Design, Synthesis, and Structure-Activity Relationships of Novel Peptide Derivatives of the Severe Acute Respiratory Syndrome-Coronavirus-2 Spike-Protein that Potently Inhibit Nicotinic Acetylcholine Receptors.

The spike-protein of SARS-CoV-2 has a distinctive amino-acid sequence (682RRARS686) that forms a cleavage site for the enzyme furin. Strikingly, the structure of the spike-protein loop containing the furin cleavage site bears substantial similarity to neurotoxin peptides found in the venoms of certain snakes and marine cone snails. Leveraging this relationship, we designed and synthesized disulfide-constrained peptides with amino-acid sequences corresponding to the furin cleavage-sites of wild-type (B.1 variant) SARS-CoV-2 or the Alpha, Delta, and Omicron variants. Remarkably, some of these peptides potently inhibited α7 and α9α10 nicotinic acetylcholine receptors (nAChR) with nM affinity and showed SARS-CoV-2 variant and nAChR subtype-dependent potencies. Nuclear magnetic resonance spectroscopy and molecular dynamics were used to rationalize structure-activity relationships between peptides and their cognate receptors. These findings delineate nAChR subtypes that can serve as high-affinity spike-protein targets in tissues central to COVID-19 pathophysiology and identify ligands and target receptors to inform the development of novel SARS-CoV-2 therapeutics.

Aspartic acid mutagenesis of αO-Conotoxin GeXIVA isomers reveals arginine residues crucial for inhibition of the α9α10 nicotinic acetylcholine receptor.

The two most active disulfide bond isomers of the analgesic αO-conotoxin GeXIVA, namely GeXIVA[1, 2] and GeXIVA[1, 4], were subjected to Asp-scanning mutagenesis to determine the key amino acid residues for activity at the rat α9α10 nicotinic acetylcholine receptor (nAChR). These studies revealed the key role of arginine residues for the activity of GeXIVA isomers towards the α9α10 nAChR. Based on these results, additional analogues with 2-4 mutations were designed and tested. The analogues [T1A,D14A,V28K]GeXIVA[1, 2] and [D14A,I23A,V28K]GeXIVA[1, 4] were developed and showed sub-nanomolar activity for the α9α10 nAChR with IC50 values of 0.79 and 0.38 nM. The latter analogue had exceptional selectivity for the α9α10 receptor subtype over other nAChR subtypes and can be considered as a drug candidate for further development. Molecular dynamics of receptor-ligand complexes allowed us to make deductions about the possible causes of increases in the affinity of key GeXIVA[1, 4] mutants for the α9α10 nAChR.

The Plant Defensin Ppdef1 Is a Novel Topical Treatment for Onychomycosis.

Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.

Structure-Activity Relationships of Alanine Scan Mutants αO-Conotoxins GeXIVA[1,2] and GeXIVA[1,4].

αO-Conotoxin GeXIVA is a selective α9α10 nicotinic acetylcholine receptor (nAChR) inhibitor displaying two disulfide bonds that can form three isomers. The bead (GeXIVA[1,2]) and ribbon (GeXIVA[1,4]) isomers possess the highest activity on rat and human α9α10 nAChRs. However, the molecular mechanism by which they inhibit the α9α10 nAChR is unknown. Here, an alanine scan of GeXIVA was used to elucidate key interactions between the peptides and the α9α10 nAChR. The majority of GeXIVA[1,2] analogues preserved affinity at α9α10 nAChR, but [R17A]GeXIVA[1,2] enhanced selectivity on the α9α10 nAChR. The I23A replacement of GeXIVA[1,4] increased activity at both rat and human α9α10 nAChRs by 10-fold. Surprisingly, these results do not support the molecular model of an interaction in the orthosteric binding site proposed previously, but rather may involve allosteric coupling with the voltage-sensitive domain of the α9α10 nAChR. These results could help to guide further development of GeXIVA analogues as analgesics.

Towards a Sustainable Management of the Spotted-Wing Drosophila: Disclosing the Effects of Two Spider Venom Peptides on Drosophila suzukii.

The spotted-wing drosophila (Drosophila suzukii) is a polyphagous pest that causes severe damage and economic losses to soft-skinned fruit production. Current control methods are dominated by inefficient cultural practices and broad-spectrum insecticides that, in addition to having toxic effects on non-target organisms, are becoming less effective due to acquired resistance. The increasing awareness of the real impact of insecticides on health and the environment has promoted the exploration of new insecticidal compounds, addressing novel molecular targets. This study explores the efficacy of two orally delivered spider venom peptides (SVPs), J-atracotoxin-Hv1c (Hv1c) and µ-theraphotoxin-Hhn2b (TRTX), to manage D. suzukii, through survival assays and the evaluation of gene expression associated with detoxification pathways. Treatment with TRTX at 111.5 µM for 48 h enhanced fly longevity compared with the control group. Gene expression analysis suggests that detoxification and stress-related mechanisms, such as expression of P450 proteins and apoptotic stimuli signaling, are triggered in D. suzukii flies in response to these treatments. Our results highlight the potential interest of SVPs to control this pest, shedding light on how to ultimately develop improved target-specific formulations.

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.

Designing antimicrobial peptides using deep learning and molecular dynamic simulations.

With the emergence of multidrug-resistant bacteria, antimicrobial peptides (AMPs) offer promising options for replacing traditional antibiotics to treat bacterial infections, but discovering and designing AMPs using traditional methods is a time-consuming and costly process. Deep learning has been applied to the de novo design of AMPs and address AMP classification with high efficiency. In this study, several natural language processing models were combined to design and identify AMPs, i.e. sequence generative adversarial nets, bidirectional encoder representations from transformers and multilayer perceptron. Then, six candidate AMPs were screened by AlphaFold2 structure prediction and molecular dynamic simulations. These peptides show low homology with known AMPs and belong to a novel class of AMPs. After initial bioactivity testing, one of the peptides, A-222, showed inhibition against gram-positive and gram-negative bacteria. The structural analysis of this novel peptide A-222 obtained by nuclear magnetic resonance confirmed the presence of an alpha-helix, which was consistent with the results predicted by AlphaFold2. We then performed a structure-activity relationship study to design a new series of peptide analogs and found that the activities of these analogs could be increased by 4-8-fold against Stenotrophomonas maltophilia WH 006 and Pseudomonas aeruginosa PAO1. Overall, deep learning shows great potential in accelerating the discovery of novel AMPs and holds promise as an important tool for developing novel AMPs.

Plant-based production of an orally active cyclotide for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is a debilitating disease that requires prolonged treatment with often severe side effects. One experimental MS therapeutic currently under development is a single amino acid mutant of a plant peptide termed kalata B1, of the cyclotide family. Like all cyclotides, the therapeutic candidate [T20K]kB1 is highly stable as it contains a cyclic backbone that is cross-linked by three disulfide bonds in a knot-like structure. This stability is much sought after for peptide drugs, which despite exquisite selectivity for their targets, are prone to rapid degradation in human serum. In preliminary investigations, it was found that [T20K]kB1 retains oral activity in experimental autoimmune encephalomyelitis, a model of MS in mice, thus opening up opportunities for oral dosing of the peptide. Although [T20K]kB1 can be synthetically produced, a recombinant production system provides advantages, specifically for reduced scale-up costs and reductions in chemical waste. In this study, we demonstrate the capacity of the Australian native Nicotiana benthamiana plant to produce a structurally identical [T20K]kB1 to that of the synthetic peptide. By optimizing the co-expressed cyclizing enzyme, precursor peptide arrangements, and transgene regulatory regions, we demonstrate a [T20K]kB1 yield in crude peptide extracts of ~ 0.3 mg/g dry mass) in whole plants and close to 1.0 mg/g dry mass in isolated infiltrated leaves. With large-scale plant production facilities coming on-line across the world, the sustainable and cost-effective production of cyclotide-based therapeutics is now within reach.

Design, synthesis, and mechanism of action of novel µ-conotoxin KIIIA analogues for inhibition of the voltage-gated sodium channel Nav1.7.

µ-Conotoxin KIIIA, a selective blocker of sodium channels, has strong inhibitory activity against several Nav isoforms, including Nav1.7, and has potent analgesic effects, but it contains three pairs of disulfide bonds, making structural modification difficult and synthesis complex. To circumvent these difficulties, we designed and synthesized three KIIIA analogues with one disulfide bond deleted. The most active analogue, KIIIA-1, was further analyzed, and its binding pattern to hNav1.7 was determined by molecular dynamics simulations. Guided by the molecular dynamics computational model, we designed and tested 32 second-generation and 6 third-generation analogues of KIIIA-1 on hNav1.7 expressed in HEK293 cells. Several analogues showed significantly improved inhibitory activity on hNav1.7, and the most potent peptide, 37, was approximately 4-fold more potent than the KIIIA Isomer I and 8-fold more potent than the wildtype (WT) KIIIA in inhibiting hNav1.7 current. Intraperitoneally injected 37 exhibited potent in vivo analgesic activity in a formalin-induced inflammatory pain model, with activity reaching ∼350-fold of the positive control drug morphine. Overall, peptide 37 has a simplified disulfide-bond framework and exhibits potent in vivo analgesic effects and has promising potential for development as a pain therapy in the future.

Discovery, Characterization, and Engineering of LvIC, an α4/4-Conotoxin That Selectively Blocks Rat α6/α3ß4 Nicotinic Acetylcholine Receptors.

α6ß4 nicotinic acetylcholine receptors (nAChRs) are expressed in the central and peripheral nervous systems, but their functions are not fully understood, largely because of a lack of specific ligands. Here, we characterized a novel α-conotoxin, LvIC, and designed a series of analogues to probe structure-activity relationships at the α6ß4 nAChR. The potency and selectivity of these conotoxins were tested using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes. One of the analogues, [D1G,ΔQ14]LvIC, potently blocked α6/α3ß4 nAChRs (α6/α3 is a chimera) with an IC50 of 19 nM, with minimal activity at other nAChR subtypes, including the structurally similar α6/α3ß2ß3 and α3ß4 subtypes. Using NMR, molecular docking, and receptor mutation, structure-activity relationships of [D1G,ΔQ14]LvIC at the α6/α3ß4 nAChR were defined. It is a potent and specific antagonist of α6ß4 nAChRs that could potentially serve as a novel molecular probe to explore α6ß4 nAChR-related neurophysiological and pharmacological functions.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

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.

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.

Modified horseshoe crab peptides target and kill bacteria inside host cells.

Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two ß-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

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.

Engineered Conotoxin Differentially Blocks and Discriminates Rat and Human α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor (nAChR) is present in the central nervous system and plays an important role in cognitive function and memory. α-Conotoxin LvIB, identified from genomic DNA of Conus lividus, its three isomers and four globular isomer analogues were synthesized and screened at a wide range of nAChR subtypes. One of the analogues, amidated [Q1G,ΔR14]LvIB, was found to be a potent blocker of rat α7 nAChRs. Importantly, it differentiates between α7 nAChRs of human (IC50: 1570 nM) and rat (IC50: 97 nM). Substitutions between rat and human α7 nAChRs at three key mutation sites revealed that no single mutant could completely change the activity profile of amidated [Q1G,ΔR14]LvIB. Rather, we found that the combined influence of Gln141, Asn184, and Lys186 determines the α7 nAChR species specificity of this peptide. This engineered α4/4 conotoxin has potential applications as a template for designing ligands to selectively block human α7 nAChRs.

In Planta Discovery and Chemical Synthesis of Bracelet Cystine Knot Peptides from Rinorea bengalensis.

Cyclotides are plant-derived peptides that have attracted interest as biocides and scaffolds for the development of stable peptide therapeutics. Cyclotides are characterized by their cyclic backbone and cystine knot framework, which engenders them with remarkably high stability. This study reports the cystine knot-related peptidome of Rinorea bengalensis, a small rainforest tree in the Violaceae family that is distributed from Australia westward to India. Surprisingly, many more acyclic knotted peptides (acyclotides) were discovered than cyclic counterparts (cyclotides), with 32 acyclotides and 1 cyclotide sequenced using combined transcriptome and proteomic analyses. Nine acyclotides were isolated and screened against a panel of mammalian cell lines, showing they had the cytotoxic properties normally associated with cyclotide-like peptides. NMR analysis of the acyclotide ribes 21 and 22 and the cyclotide ribe 33 confirmed that these peptides contained the cystine knot structural motif. The bracelet-subfamily cyclotide ribe 33 was amenable to chemical synthesis in reasonable yield, an achievement that has long eluded previous attempts to synthetically produce bracelet cyclotides. Accordingly, ribe 33 represents an exciting new bracelet cyclotide scaffold that can be subject to chemical modification for future molecular engineering applications.

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.

Production of a structurally validated cyclotide in rice suspension cells is enabled by a supporting biosynthetic enzyme.

MAIN CONCLUSION: We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides. Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1b from O. affinis. Without co-expression, kB1 was predominantly produced as linear peptide. Through HPLC-MS co-elution, reduction, alkylation, enzymatic digestion, and proton NMR analysis, kB1 produced in rice was shown to be structurally identical to native kB1. This study reports the first example of an engineered plant suspension cell culture with the required molecular machinery for efficient production and cyclisation of a heterologous cyclotide.

Neurotoxic peptides from the venom of the giant Australian stinging tree.

Stinging trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. Dendrocnide-induced acute pain typically lasts for several hours, and intermittent painful flares can persist for days and weeks. Pharmacological activity has been attributed to small-molecule neurotransmitters and inflammatory mediators, but these compounds alone cannot explain the observed sensory effects. We show here that the venoms of Australian Dendrocnide species contain heretofore unknown pain-inducing peptides that potently activate mouse sensory neurons and delay inactivation of voltage-gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are miniproteins of 4 kDa, whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure, and pharmacology.