances in discovery platforms, the pharmaceutical industry has a growing interest in cyclic peptides and has pushed several into clinical trials. However, structural complexity of cyclic peptides brings extreme challenges for structure elucidation efforts. Isotopic fine structure analysis, Nuclear magnetic resonance (NMR), and detailed tandem mass spectrometry rapidly provided peptide sequence for streptnatamide A, a cyclic peptide isolated from a marine-derived Streptomyces sp. Marfey's analysis determined the stereochemistry of all amino acids, enabling the unambiguous structure determination of this compound. A non-ribosomal peptide synthetase biosynthetic gene cluster (stp) was tentatively identified and annotated for streptnatamide A based on the in silico analysis of whole genome sequencing data. These analytical tools will be powerful tools to overcome the challenges for cyclic peptide structure elucidation and accelerate the development of bioactive cyclic peptides.

Thioamides have structural and chemical similarity to peptide bonds, and therefore offer valuable insights when probing peptide backbone interactions, including hydrogen bonding, stereoelectronic, and hydrophobicity effects. There is a perception that methods to install thioamides within peptides are sufficient, yet anecdotal reports indicate that many labs have sought to employ thioamides in a variety of studies but the results of many synthetic campaigns do not yield the intended products, leading researchers to abandon such projects and any information these structural probes would provide. We catalogue and provide evidence for the major pitfalls associated with current methods to synthesize thioamide-containing peptides during each stage of solid-phase peptide synthesis (SPPS), including (A) thioamide coupling, (B) peptide elongation, and (C) peptide cleavage from resin. We then demonstrate the utility of thioimidate protecting groups as a means to side-step each of these problematic synthetic difficulties. Our approach is generally applicable to all peptides and ultimately permits access to an important benchmark $\alpha$-helical peptide that had previously eluded synthesis and isolation. With the process of thionopeptide synthesis demystified, a broader range of researchers should find it easier to employ thioamides in the study of peptide-based biomolecules.

Reciprocal stapling of two helices, an aromatic foldamer helix and a peptide α-helix, was observed in the conformation of a hybrid foldamer–peptide macrocycle when bound to the protein target against which it was selected. This intriguing shape and the possible contribution of the foldamer to protein binding highlight potential benefits of inserting a foldamer segment in display selection of macrocyclic peptides.

Abstract

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer–peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.

Isonitrileproline (Inp) is introduced as the first isonitrile-containing amino acid for solid-phase peptide synthesis. The conformation directing properties of the isonitrile group and its effect on the collagen triple helix were elucidated. The value of Inp for peptide derivatization was showcased by a chemoselective ligation with a functional chlorooxime.

Abstract

Functional groups that allow for chemoselective and bioorthogonal derivatization are valuable tools for the labelling of peptides and proteins. The isonitrile is such a group but synthetic methods for its incorporation into peptides by solid-phase peptide synthesis are not known. Here, we introduce (4S)- and (4R)-isonitrileproline (Inp) as building blocks for solid-phase peptide synthesis. Conformational studies of (4S)- and (4R)-Inp and thermal stability analysis of Inp-containing collagen triple helices revealed that the isonitrile group exerts a stereoelectronic gauche effect. We showcase the value of Inp for bioorthogonal labelling by derivatization of Inp-containing collagen model peptides (CMPs). Dual labelling with a pair of bioorthogonal reactions of a CMP containing Inp and azidoproline residues further highlights the versatility of the new isonitrile-containing amino acids.

In phage display selection using M13 bacteriophage, the M13 main body, which is a huge filament>10³ time larger than the displayed peptide, has been considered to interfere with the result of affinity selection. This interference can be attenuated by magnetically orienting the M13 main body vertical to the target surface, which results in the suppression of nonspecific adhesion and the change in the population of selected M13 clones.

Abstract

Although phage display selection using a library of M13 bacteriophage has become a powerful tool for finding peptides that bind to target materials on demand, a remaining concern of this method is the interference by the M13 main body, which is a huge filament >10³ times larger than the displayed peptide, and therefore would nonspecifically adhere to the target or sterically inhibit the binding of the displayed peptide. Meanwhile, filamentous phages are known to be orientable by an external magnetic field. If M13 filaments are magnetically oriented during the library selection, their angular arrangement relative to the target surface would be changed, being expected to control the interference by the M13 main body. This study reports that the magnetic orientation of M13 filaments vertical to the target surface significantly affects the selection. When the target surface was affinitive to the M13 main body, this orientation notably suppressed the nonspecific adhesion. Furthermore, when the target surface was less affinitive to the M13 main body and intrinsically free from the nonspecific adhesion, this orientation drastically changed the population of M13 clones obtained through library selection. The method of using no chemicals but only a physical stimulus is simple, clean, and expected to expand the scope of phage display selection.

Novel boronic acids have been developed for the catalyzed amide synthesis. Lewis acidity estimation using DFT, associated with collected kinetic data, allowed to uncover ortho-(sulfonyloxy)benzeneboronic acids that compared favorably with the established state-of-the-art boronic acids. Efficient coupling of aliphatic, aromatic, and heteroaromatic carboxylic acids as well as primary and secondary amines were obtained.

Abstract

This study outlines the development of novel boronic acids as catalysts for the direct synthesis of amides from carboxylic acids and amines. The Lewis acidity of the boronic acids was estimated by means of computational techniques, and the observed increase in catalytic activity was corroborated by kinetic data derived from a model reaction. Our investigations led to the discovery of a set of ortho-(sulfonyloxy)benzeneboronic acids that compared favorably with the established state-of-the-art. These newly developed catalysts demonstrated efficacy in the coupling of aliphatic, aromatic, and heteroaromatic acids, as well as primary and secondary amines.

Herein, the application of diethylazodicarboxylate as an efficient and highly chemoselective oxidising reagent for the cyclisation of peptides through a disulfide bond is demonstrated. The scope of this application was demonstrated both in solution and on solid phase, and can be extended to the cyclisation of peptides containing oxidation-sensitive residues, such as methionine and tryptophan.

Abstract

Cyclic peptides are important molecules, playing key roles in protein architecture, as chemical probes, and increasingly as crucial structural elements of clinically-useful therapeutics. Herein we report methodology using azodicarboxylates as efficient reagents for the facile synthesis of cyclic peptides through a disulfide bridge. The utility of this approach in both solution and solid-phase, and compatibility with common amino acid side chain functionalities is demonstrated, resulting in cyclic peptides in good yield and purity. This approach has significant potential application for synthesis of molecules of biological or therapeutic significance.

Phage display is commonly employed for the discovery of high affinity ligands to biomolecular targets. However, ranking the discovered ligands for their affinity and specificity to the target is obscured by genetic amplification bias and amplification of target-unrelated phage, resulting in inefficient experimental validation and potentially intractable discovery. Here, we describe the use of indirect machine learning (ML) to improve the efficient discovery of target-specific peptide ligands from next-generation sequencing (NGS) data. We combine peptide sequence information (input) with experimental fitness scores (output) of the individual peptide performance across the rounds of bio-panning in a bidirectional long short-term memory (BiLSTM) architecture. Because the fitness scores contain bias, we use regularization to facilitate limited indirect learning and effectively process the peptide sequence information, while still using the predicted fitness scores to rank the peptides. Peptides containing high-affinity binding motifs to our target were ranked by the regularized model more than threefold higher, compared to any combination of experimental fitness scores. Baseline models of random forest (RF) and -nearest neighbor (KNN) demonstrated slightly lower performance but also demonstrated the importance of regularization. However, the BiLSTM model emerged as the most robust, as it was less sensitive to the peptide representation and the specific fitness score used. Shapley residue analysis generated interpretable structure-activity-relationship (SAR) by providing insight into predicted affinity-driving residues and physicochemical properties across the entire peptide and as well as at motif-specific positions. We expect that this approach will elucidate high-affinity ligands against a multitude of targets, vastly improving the discovery capability of phage display.

Photoredox-catalysed reaction of activated thioethers unveils alanyl radical intermediates in a stereoretentive and site-selective manner. Installation of the activating group is simple, and the intermediate radical can react with non-activated alkenes to form non-natural residues bearing aliphatic, hydrophobic units. The method is applicable to late-stage peptide functionalisation and expands the scope of analogues that can be accessed.

Abstract

Late-stage functionalisation is an attractive method to generate peptide analogues containing non-natural residues. It is shown that cysteine residues can be activated as Crich-type thioethers, either by alkylation of a synthetic cysteine-continuing peptide or by incorporation of a modified cysteine unit into solid phase or solution phase peptide synthesis. Photoredox catalysed reaction of the thioether generates an alanyl radical intermediate in a stereoretentive and site-selective manner, even in the presence of free cysteine residues. The radical can react with non-activated alkenes to form non-natural residues bearing aliphatic, hydrophobic units. A method to avoid unwanted alkylation of amine residues was identified and the process was applied to the functionalization of both linear and cyclic synthetic peptides.

Straightforward access to seleno-based light-cleavable auxiliaries, including diverse amino acids, facilitates access to challenging protein targets via diselenide–selenoester ligations (DSL) and expressed protein selenoester ligations (EPSL).

Abstract

Diselenide–selenoester ligations are increasingly used for the synthesis of proteins. Excellent ligation rates, even at low concentrations, in combination with mild and selective deselenization conditions can overcome some of the most severe challenges in chemical protein synthesis. Herein, the versatile multicomponent synthesis and application of a new ligation auxiliary that combines a photocleavable scaffold with the advantages of selenium-based ligation strategies are presented. Its use was investigated with respect to different ligation junctions and describe a novel para-methoxybenzyl deprotection reaction for the selenol moiety. The glycine-based auxiliary enabled successful synthesis of the challenging target protein G-CSF.

This work presents a detailed computational investigation on reaction mechanisms of the macrocyclization of linear peptide aldehydes to selectively form imidazolidinone cyclic peptides. Our work demonstrates that intramolecular hydrogen bonds (IMHBs) act as transient internal factors to control stereoselectivity by promoting a kinetically facile zwitterionic mechanism.

Abstract

Macrocyclic peptides have become increasingly important in the pharmaceutical industry. We present a detailed computational investigation of the reaction mechanism of the recently developed “CyClick” chemistry to selectively form imidazolidinone cyclic peptides from linear peptide aldehydes, without using catalysts or directing groups (Angew. Chem. Int. Ed. 2019, 58, 19073–19080). We conducted computational mechanistic to investigate the effects of intramolecular hydrogen bonds (IMHBs) in promoting a kinetically facile zwitterionic mechanism in “CyClick” of pentapeptide aldehyde AFGPA. Our DFT calculations highlighted the importance of IMHB in pre-organization of the resting state, stabilization of the zwitterion intermediate, and the control of the product stereoselectivity. Furthermore, we have also identified that the low ring strain energy promotes the “CyClick” of hexapeptide aldehyde AAGPFA to form a thermodynamically more stable 15+5 imidazolidinone cyclic peptide product. In contrast, large ring strain energy suppresses “CyClick” reactivity of tetra peptide aldehyde AFPA from forming the 9+5 imidazolidinone cyclic peptide product.