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. The chemical engineering of cyclotides has generated novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. However key challenges potentially limit the widespread use of cyclotides in molecular grafting applications. Folding of cyclotides containing bioactive epitopes remains a major bottleneck in cyclotide synthesis. Here we present a modular 'plug and play' approach that effectively bypasses problems associated with the oxidative folding of cyclotides. By grafting onto a pre-formed acyclic cyclotide-like scaffold we show that difficult-to-graft sequences can be easily obtained and can target GPCRs with nanomolar affinities and potencies. We further show the suitability of this new method to graft other complex epitopes including structures with additional disulfide bonds that are not readily available via currently employed chemical methods, thus fully unlocking cyclotides to be used in drug design applications.

Isolation and Characterization of Insecticidal Cyclotides from Viola communis.

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. The presence of cyclotides in Viola communis was inferred by mass spectroscopy previously, but their sequences and properties had yet to be explored. In this study, the occurrence of cyclotides in this plant was investigated using proteomics and transcriptomics. Twenty cyclotides were identified at the peptide level, including two new members from the bracelet (Vcom1) and Möbius (Vcom2) subfamilies. Structural analysis of these newly identified peptides demonstrated a similar fold compared with cyclotides from the same respective subfamilies. Biological assays of Vcom1 and Vcom2 revealed them to be cytotoxic to Sf9 insect cell lines, with Vcom1 demonstrating higher potency than Vcom2. The results suggest that they could be further explored as insecticidal agents and confirm earlier general findings that bracelet cyclotides have more potent insecticidal activity than their Möbius relatives. Seven new cyclotide-like sequences were observed in the transcriptome of V. communis, highlighting the Violaceae as a rich source for new cyclotides with potential insecticidal activity. An analysis of sequences flanking the cyclotide domain in the various precursors from V. communis and other Violaceae plants revealed new insights into cyclotide processing and suggested the possibility of two alternative classes of N-terminal processing enzymes for cyclotide biosynthesis.

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. We find that internal lysines with an adjacent leucine residue mimic the conventional N-terminal glycine-leucine substrate. This dipeptide motif enables efficient intra- or intermolecular ligation through internal lysine side chains, minimally leaving an asparagine C-terminally linked to the lysine side chain via an isopeptide bond. The versatility of this approach is demonstrated by the chemoenzymatic synthesis of peptides with non-native C terminus-to-side chain topology and the conjugation of chemically modified peptides to recombinant proteins.

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. These reactions proceed efficiently under mild conditions (near-neutral pH) and allow the optimal recognition elements for asparaginyl ligases (P1 Asn and P2'' Leu) to be preserved. Certain products, particularly proline-containing products, were found to be protected from recognition by the enzyme, allowing for straightforward sequential labelling of proteins. Additionally, incorporation of 4-azidoproline enables one-pot dual labelling directly at the ligation junction. These capabilities further expand the chemical diversity of asparaginyl ligase-catalysed reactions and provide an alternative approach for straightforward, successive modification of protein substrates.

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 ß-turn nucleation on cyclotide oxidative folding. Two types of ß-turn mimics were grafted into kalata B1, individually replacing each of the four ß-turns in the folded cyclotide. Insertion of d-Pro-Gly into loop 5 was beneficial to the folding of both cyclic kB1 and a linear form of the peptide. The linear grafted analog folded four-times faster in aqueous conditions than cyclic kB1 in optimized conditions. Additionally, the cyclic analogue folded without the need for redox agents by transitioning through a native-like intermediate that was on-pathway to product formation. Kalata B1 is from the Möbius subfamily of cyclotides. Grafting d-Pro-Gly into loop 5 of cyclotides from two other subfamilies also had a beneficial effect on folding. Our findings demonstrate the importance of a ß-turn nucleation site for cyclotide oxidative folding, which could be adopted as a chemical strategy to improve the in vitro folding of diverse cystine-rich peptides.

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. Notably, these previously described strategies leave the polypeptide backbone orientation unaltered. Here we describe an unnatural protein permutation, the protein domain inversion, whereby a C-terminal portion of a protein is enzymatically inverted from the canonical N-to-C to a C-to-C configuration with respect to the N-terminal part of the protein. The closest conceptually analogous biological process is perhaps the inversion of DNA segments as catalyzed by recombinases. We achieve these inversions using an engineered sortase A, a widely used transpeptidase. Our reactions proceed efficiently under mild conditions at 4-25 °C and are compatible with entirely heterologously-produced protein substrates.

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. Endogenously, the receptor is activated by peptide ligands, i.e., three melanocyte-stimulating hormones (α-MSH, ß-MSH, and γ-MSH) and by adrenocorticotropic hormone. Therefore, we utilized a peptide drug design approach, utilizing "molecular grafting" of pharmacophore peptide sequence motifs onto a stable nature-derived peptide scaffold. Specifically, protegrin-4-like-peptide-1 (Pr4LP1) and arenicin-1-like-peptide-1 (Ar3LP1) fully activated MC4R in a functional cAMP assay with potencies of 3.7 and 1.0 nM, respectively. In a nanoluciferase complementation assay with less signal amplification, the designed peptides fully recruited mini-Gs with subnanomolar and nanomolar potencies. Interestingly, these novel peptide MC4R ligands recruited ß-arrestin-2 with ∼2-fold greater efficacies and ∼20-fold increased potencies as compared to the endogenous α-MSH. The peptides were inactive at related MC1R and MC3R in a cAMP accumulation assay. These findings highlight the applicability of animal-derived disulfide-rich scaffolds to design pathway and subtype selective MC4R pharmacological probes. In the future, this approach could be exploited to develop functionally selective ligands that could offer safer and more effective obesity 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. In this study, an asparaginyl endopeptidase (AEP)-mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six- to nine-residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage-gated calcium channels (CaV 2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins.

Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function.

Voltage-gated sodium (NaV) 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 NaV 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 NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.

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., short biological half-lives and poor cell permeability. In this review, we examine the utility of molecular engineering to insert bioactive sequences into constrained scaffolds with desired pharmaceutical properties. Applying lessons learnt from nature, we focus on molecular grafting of cyclic disulfide-rich scaffolds (naturally derived or engineered), shown to be intrinsically stable and amenable to sequence modifications, and their utility as privileged frameworks in drug design.

Engineering the Cyclization Loop of MCoTI-II Generates Targeted Cyclotides that Potently Inhibit Factor XIIa.

Factor XIIa (FXIIa) is a promising target for developing new drugs that prevent thrombosis without causing bleeding complications. A native cyclotide (MCoTI-II) is gaining interest for engineering FXIIa-targeted anticoagulants as this peptide inhibits FXIIa but not other coagulation proteases. Here, we engineered the native biosynthetic cyclization loop of MCoTI-II (loop 6) to generate improved FXIIa inhibitors. Decreasing the loop length led to gains in potency up to 7.7-fold, with the most potent variant having five residues in loop 6 (Ki = 25 nM). We subsequently examined sequence changes within loop 6 and an adjacent loop, with substitutions at P4 and P2' producing a potent FXIIa inhibitor (Ki = 2 nM) that displayed more than 700-fold selectivity, was stable in human serum, and blocked the intrinsic coagulation pathway in human plasma. These findings demonstrate that engineering the biosynthetic cyclization loop can generate improved cyclotide variants, expanding their potential for drug discovery.

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 (pKi) and functional activation (pEC50) of the melanocortin receptor subtypes. Stapled peptides generally had improved activity, with aromatic stapled peptides yielding selective MC1R agonists, including a xylene-stapled peptide (2) with an EC50 of 1.9 nM for MC1R and >150-fold selectivity for MC3R and MC4R. Selected stapled peptides were further functionalized with linkers and payloads, generating a series of conjugated peptides with potent MC1R activity, including one pyridazine-functionalized peptide (21) with picomolar activity at MC1R (Ki 58 pM; EC50 < 9 pM). This work demonstrates that staples can be used as modular synthetic tools to tune potency and selectivity in peptide-based drug design.

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. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.

Low potency inhibition of NaV1.7 by externally applied QX-314 via a depolarizing shift in the voltage-dependence of activation.

QX-314 is a quaternary permanently charged lidocaine derivative that inhibits voltage-gated sodium channels (NaV). As it is membrane impermeable, it is generally considered that QX-314 applied externally is inactive, unless it can gain access to the internal local anesthetic binding site via another entry pathway. Here, we characterized the electrophysiological effects of QX-314 on NaV1.7 heterologously expressed in HEK293 cells, and found that at high concentrations, external QX-314 inhibited NaV1.7 current (IC50 2.0 ± 0.3 mM) and shifted the voltage-dependence to more depolarized potentials (ΔV50 +10.6 mV). Unlike lidocaine, the activity of external QX-314 was not state- or use-dependent. The effect of externally applied QX-314 on NaV1.7 channel biophysics differed to that of internally applied QX-314, suggesting QX-314 has an additional externally accessible site of action. In line with this hypothesis, disruption of the local anesthetic binding site in a [F1748A]NaV1.7 mutant reduced the potency of lidocaine by 40-fold, but had no effect on the potency or activity of externally applied QX-314. Therefore, we conclude, using an expression system where QX-314 was unable to cross the membrane, that externally applied QX-314 is able to inhibit NaV1.7 peak current at low millimolar concentrations.

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. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function.

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. This disulfide-bridged peptide, showed an IC50 value in the low micromolar range. Upon further characterization, cyclisation was found to be essential for its activity. To increase metabolic stability, a series of disulfide mimetics were designed and a redox-stable 1,4-disubstituted 1,2,3-triazole analogue displayed activity in the double-digit micromolar range. Further experiments revealed that this triazole peptidomimetic is also active against CsrA from Escherichia coli and RsmA from Pseudomonas aeruginosa. This study provides an ideal starting point for medicinal chemistry optimization of this macrocyclic peptide and might pave the way towards broad-acting virulence modulators.

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. In addition to directly catalyzing formation of l-/d- or α-/ß-amino acid junctions, we find C-terminal Leu-ethylenediamine (Leu-Eda) motifs to be bona fide mimetics of native N-terminal Gly-Leu sequences. Appending a C-terminal Leu-Eda to synthetic peptides or, via an intein-splicing approach, to recombinant proteins enables direct transpeptidase-catalyzed C-to-C ligations. This work significantly expands the synthetic scope of enzyme-catalyzed protein transpeptidation reactions.

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. To demonstrate the utility of this approach, we prepare a protein-drug conjugate, generate a genetically inaccessible C-to-C protein fusion, and site specifically label both termini of a single protein in sequential steps.

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 in vitro binding and functional assays, the most potent molecule, compound 6, displayed low picomolar agonist activity at hMC1R (pEC50 > 10.3; EC50 < 50 pM; pKi: 10.16 ± 0.04; Ki: 69 ± 5 pM) and is at least 30-fold more selective for this receptor than for hMC3R, hMC4R, or hMC5R. The results are discussed in the context of structural homology models of hMCRs in complex with the developed bivalent ligands.

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. We describe the progress that has been made to engineer highly efficient ligases as well as efforts to chemically manipulate the enzyme reaction to favour product formation. These enzymes are powerful additions to the protein engineer's toolbox.