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Messenger RNA (mRNA) display is being increasingly adopted for peptide drug candidate discovery. While many conditions have been reported for the affinity enrichment step and in some cases for peptide modification, there is still limited understanding about the versatility of peptide–puromycin–mRNA/cDNA (complementary DNA) complexes. This work explores the chemical stability of mRNA/cDNA hybrid complexes under a range of different fundamental chemical conditions as well as with peptide modification conditions reported in an mRNA display setting. We further compare the stability of full complexes originating from two different mRNA display systems (RaPID and cDNA‐TRAP). Overall, these complexes were found to be stable under a broad range of conditions, with some edge conditions benefitting from encoding directly in cDNA rather than mRNA. This should allow for more and broader exploitation of late‐stage peptide modification chemistry in mRNA display, with confidence regarding the stability of encoding, and potentially better hit‐finding campaigns as a result. [ABSTRACT FROM AUTHOR]

In ribosomal synthesis of peptides and proteins, genetic information is translated into an amino acid polymer according to the genetic code, which describes the translational command encoded by each codon. However, parts of the genetic code can be adjusted to customize translations. One option is to remove decoding for a specific codon, resulting in a vacant codon. Such vacant codons can be used to stall the ribosome for mechanistic studies and display techniques. Alternatively, the liberated codon can be assigned to encode for incorporation of a noncanonical building block for expansion of the genetic code. In this review we provide an overview of the methods currently available for vacating codons in prokaryotic translation (agnostic of how these are later applied), targeting factors such as amino‐acyl tRNA synthetases, tRNA, release factors, and the initiation machinery. Moreover, we assess applicability and compatibility of the currently available techniques and discuss which have the potential to develop into even more powerful approaches in the future. [ABSTRACT FROM AUTHOR]

Propionibacterium acnes , though generally considered part of the normal flora of human skin, is an opportunistic pathogen associated with acne vulgaris as well as other diseases, including endocarditis, endophthalmitis and prosthetic joint infections. Its virulence potential is also supported by knowledge gained from its sequenced genome. Indeed, a vaccine targeting a putative cell wall-anchored P. acnes sialidase has been shown to suppress cytotoxicity and pro-inflammatory cytokine release induced by the organism, and is proposed as an alternative treatment for P. acnes -associated diseases. Here, we report the crystal structures of the surface sialidase and its complex with the transition-state mimic Neu5Ac2en. Our structural and kinetic analyses, together with insight from a glycan array screen, which probes subtle specificities of the sialidase for α-2,3-sialosides, provide a basis for the structure-based design of novel small-molecule therapeutics against P. acnes infections. [ABSTRACT FROM AUTHOR]

Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct 1HNMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2. [ABSTRACT FROM AUTHOR]

Over the sixty years since Koshland initially formulated the classical mechanisms for retaining and inverting glycosidases, researchers have assembled a large body of supporting evidence and have documented variations of these mechanisms. Recently, however, researchers have uncovered a number of completely distinct mechanisms for enzymatic cleavage of glycosides involving elimination and/or hydration steps. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) display is being increasingly adopted for peptide drug candidate discovery. While many conditions have been reported for the affinity enrichment step and in some cases for peptide modification, there is still limited understanding about the versatility of peptide-puromycin-mRNA/cDNA (complementary DNA) complexes. This work explores the chemical stability of mRNA/cDNA hybrid complexes under a range of different fundamental chemical conditions as well as with peptide modification conditions reported in an mRNA display setting. We further compare the stability of full complexes originating from two different mRNA display systems (RaPID and cDNA-TRAP). Overall, these complexes were found to be stable under a broad range of conditions, with some edge conditions benefitting from encoding directly in cDNA rather than mRNA. This should allow for more and broader exploitation of late-stage peptide modification chemistry in mRNA display, with confidence regarding the stability of encoding, and potentially better hit-finding campaigns as a result., (© 2024 The Authors. Chemistry - An Asian Journal published by Wiley-VCH GmbH.)

The human complement pathway plays a pivotal role in immune defence, homeostasis, and autoimmunity regulation, and complement-based therapeutics have emerged as promising interventions, with both antagonistic and agonistic approaches being explored. The classical pathway of complement is initiated when the C1 complex binds to hexameric antibody platforms. Recent structural data revealed that C1 binds to small, homogeneous interfaces at the periphery of the antibody platforms. Here, we have developed a novel strategy for complement activation using macrocyclic peptides designed to mimic the interface between antibodies and the C1 complex. In vitro selection utilizing the RaPID system identified a cyclic peptide (cL3) that binds to the C1 complex via the globular head domains of C1q. Notably, when immobilized on surfaces, cL3 effectively recruits C1 from human serum, activates C1s proteases, and induces lysis of cell-mimetic lipid membranes. This represents the first instance of a peptide capable of activating complement by binding C1 when immobilized. Further characterization and synthesis of deletion mutants revealed a critical cycle size of cL3 essential for C1 binding and efficient complement activation. Importantly, cL3 also demonstrated the ability to inhibit complement-mediated lysis without affecting C1 binding, highlighting its potential as a therapeutic modality to prevent complement-dependent cytotoxicity whilst promoting cellular phagocytosis and cell clearance. In summary, this study introduces the concept of " Peptactins " - peptide-based activators of complement - and underscores the potential of macrocyclic peptides for complement modulation, offering potential advantages over traditional biologicals in terms of size, production, and administration., Competing Interests: SMWRH, LA, ALB and THS are co-authors on a patent describing the development and use of peptactins. SAKJ declares no competing interests., (This journal is © The Royal Society of Chemistry.)

Herpes simplex virus (HSV-1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late-stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV-1. A range of HS-oligosaccharides was prepared to examine the importance of chain length and 2-O-sulfation of iduronic moieties for Hpse inhibition. It was found that hexa- and octasaccharides potently inhibited the enzyme and that 2-O-sulfation of iduronic acid is tolerated. Computational studies provided a rationale for the observed structure-activity relationship. Treatment of human corneal epithelial cells (HCEs) infected with HSV-1 with the hexa- and octasaccharide blocked viral induced shedding of HS which significantly reduced spread of virions. The compounds also inhibited migration and proliferation of immortalized HCEs thereby providing additional therapeutic properties., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)