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The development of novel antivirals is crucial not only for managing current COVID‐19 infections but for addressing potential future zoonotic outbreaks. SARS‐CoV‐2 main protease (Mpro) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS‐CoV‐2 Mpro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation‐tolerant amino acid residues within the peptide sequence, we describe the development of a second‐generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS‐CoV‐2 Mpro. [ABSTRACT FROM AUTHOR]

The non-heme iron-dependent dioxygenase 2-aminoethanethiol (aka cysteamine) dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyzes O2-dependent Nt-cys sulfinylation, which promotes proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signaling (RGS) 4 and 5, and the proinflammatory cytokine interleukin-32, all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. However, the role of individual chemical groups, active site metal, amino acid composition, and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and interleukin-32. We demonstrate, using surface plasmon response and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through ¹H-15N heteronuclear single quantum coherence nuclear magnetic resonance titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation. [ABSTRACT FROM AUTHOR]

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD--the N-terminal BD of BRD3--using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution. [ABSTRACT FROM AUTHOR]

The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (M pro ) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 M pro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation M pro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 M pro ., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e-SE) platform for the efficient site-selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e-SE is highlighted through late-stage C-terminal modification of the FDA-approved cancer drug leuprolide and assembly of a library of anti-HER2 affibody conjugates bearing complex cargoes. Following assembly by e-SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The decoration of proteins with post-translational modifications (PTMs) serves as a mechanism to expand the functional repertoire of the proteome. Tyrosine sulfation is a PTM that has been shown to be a key regulator of extracellular protein-protein interactions in a select number of examples. However, the challenges associated with identifying and characterising the functional consequences of tyrosine sulfation have hindered our ability to understand the full scope of its role in the wider proteome when compared with that of other PTMs, for example, phosphorylation and glycosylation. In this account, we highlight recent advances in the prediction and detection of tyrosine sulfation and outline the need for continued innovation in this area. We also discuss the utility of emerging solid-phase synthesis and peptide ligation strategies for accessing libraries of homogeneously sulfated peptides and proteins to help reveal functional aspects of the sulfoproteome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)