Revealing molecular determinants governing mambalgin-3 pharmacology at acid-sensing ion channel 1 variants.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a.

Chemical synthesis of grafted cyclotides using a "plug and play" approach.

Cyclotides are a diverse class of plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology. Their remarkable structural stability and resistance to proteolytic degradation can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. Thus, cyclotides have emerged as powerful scaffold molecules for designing peptide-based therapeutics.

Repurposing a plant peptide cyclase for targeted lysine acylation.

Transpeptidases are powerful tools for protein engineering but are largely restricted to acting at protein backbone termini. Alternative enzymatic approaches for internal protein labelling require bulky recognition motifs or non-proteinogenic reaction partners, potentially restricting which proteins can be modified or the types of modification that can be installed. Here we report a strategy for labelling lysine side chain ε-amines by repurposing an engineered asparaginyl ligase, which naturally catalyses peptide head-to-tail cyclization, for versatile isopeptide ligations that are compatible with peptidic substrates.

Isolation and Characterization of Insecticidal Cyclotides from .

Cyclotides are cysteine-rich plant-derived peptides composed of 28-37 amino acids with a head-to-tail cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Their beneficial biophysical properties make them promising molecules for pharmaceutical and agricultural applications. The Violaceae plant family is the major cyclotide-producing family, and to date, every examined plant from this family has been found to contain cyclotides.

Tertiary amide bond formation by an engineered asparaginyl ligase.

Transpeptidases are powerful tools for site-specific protein modification, enabling the production of tailored biologics to investigate protein function and aiding the development of next-generation therapeutics and diagnostics. Although protein labelling at the N- or C-terminus is readily accomplished using a range of established transpeptidases, these reactions are generally limited to forming products that are linked by a standard (secondary) amide bond. Here we show that, unlike other widely used transpeptidases, an engineered asparaginyl ligase is able to efficiently synthesise tertiary amide bonds by accepting diverse secondary amine nucleophiles.

Nucleation of a key beta-turn promotes cyclotide oxidative folding.

Cyclotides are plant-derived peptides characterized by a head-to-tail cyclic backbone and a cystine knot motif comprised of three disulfide bonds. Formation of this motif via in vitro oxidative folding can be challenging and can result in misfolded isomers with nonnative disulfide connectivities. Here, we investigated the effect of β-turn nucleation on cyclotide oxidative folding.

Sortase-Catalyzed Protein Domain Inversion.

Topological transformations and permutations of proteins have attracted significant interest as strategies to generate new protein functionalities or stability. These efforts have mainly been inspired by naturally occurring post-translational modifications, such as head-to-tail cyclization, circular permutation, or lasso-like entanglement. Such approaches can be realized experimentally via genetic encoding, in the case of circular permutation, or via enzymatic processing, in the case of cyclization.

Development of Melanocortin 4 Receptor Agonists by Exploiting Animal-Derived Macrocyclic, Disulfide-Rich Peptide Scaffolds.

G protein-coupled receptors are among the most widely studied classes of drug targets. A major challenge in this field is to develop ligands that will selectively modulate a single receptor subtype to overcome the disadvantages of undesired "off target" effects caused by lack of target and thus signaling specificity. In the current study, we explored ligand design for the melanocortin 4 receptor (MC4R) since it is an attractive target for developing antiobesity drugs.

A Chemoenzymatic Approach To Produce a Cyclic Analogue of the Analgesic Drug MVIIA (Ziconotide).

Ziconotide (ω-conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA.

Pain-causing stinging nettle toxins target TMEM233 to modulate Na1.7 function.

Voltage-gated sodium (Na) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived Na channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at Na channels, and that co-expression of TMEM233 modulates the gating properties of Na1.

Native and Engineered Cyclic Disulfide-Rich Peptides as Drug Leads.

Bioactive peptides are a highly abundant and diverse group of molecules that exhibit a wide range of structural and functional variation. Despite their immense therapeutic potential, bioactive peptides have been traditionally perceived as poor drug candidates, largely due to intrinsic shortcomings that reflect their endogenous heritage, i.e.

Late-Stage Functionalization with Cysteine Staples Generates Potent and Selective Melanocortin Receptor-1 Agonists.

In this work, cysteine staples were used as a late-stage functionalization strategy to diversify peptides and build conjugates targeting the melanocortin G-protein-coupled receptors [melanocortin receptor-1 (MC1R) and MC3R-MC5R]. Monocyclic and bicyclic agonists based on sunflower trypsin inhibitor-1 were used to generate a selection of stapled peptides that were evaluated for binding (p) and functional activation (pEC) of the melanocortin receptor subtypes. Stapled peptides generally had improved activity, with aromatic stapled peptides yielding selective MC1R agonists, including a xylene-stapled peptide () with an EC of 1.

Neurotoxic and cytotoxic peptides underlie the painful stings of the tree nettle Urtica ferox.

The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U.

Cystine Knot Peptides with Tuneable Activity and Mechanism.

Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin.

Phage display-based discovery of cyclic peptides against the broad spectrum bacterial anti-virulence target CsrA.

Small macrocyclic peptides are promising candidates for new anti-infective drugs. To date, such peptides have been poorly studied in the context of anti-virulence targets. Using phage display and a self-designed peptide library, we identified a cyclic heptapeptide that can bind the carbon storage regulator A (CsrA) from Yersinia pseudotuberculosis and displace bound RNA.

Enzymatic C-to-C Protein Ligation.

Transpeptidase-catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N-to-C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed.

Enzymatic C-Terminal Protein Engineering with Amines.

Chemoenzymatic protein and peptide modification is a powerful means of generating defined, homogeneous conjugates for a range of applications. However, the use of transpeptidases is limited by the need to prepare synthetic peptide conjugates to be ligated, bulky recognition tags remaining in the product, and inefficient substrate turnover. Here, we report a peptide/protein labeling strategy that utilizes a promiscuous, engineered transpeptidase to irreversibly incorporate diverse, commercially available amines at a C-terminal asparagine.

Melanocortin 1 Receptor Agonists Based on a Bivalent, Bicyclic Peptide Framework.

We have designed a new class of highly potent bivalent melanocortin receptor ligands based on the nature-derived bicyclic peptide sunflower trypsin inhibitor 1 (SFTI-1). Incorporation of melanotropin pharmacophores in each of the two turn regions of SFTI-1 resulted in substantial gains in agonist activity particularly at human melanocortin receptors 1 and 3 (hMC1R/hMC3R) compared to monovalent analogues. In binding and functional assays, the most potent molecule, compound , displayed low picomolar agonist activity at hMC1R (pEC > 10.

Asparaginyl Ligases: New Enzymes for the Protein Engineer's Toolbox.

Enzyme-catalysed site-specific protein modifications enable the precision manufacture of conjugates for the study of protein function and/or for therapeutic or diagnostic applications. Asparaginyl ligases are a class of highly efficient transpeptidases with the capacity to modify proteins bearing only a tripeptide recognition motif. Herein, we review the types of protein modification that are accessible using these enzymes, including N- and C-terminal protein labelling, head-to-tail cyclisation, and protein-protein conjugation.

Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases.

Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography.

Improved Asparaginyl-Ligase-Catalyzed Transpeptidation via Selective Nucleophile Quenching.

The use of enzymes for the site-specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields.

Make it or break it: Plant AEPs on stage in biotechnology.

Asparaginyl endopeptidases (AEPs) are cysteine proteases that control a myriad of cellular functions in plants, including maturation of seed storage proteins and programmed cell death. Recently, several noteworthy AEPs have been discovered that primarily function as transpeptidases rather than hydrolases, to instead catalyse the formation of new peptide bonds. These AEPs appear to have evolved for the cyclisation of a large class of plant defence peptides called cyclotides.