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.

Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor.

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-ß-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-ß-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-ß-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.

Exploring the therapeutic potential of an antinociceptive and anti-inflammatory peptide from wasp venom.

Animal venoms are rich sources of neuroactive compounds, including anti-inflammatory, antiepileptic, and antinociceptive molecules. Our study identified a protonectin peptide from the wasp Parachartergus fraternus' venom using mass spectrometry and cDNA library construction. Using this peptide as a template, we designed a new peptide, protonectin-F, which exhibited higher antinociceptive activity and less motor impairment compared to protonectin. In drug interaction experiments with naloxone and AM251, Protonectin-F's activity was decreased by opioid and cannabinoid antagonism, two critical antinociception pathways. Further experiments revealed that this effect is most likely not induced by direct action on receptors but by activation of the descending pain control pathway. We noted that protonectin-F induced less tolerance in mice after repeated administration than morphine. Protonectin-F was also able to decrease TNF-α production in vitro and modulate the inflammatory response, which can further contribute to its antinociceptive activity. These findings suggest that protonectin-F may be a potential molecule for developing drugs to treat pain disorders with fewer adverse effects. Our results reinforce the biotechnological importance of animal venom for developing new molecules of clinical interest.

Development of a Selective Peptide κ-Opioid Receptor Antagonist by Late-Stage Functionalization with Cysteine Staples.

The κ-opioid receptor (KOR) is an attractive target for the development of novel drugs. KOR agonists are potentially safer pain medications, whereas KOR antagonists are promising drug candidates for the treatment of neuropsychiatric disorders. Hitherto, the vast majority of selective drug leads that have been developed for KOR are small molecules. In this study, novel peptide probes were designed by using an endogenous dynorphin A1-13 sequence as a template for peptide stapling via late-stage cysteine functionalization. Leveraging this strategy, we developed a stable and potent KOR antagonist, CSD-CH2(1,8)-NH2, with approximately 1000-fold improved selectivity for KOR over µ- and δ-opioid receptors. Its potent competitive KOR antagonism was verified in KOR-expressing cells, peripheral dorsal root ganglion neurons, and using the tail-flick and rotarod tests in mice. This work highlights the value of cysteine stapling to develop selective peptide probes to modulate central KOR function, as innovative peptide drug candidates for the treatment of KOR-related illnesses.

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.

Development of a novel angiotensin converting enzyme 2 stimulator with broad implications in SARS-CoV2 and type 1 diabetes.

Angiotensin-converting enzyme 2 (ACE2) is protective in cardiovascular disease, lung injury and diabetes yet paradoxically underlies our susceptibility to SARs-CoV2 infection and the fatal heart and lung disease it can induce. Furthermore, diabetic patients have chronic, systemic inflammation and altered ACE2 expression resulting in increased risk of severe COVID-19 and the associated mortality. A drug that could increase ACE2 activity and inhibit cellular uptake of severe acute respiratory syndrome coronavirus 2 (SARs-CoV2), thus decrease infection, would be of high relevance to cardiovascular disease, diabetes and SARs-CoV2 infection. While the need for such a drug lead was highlighted over a decade ago receiving over 600 citations,1 to date, no such drugs are available.2 Here, we report the development of a novel ACE2 stimulator, designated '2A'(international PCT filed), which is a 10 amino acid peptide derived from a snake venom, and demonstrate its in vitro and in vivo efficacy against SARs-CoV2 infection and associated lung inflammation. Peptide 2A also provides remarkable protection against glycaemic dysregulation, weight loss and disease severity in a mouse model of type 1 diabetes. No untoward effects of 2A were observed in these pre-clinical models suggesting its strong clinical translation potential.

Importance of the Cyclic Cystine Knot Structural Motif for Immunosuppressive Effects of Cyclotides.

The cyclotide T20K inhibits the proliferation of human immune cells and is currently in clinical trials for multiple sclerosis. Here, we provide novel functional data and mechanistic insights into structure-activity relationships of T20K. Analogs with partial or complete reduction of the cystine knot had loss of function in proliferation experiments. Similarly, an acyclic analog of T20K was inactive in lymphocyte bioassays. The lack of activity of non-native peptide analogs appears to be associated with the ability of cyclotides to interact with and penetrate cell membranes, since cellular uptake studies demonstrated fast fractional transfer only of the native peptide into the cytosol of human immune cells. Therefore, structural differences between cyclic and linear native folded peptides were investigated by NMR to elucidate structure-activity relationships. Acyclic T20K had a less rigid backbone and considerable structural changes in loops 1 and 6 compared to the native cyclic T20K, supporting the idea that the cyclic cystine knot motif is a unique bioactive scaffold. This study provides evidence that this structural motif in cyclotides governs bioactivity, interactions with and transport across biological membranes, and the structural integrity of these peptides. These observations could be useful to understand the structure-activity of other cystine knot proteins due to the structural conservation of the cystine knot motif across evolution and to provide guidance for the design of novel cyclic cysteine-stabilized molecules.

Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens.

According to the World Health Organization (WHO) the development of resistance against antibiotics by microbes is one of the most pressing health concerns. The situation will intensify since only a few pharmacological companies are currently developing novel antimicrobial compounds. Discovery and development of novel antimicrobial compounds with new modes of action are urgently needed. Antimicrobial peptides (AMPs) are known to be able to kill multidrug-resistant bacteria and, therefore, of interest to be developed into antimicrobial drugs. Proteolytic stability and toxicities of these peptides are challenges to overcome, and one strategy frequently used to address stability is cyclization. Here we introduced a disulfide-bond to cyclize a potent and nontoxic 9mer peptide and, in addition, as a proof-of-concept study, grafted this peptide into loop 6 of the cyclotide MCoTI-II. This is the first time an antimicrobial peptide has been successfully grafted onto the cyclotide scaffold. The disulfide-cyclized and grafted cyclotide showed moderate activity in broth and strong activity in 1/5 broth against clinically relevant resistant pathogens. The linear peptide showed superior activity in both conditions. The half-life time in 100% human serum was determined, for the linear peptide, to be 13 min, for the simple disulfide-cyclized peptide, 9 min, and, for the grafted cyclotide 7 h 15 min. The addition of 10% human serum led to a loss of antimicrobial activity for the different organisms, ranging from 1 to >8-fold for the cyclotide. For the disulfide-cyclized version and the linear version, activity also dropped to different degrees, 2 to 18-fold, and 1 to 30-fold respectively. Despite the massive difference in stability, the linear peptide still showed superior antimicrobial activity. The cyclotide and the disulfide-cyclized version demonstrated a slower bactericidal effect than the linear version. All three peptides were stable at high and low pH, and had very low hemolytic and cytotoxic activity.

Design of a Stable Cyclic Peptide Analgesic Derived from Sunflower Seeds that Targets the κ-Opioid Receptor for the Treatment of Chronic Abdominal Pain.

The rising opioid crisis has become a worldwide societal and public health burden, resulting from the abuse of prescription opioids. Targeting the κ-opioid receptor (KOR) in the periphery has emerged as a powerful approach to develop novel pain medications without central side effects. Inspired by the traditional use of sunflower (Helianthus annuus) preparations for analgesic purposes, we developed novel stabilized KOR ligands (termed as helianorphins) by incorporating different dynorphin A sequence fragments into a cyclic sunflower peptide scaffold. As a result, helianorphin-19 selectively bound to and fully activated the KOR with nanomolar potency. Importantly, helianorphin-19 exhibited strong KOR-specific peripheral analgesic activity in a mouse model of chronic visceral pain, without inducing unwanted central effects on motor coordination/sedation. Our study provides a proof of principle that cyclic peptides from plants may be used as templates to develop potent and stable peptide analgesics applicable via enteric administration by targeting the peripheral KOR for the treatment of chronic abdominal pain.

Cyclotides from Brazilian Palicourea sessilis and Their Effects on Human Lymphocytes.

Cyclotides are plant-derived peptides found within five families of flowering plants (Violaceae, Rubiaceae, Fabaceae, Solanaceae, and Poaceae) that have a cyclic backbone and six conserved cysteine residues linked by disulfide bonds. Their presence within the Violaceae species seems ubiquitous, yet not all members of other families produce these macrocyclic peptides. The genus Palicourea Aubl. (Rubiaceae) contains hundreds of neotropical species of shrubs and small trees; however, only a few cyclotides have been discovered hitherto. Herein, five previously uncharacterized Möbius cyclotides within Palicourea sessilis and their pharmacological activities are described. Cyclotides were isolated from leaves and stems of this plant and identified as pase A-E, as well as the known peptide kalata S. Cyclotides were de novo sequenced by MALDI-TOF/TOF mass spectrometry, and their structures were solved by NMR spectroscopy. Because some cyclotides have been reported to modulate immune cells, pase A-D were assayed for cell proliferation of human primary activated T lymphocytes, and the results showed a dose-dependent antiproliferative function. The toxicity on other nonimmune cells was also assessed. This study reveals that pase cyclotides have potential for applications as immunosuppressants and in immune-related disorders.

Antimicrobial and Anticancer Properties of Synthetic Peptides Derived from the Wasp Parachartergus fraternus.

Agelaia-MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin-F, were designed to reduce nonspecific toxicity and improve potency. Here, the three-dimensional structures of neuroVAL, protonectin and protonectin-F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram-positive bacteria, with agelaia-MPI being the most active. Protonectin and protonectin-F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell-penetrating peptide, while protonectin-F needs further modification to realize its potential as an antitumor peptide.

Harnessing cyclotides to design and develop novel peptide GPCR ligands.

Cyclotides are plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology that confers them with remarkable structural stability and resistance to proteolytic degradation. Recently, cyclotides have emerged as promising scaffold molecules for designing peptide-based therapeutics. Here, we provide examples of how engineering cyclotides using molecular grafting may lead to the development of novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. Integrating bioactive epitopes into stable cyclotide scaffolds can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. We also discuss and highlight the importance of engineered cyclotides as novel tools to study GPCR signaling.

The Vast Structural Diversity of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) occur in all kingdoms of life and are integral to host defense. They have diverse structures and target a variety of organisms, both by nonspecific membrane interactions and via specific targets. Here we discuss the structures of AMPs from the four main classes currently recognized - that is, peptides with (i) α-helical, (ii) ß-sheet, (iii) αß, or (iv) non-αß elements - as well as the growing pool of complex topologies including various post-translational modifications (PTMs). We propose to group these latter peptides into a fifth class of AMPs. Such peptides exhibit high stability and amenability to chemical engineering, making them of interest for the development of novel antimicrobial agents. Advances and challenges in the development of these peptides towards therapeutic leads are presented.

Inhibition of Breast Cancer Cell Migration by Cyclotides Isolated from Pombalia calceolaria.

Two new bracelet cyclotides from roots of Pombalia calceolaria with potential anticancer activity have been characterized in this work. The cyclotides Poca A and B (1 and 2) and the previously known CyO4 (3) were de novo sequenced by MALDI-TOF/TOF mass spectrometry (MS). The MS2 spectra were examined and the amino acid sequences were determined. The purified peptides were tested for their cytotoxicity and effects on cell migration of MDA-MB-231, a triple-negative breast cancer cell line. The isolated cyclotides reduced the number of cancer cells by more than 80% at 20 µM, and the concentration-related cytotoxic responses were observed with IC50 values of 1.8, 2.7, and 9.8 µM for Poca A (1), Poca B (2), and CyO4 (3), respectively. Additionally, the inhibition of cell migration (wound-healing assay) exhibited that CyO4 (3) presents an interesting activity profile, in being able to inhibit cell migration (50%) at a subtoxic concentration (2 µM). The distribution of these cyclotides in the roots was analyzed by MALDI imaging, demonstrating that all three compounds are present in the phloem and cortical parenchyma regions.

Transcriptome and toxin family analysis of the paralysis tick, Ixodes holocyclus.

The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks collected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced using Illumina HiSeq 100 bp pair end read technologies. Unique and non-redundant holocyclotoxin sequences were designated as HT2-HT19, as none were identical to the previously described HT1. Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particularly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first known genomic database developed for the Australian paralysis tick. The database contributed to the identification and subsequent characterization of the holocyclotoxin family that will inform the development of novel anti-paralysis control methods.

Structure-Activity Relationships of Insect Defensins.

Insects make up the largest and most diverse group of organisms on earth with several million species to exist in total. Considering the sheer number of insect species and the vast variety of ways they interact with their environment through chemistry, it is clear that they have significant potential as a source of new lead molecules. They have adapted to a range of ecological habitats and exhibit a symbiotic lifestyle with various microbes such as bacteria and fungi. Accordingly, numerous antimicrobial compounds have been identified including for example defensin peptides. Insect defensins were found to have broad-spectrum activity against various gram-positive/negative bacteria as well as fungi. They exhibit a unique structural topology involving the complex arrangement of three disulfide bonds as well as an alpha helix and beta sheets, which is known as cysteine-stabilized αß motif. Their stability and amenability to peptide engineering make them promising candidates for the development of novel antibiotics lead molecules. This review highlights the current knowledge regarding the structure-activity relationships of insect defensin peptides and provides basis for future studies focusing on the rational design of novel cysteine-rich antimicrobial peptides.

Global map of oxytocin/vasopressin-like neuropeptide signalling in insects.

Oxytocin and vasopressin mediate a range of physiological functions that are important for osmoregulation, reproduction, social behaviour, memory and learning. The origin of this signalling system is thought to date back ~600 million years. Oxytocin/vasopressin-like peptides have been identified in several invertebrate species and they appear to be functionally related across the entire animal kingdom. There is little information available about the biology of this peptide G protein-coupled receptor signalling system in insects. Recently over 200 insect genome/transcriptome datasets were released allowing investigation of the molecular structure and phylogenetic distribution of the insect oxytocin/vasopressin orthologue - inotocin peptides and their receptors. The signalling system is present in early arthropods and representatives of some early-diverging lineages. However, Trichoptera, Lepidoptera, Siphonaptera, Mecoptera and Diptera, lack the presence of inotocin genes, which suggests the peptide-receptor system was probably lost in their common ancestor ~280 million-years-ago. In addition we detected several losses of the inotocin signalling system in Hemiptera (white flies, scale insects and aphids), and the complete absence in spiders (Chelicerata). This unique insight into evolutionarily patterns and sequence diversity of neuroendocrine hormones will provide opportunities to elucidate the physiology of the inotocin signalling system in one of the largest group of animals.

Tick holocyclotoxins trigger host paralysis by presynaptic inhibition.

Ticks are important vectors of pathogens and secreted neurotoxins with approximately 69 out of 692 tick species having the ability to induce severe toxicoses in their hosts. The Australian paralysis tick (Ixodes holocyclus) is known to be one of the most virulent tick species producing a flaccid paralysis and fatalities caused by a family of neurotoxins known as holocyclotoxins (HTs). The paralysis mechanism of these toxins is temperature dependent and is thought to involve inhibition of acetylcholine levels at the neuromuscular junction. However, the target and mechanism of this inhibition remain uncharacterised. Here, we report that three members of the holocyclotoxin family; HT-1 (GenBank AY766147), HT-3 (GenBank KP096303) and HT-12 (GenBank KP963967) induce muscle paralysis by inhibiting the dependence of transmitter release on extracellular calcium. Previous study was conducted using extracts from tick salivary glands, while the present study is the first to use pure toxins from I. holocyclus. Our findings provide greater insight into the mechanisms by which these toxins act to induce paralysis.

Unveiling the diversity of cyclotides by combining peptidome and transcriptome analysis.

Circular peptides have attracted much interest in recent drug development efforts, particularly due to their increased stability over linear counterparts. The family of plant cyclotides represents one of the largest classes of naturally-occurring backbone-cyclized peptides displaying exceptional sequence variability and plasticity around three knotted disulfide bonds. Accordingly, a multitude of pharmaceutically as well as agrochemically relevant bioactivities has been ascribed to them. Their abundance across various species within flowering plants is highlighted by estimated numbers of up to 150,000 different sequences present in single plant families and over 160 at the species level. However, this vast diversity impedes thorough sequence characterization by standard analytical methods using mass spectrometry and thus limits access to a wealth of potentially bioactive compounds that may represent novel lead molecules. Recently the ribosomal origin of cyclotides has been exploited as an alternative way to discover novel sequences. The analysis at nucleotide level allows not only the identification of peptides but also their parent precursor proteins. This combined approach opens access to the discovery of sequences that can provide novel structural templates for a variety of pharmaceutical as well as agrochemical applications. Here we review recent literature related to the discovery of cyclotides. Challenges and opportunities using classical mass spectrometry workflows and novel approaches such as in silico mining will be discussed. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 774-783, 2016.