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Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type Ca V 3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type Ca V s but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH 2 ) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH 2 to be a concentration-dependent partial inhibitor of Ca V 3.2 (IC 50 = 1.18 μM) and Ca V 3.3 (IC 50 = 0.49 μM) depolarized currents but was ineffective at Ca V 3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type Ca V 2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH 2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at Ca V 3.2 and Ca V 3.3. As both Ca V 3.2 and Ca V 3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH 2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH 2 produced antiallodynia in both mechanical and thermal pain.

Breast cancer is a leading cause of death in women, and its management highly depends on early disease diagnosis and monitoring. This remains challenging due to breast cancer's heterogeneity and a scarcity of specific biomarkers that could predict responses to therapy and enable personalized treatment. This Perspective describes the diagnostic landscape for breast cancer management, molecular strategies targeting receptors overexpressed in tumors, the theranostic potential of the oxytocin receptor (OTR) as an emerging breast cancer target, and the development of OTR-specific optical and nuclear tracers to study, visualize, and treat tumors. A special focus is on the chemistry and pharmacology underpinning OTR tracer development, preclinical in vitro and in vivo studies, challenges, and future directions. The use of peptide-based tracers targeting upregulated receptors in cancer is a highly promising strategy complementing current diagnostics and therapies and providing new opportunities to improve cancer management and patient survival.

Cardiovascular ailments are a major cause of mortality where over 1.3 billion people suffer from hypertension leading to heart-disease related deaths. Snake venoms possess a broad repertoire of natriuretic peptides with therapeutic potential for treating hypertension, congestive heart failure, and related cardiovascular disease. We now describe several taipan ( Oxyuranus microlepidotus ) natriuretic peptides TNPa-e which stimulated cGMP production through the natriuretic peptide receptor A (NPR-A) with higher potencies for the rat NPR-A (rNPR-A) over human NPR-A (hNPR-A). TNPc and TNPd were the most potent, demonstrating 100- and 560-fold selectivity for rNPR-A over hNPR-A. In vivo studies found that TNPc decreased diastolic and systolic blood pressure (BP) and increased heart rate (HR) in conscious normotensive rabbits, to a level that was similar to that of human atrial natriuretic peptide (hANP). TNPc also enhanced the bradycardia due to cardiac afferent stimulation (Bezold-Jarisch reflex). This indicated that TNPc possesses the ability to lower blood pressure and facilitate cardiac vagal afferent reflexes but unlike hANP does not produce tachycardia. The 3-dimensional structure of TNPc was well defined within the pharmacophoric disulfide ring, displaying two turn-like regions (RMSD = 1.15 Å). Further, its much greater biological stability together with its selectivity and potency will enhance its usefulness as a biological tool.

Australian funnel-web spiders are amongst the most dangerous venomous animals. Their venoms induce potentially deadly symptoms, including hyper- and hypotension, tachycardia, bradycardia and pulmonary oedema. Human envenomation is more frequent with the ground-dwelling species, including the infamous Sydney funnel-web spider ( Atrax robustus ); although, only two tree-dwelling species induce more severe envenomation. To unravel the mechanisms that lead to this stark difference in clinical outcomes, we investigated the venom transcriptome and proteome of arboreal Hadronyche cerberea and H. formidabilis . Overall, Hadronyche venoms comprised 44 toxin superfamilies, with 12 being exclusive to tree-dwellers. Surprisingly, the major venom components were neprilysins and uncharacterized peptides, in addition to the well-known ω- and δ-hexatoxins and double-knot peptides. The insecticidal effects of Hadronyche venom on sheep blowflies were more potent than Atrax venom, and the venom of both tree- and ground-dwelling species potently modulated human voltage-gated sodium channels, particularly Na V 1.2. Only the venom of tree-dwellers exhibited potent modulation of voltage-gated calcium channels. H. formidabilis appeared to be under less diversifying selection pressure compared to the newly adapted tree-dweller, H. cerberea . Thus, this study contributes to unravelling the fascinating molecular and pharmacological basis for the severe envenomation caused by the Australian tree-dwelling funnel-web spiders.

Background and Purpose: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma., Experimental Approach: We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish)., Key Results: Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAF V600E -mutated cells with minimal effects on fibroblasts. In melanoma-treated cells, Octpep-1 increased ROS with unaltered mitochondrial membrane potential and promoted non-mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep-1 depends upon the α-helix and polyproline conformation in the C-terminal region of the peptide. A truncated form of the C-terminal end of Octpep-1 displayed enhanced potency and efficacy against melanoma. Octpep-1 reduced the progression of tumours in xenograft melanoma mice and zebrafish., Conclusion and Implications: We unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma., (© 2022 British Pharmacological Society.)

Inhibition of T-type calcium channels (Ca V 3) prevents development of diseases related to cardiovascular and nerve systems. Further, knockout animal studies have revealed that some diseases are mediated by specific subtypes of Ca V 3. However, subtype-specific Ca V 3 inhibitors for therapeutic purposes or for studying the physiological roles of Ca V 3 subtypes are missing. To bridge this gap, we employed our spider venom library and uncovered that Avicularia spec. ("Amazonas Purple", Peru) tarantula venom inhibited specific T-type Ca V channel subtypes. By using chromatographic and mass-spectrometric techniques, we isolated and sequenced the active toxin ω-Avsp1a, a C-terminally amidated 36 residue peptide with a molecular weight of 4224.91 Da, which comprised the major peak in the venom. Both native (4.1 μM) and synthetic ω-Avsp1a (10 μM) inhibited 90% of Ca V 3.1 and Ca V 3.3, but only 25% of Ca V 3.2 currents. In order to investigate the toxin binding site, we generated a range of chimeric channels from the less sensitive Ca V 3.2 and more sensitive Ca V 3.3. Our results suggest that domain-1 of Ca V 3.3 is important for the inhibitory effect of ω-Avsp1a on T-type calcium channels. Further studies revealed that a leucine of T-type calcium channels is crucial for the inhibitory effect of ω-Avsp1a.

α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 3 10 -helix frequently employed by framework I α-conotoxins; α-SII acted at the muscle nAChR with half-maximal inhibitory concentrations (IC 50 ) of 120 nM (adult) and 370 nM (fetal) though weakly at neuronal nAChRs. Truncation of α-SII to a two disulfide bond amidated peptide with framework I disulfide connectivity led to similar activity. Surprisingly, the more constrained α-SII was less stable under mild reducing conditions and displayed a unique docking mode at the nAChR.

The King Baboon spider, Pelinobius muticus , is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus , but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including Na V 1.8, K V 2.1, and tetrodotoxin-sensitive Na V channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The short disulfide-rich α-conotoxins derived from the venom of Conus snails comprise a conserved C I C II (m)C III (n)C IV cysteine framework (m and n, number of amino acids) and the majority antagonize nicotinic acetylcholine receptors (nAChRs). Depending on disulfide connectivity, α-conotoxins can exist as either globular (C I -C III , C II -C IV ), ribbon (C I -C IV , C II -C III ) or bead (C I -C II , C III -C IV ) isomers. In the present study, C. geographus α-conotoxins GI, GIB, G1.5 and G1.9 were chemically synthesized as globular and ribbon isomers and their activity investigated at human nAChRs expressed in Xenopus oocytes using the two-electrode voltage clamp recording technique. Both the globular and ribbon isomers of the 3/5 (m/n) α-conotoxins GI and GIB selectively inhibit heterologous human muscle-type α1β1δε nAChRs, whereas G1.5, a 4/7 α-conotoxin, selectively antagonizes neuronal (non-muscle) nAChR subtypes particularly human α3β2, α7 and α9α10 nAChRs. In contrast, globular and ribbon isomers of G1.9, a novel C-terminal elongated 4/8 α-conotoxin exhibited no activity at the human nAChR subtypes studied. This study reinforces earlier observations that 3/5 α-conotoxins selectively target the muscle nAChR subtypes, although interestingly, GIB is also active at α7 and α9 α10 nAChRs. The 4/7 α-conotoxins target human neuronal nAChR subtypes whereas the pharmacology of the 4/8 α-conotoxin remains unknown., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides., Competing Interests: The authors declare no competing interest.

Since the introduction of insulin almost a century ago, more than 80 peptide drugs have reached the market for a wide range of diseases, including diabetes, cancer, osteoporosis, multiple sclerosis, HIV infection and chronic pain. In this Perspective, we summarize key trends in peptide drug discovery and development, covering the early efforts focused on human hormones, elegant medicinal chemistry and rational design strategies, peptide drugs derived from nature, and major breakthroughs in molecular biology and peptide chemistry that continue to advance the field. We emphasize lessons from earlier approaches that are still relevant today as well as emerging strategies such as integrated venomics and peptide-display libraries that create new avenues for peptide drug discovery. We also discuss the pharmaceutical landscape in which peptide drugs could be particularly valuable and analyse the challenges that need to be addressed for them to reach their full potential.

Melanoma is the main cause of skin cancer deaths, with special emphasis in those cases carrying BRAF mutations that trigger the mitogen-activated protein kinases (MAPK) signaling and unrestrained cell proliferation in the absence of mitogens. Current therapies targeting MAPK are hindered by drug resistance and relapse that rely on metabolic rewiring and Akt activation. To identify new drug candidates against melanoma, we investigated the molecular mechanism of action of the Octopus Kaurna -derived peptide, Octpep-1, in human BRAF(V600E) melanoma cells using proteomics and RNAseq coupled with metabolic analysis. Fluorescence microscopy verified that Octpep-1 tagged with fluorescein enters MM96L and NFF cells and distributes preferentially in the perinuclear area of MM96L cells. Proteomics and RNAseq revealed that Octpep-1 targets PI3K/AKT/mTOR signaling in MM96L cells. In addition, Octpep-1 combined with rapamycin (mTORC1 inhibitor) or LY3214996 (ERK1/2 inhibitor) augmented the cytotoxicity against BRAF(V600E) melanoma cells in comparison with the inhibitors or Octpep-1 alone. Octpep-1-treated MM96L cells displayed reduced glycolysis and mitochondrial respiration when combined with LY3214996. Altogether these data support Octpep-1 as an optimal candidate in combination therapies for melanoma BRAF(V600E) mutations.

Vasopressin (VP) and oxytocin (OT) are cyclic neuropeptides that regulate fundamental physiological functions via four G protein-coupled receptors, V 1a R, V 1b R, V 2 R, and OTR. Ligand development remains challenging for these receptors due to complex structure-activity relationships. Here, we investigated dimerization as a strategy for developing ligands with novel pharmacology. We regioselectively synthesised and systematically studied parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimer constructs of VP, OT and dVDAVP (1-deamino-4-valine-8-d-arginine-VP). All disulfide-linked dimers, except for the head-to-tail cyclized constructs, retained nanomolar potency despite the structural implications of dimerization. Our results support a single chain interaction for receptor activation. Dimer orientation had little impact on activity, except for the dVDAVP homodimers, where an antagonist to agonist switch was observed at the V 1a R. This study provides novel insights into the structural requirements of VP/OT receptor activation and spotlights dimerization as a strategy to modulate pharmacology, a concept also frequently observed in nature., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I 3 -subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus . Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I 3 -subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded β-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

Management of chronic pain presents a major challenge, since many currently available treatments lack efficacy and have problems such as addiction and tolerance. Loss of function mutations in the SCN9A gene lead to a congenital inability to feel pain, with no other sensory deficits aside from anosmia. SCN9A encodes the voltage-gated sodium (Na V ) channel 1.7 (Na V 1.7), which has been identified as a primary pain target. However, in developing Na V 1.7-targeted analgesics, extreme care must to be taken to avoid off-target activity on other Na V subtypes that are critical for survival. Since spider venoms are an excellent source of Na V channel modulators, we screened a panel of spider venoms to identify selective Na V 1.7 inhibitors. This led to identification of two novel Na V modulating venom peptides (β/μ-theraphotoxin-Pe1a and β/μ-theraphotoxin-Pe1b (Pe1b) from the arboreal tarantula Phormingochilus everetti. A third peptide isolated from the tarantula Bumba pulcherrimaklaasi was identical to the well-known ProTx-I (β/ω-theraphotoxin-Tp1a) from the tarantula Thrixopelma pruriens. A tethered toxin (t-toxin)-based alanine scanning strategy was used to determine the Na V 1.7 pharmacophore of ProTx-I. We designed several ProTx-I and Pe1b analogues, and tested them for activity and Na V channel subtype selectivity. Several analogues had improved potency against Na V 1.7, and altered specificity against other Na V channels. These analogues provide a foundation for development of Pe1b as a lead molecule for therapeutic inhibition of Na V 1.7., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Australian funnel-web spiders are infamous for causing human fatalities, which are induced by venom peptides known as δ-hexatoxins (δ-HXTXs). Humans and other primates did not feature in the prey or predator spectrum during evolution of these spiders, and consequently the primate lethality of δ-HXTXs remains enigmatic. Funnel-web envenomations are mostly inflicted by male spiders that wander from their burrow in search of females during the mating season, which suggests a role for δ-HXTXs in self-defense since male spiders rarely feed during this period. Although 35 species of Australian funnel-web spiders have been described, only nine δ-HXTXs from four species have been characterized, resulting in a lack of understanding of the ecological roles and molecular evolution of δ-HXTXs. Here, by profiling venom-gland transcriptomes of 10 funnel-web species, we report 22 δ-HXTXs. Phylogenetic and evolutionary assessments reveal a remarkable sequence conservation of δ-HXTXs despite their deep evolutionary origin within funnel-web spiders, consistent with a defensive role. We demonstrate that δ-HXTX-Ar1a, the lethal toxin from the Sydney funnel-web spider Atrax robustus , induces pain in mice by inhibiting inactivation of voltage-gated sodium (Na V ) channels involved in nociceptive signaling. δ-HXTX-Ar1a also inhibited inactivation of cockroach Na V channels and was insecticidal to sheep blowflies. Considering their algogenic effects in mice, potent insecticidal effects, and high levels of sequence conservation, we propose that the δ-HXTXs were repurposed from an initial insecticidal predatory function to a role in defending against nonhuman vertebrate predators by male spiders, with their lethal effects on humans being an unfortunate evolutionary coincidence., Competing Interests: The authors declare no competing interest.

Breast cancer is making up one-quarter of all new female cancer cases diagnosed worldwide. Breast cancer surgeries, radiation therapies, cytotoxic chemotherapies and targeted therapies have made significant progress and play a dominant role in breast cancer patient management. However, many challenges remain, including resistance to systemic therapies, tumour recurrence and metastasis. The cyclic neuropeptide oxytocin (OT) elicits a plethora of biological responses via the oxytocin receptor (OTR) in both the central and peripheral nervous system, including social bonding, stress, maternal behaviour, sexual activity, uterus contraction, milk ejection and cancer. As a typical member of the G protein-coupled receptor family, OTR represents also an intriguing target for cancer therapy. There is emerging evidence that OTR plays a role in breast cancer development and progression, and several breast cancer cell lines express OTR. However, despite supporting evidence that OT lowers breast cancer risks, its mechanistic role in breast cancer development and the related signalling pathways are not fully understood. Here, we review the current knowledge of the OT/OTR signalling system in healthy breast tissue as well as in breast cancer, and discuss OTR as a potential therapeutic target for breast cancer management.

Ant venoms have recently attracted increased attention due to their chemical complexity, novel molecular frameworks, and diverse biological activities. The heterodimeric peptide ∆-myrtoxin-Mp1a (Mp1a) from the venom of the Australian jack jumper ant, Myrmecia pilosula , exhibits antimicrobial, membrane-disrupting, and pain-inducing activities. In the present study, we examined the activity of Mp1a and a panel of synthetic analogues against the gastrointestinal parasitic nematode Haemonchus contortus , the fruit fly Drosophila melanogaster , and for their ability to stimulate pain-sensing neurons. Mp1a was found to be both insecticidal and anthelmintic, and it robustly activated mammalian sensory neurons at concentrations similar to those reported to elicit antimicrobial and cytotoxic activity. The native antiparallel Mp1a heterodimer was more potent than heterodimers with alternative disulfide connectivity, as well as monomeric analogues. We conclude that the membrane-disrupting effects of Mp1a confer broad-spectrum biological activities that facilitate both predation and defense for the ant. Our structure-activity data also provide a foundation for the rational engineering of analogues with selectivity for particular cell types., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: Animal toxins have contributed significantly to our understanding of the neurobiology of receptors and ion channels. We studied the venom of the coral snake Micrurus fulvius fulvius and identified and characterized the structure and pharmacology of a new homodimeric neurotoxin, fulditoxin, that exhibited novel pharmacology at nicotinic ACh receptors (nAChRs)., Experimental Approach: Fulditoxin was isolated by chromatography, chemically synthesized, its structure determined by X-ray crystallography, and its pharmacological actions on nAChRs characterized by organ bath assays and two-electrode voltage clamp electrophysiology., Key Results: Fulditoxin's distinct 1.95-Å quaternary structure revealed two short-chain three-finger α-neurotoxins (α-3FNTxs) non-covalently bound by hydrophobic interactions and an ability to bind metal and form tetrameric complexes, not reported previously for three-finger proteins. Although fulditoxin lacked all conserved amino acids canonically important for inhibiting nAChRs, it produced postsynaptic neuromuscular blockade of chick muscle at nanomolar concentrations, comparable to the prototypical α-bungarotoxin. This neuromuscular blockade was completely reversible, which is unusual for snake α-3FNTxs. Fulditoxin, therefore, interacts with nAChRs by utilizing a different pharmacophore. Unlike short-chain α-3FNTxs that bind only to muscle nAChRs, fulditoxin utilizes dimerization to expand its pharmacological targets to include human neuronal α4β2, α7, and α3β2 nAChRs which it blocked with IC 50 values of 1.8, 7, and 12 μM respectively., Conclusions and Implications: Based on its distinct quaternary structure and unusual pharmacology, we named this new class of dimeric Micrurus neurotoxins represented by fulditoxin as Σ-neurotoxins, which offers greater insight into understanding the interactions between nAChRs and peptide antagonists., (© 2019 The British Pharmacological Society.)

Compelling human genetic studies have identified the voltage-gated sodium channel Na V 1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na V 1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa V 1.7 using patch-clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNa V 1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na V 1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets Na V 1.4, Na V 1.5, and Na V 1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo , significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na V 1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na V 1.7 in pain., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (Na V ). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/ δ -theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate Na V channels. Pme1a is a 35 residue peptide that inhibits Na V 1.7 peak current (IC 50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V 1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits Na V 1.7 peak current (EC 50 > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at Na V 1.7 (IC 50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at Na V channels in the future., Competing Interests: The authors declare no conflict of interest

Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC 50  = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α 1 -adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.

The venom of the marine predatory cone snails (genus Conus ) has evolved for prey capture and defense, providing the basis for survival and rapid diversification of the now estimated 750+ species. A typical Conus venom contains hundreds to thousands of bioactive peptides known as conotoxins. These mostly disulfide-rich and well-structured peptides act on a wide range of targets such as ion channels, G protein-coupled receptors, transporters, and enzymes. Conotoxins are of interest to neuroscientists as well as drug developers due to their exquisite potency and selectivity, not just against prey but also mammalian targets, thereby providing a rich source of molecular probes and therapeutic leads. The rise of integrated venomics has accelerated conotoxin discovery with now well over 10,000 conotoxin sequences published. However, their structural and pharmacological characterization lags considerably behind. In this review, we highlight the diversity of new conotoxins uncovered since 2014, their three-dimensional structures and folds, novel chemical approaches to their syntheses, and their value as pharmacological tools to unravel complex biology. Additionally, we discuss challenges and future directions for the field.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak ( K i low micromolar) μ-opioid agonists, which led to the design of bilorphin, a potent and selective μ-opioid receptor (MOPr) agonist ( K i 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit β-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting β-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics., Competing Interests: Competing interest statement: Patent Application(s) corresponding to Australian Patent Application 2018901944 The University of Sydney and The University of Queensland has been filed concerning this peptide. Title: Analgesics and Methods of Use Thereof., (Copyright © 2019 the Author(s). Published by PNAS.)

Pain is the leading cause of disability in the developed world but remains a poorly treated condition. Specifically, postsurgical pain continues to be a frequent and undermanaged condition. Here, we investigate the analgesic potential of pharmacological NaV1.7 inhibition in a mouse model of acute postsurgical pain, based on incision of the plantar skin and underlying muscle of the hind paw. We demonstrate that local and systemic treatment with the selective NaV1.7 inhibitor μ-theraphotoxin-Pn3a is effectively antiallodynic in this model and completely reverses mechanical hypersensitivity in the absence of motor adverse effects. In addition, the selective NaV1.7 inhibitors ProTx-II and PF-04856264 as well as the clinical candidate CNV1014802 also reduced mechanical allodynia. Interestingly, co-administration of the opioid receptor antagonist naloxone completely reversed analgesic effects of Pn3a, indicating an involvement of endogenous opioids in the analgesic activity of Pn3a. In addition, we found superadditive antinociceptive effects of subtherapeutic Pn3a doses not only with the opioid oxycodone but also with the GABAB receptor agonist baclofen. Transcriptomic analysis of gene expression changes in dorsal root ganglia of mice after surgery did not reveal any changes in mRNA expression of endogenous opioids or opioid receptors; however, several genes involved in pain, including Runx1 (Runt related transcription factor 1), Cacna1a (CaV2.1), and Cacna1b (CaV2.2), were downregulated. In summary, these findings suggest that pain after surgery can be successfully treated with NaV1.7 inhibitors alone or in combination with baclofen or opioids, which may present a novel and safe treatment strategy for this frequent and poorly managed condition.

The Estuarine stonefish (Synanceia horrida) is recognised as one of the most venomous fish species in the world but the overall venom composition has yet to be investigated using in-depth transcriptomic and proteomic methods. To date, known venom components are restricted to a hyaluronidase and a large, pore-forming toxin known as Stonustoxin (SNTX). Transcriptomic sequencing of the venom gland resulted in over 170,000 contigs with only 0.4% that were homologous to putative venom proteins. Integration of the transcriptomic data with proteomic data from the S. horrida venom confirmed the hyaluronidase and SNTX to be present, together with several other protein families including major contributions from C-type lectins. Other protein families observed included peroxiredoxin and several minor protein families such as Golgi-associated plant pathogenesis related proteins, tissue pathway factor inhibitors, and Kazal-type serine protease inhibitors that, although not putative venom proteins, may contribute to the venom's adverse effects. BIOLOGICAL SIGNIFICANCE: Proteomic analysis of milked Synanceia horrida venom, paired with transcriptomic analysis of the venom gland tissue revealed for the first time the composition of one of the world's most dangerous fish venoms. The results demonstrate that the venom is relatively less complex compared to other well-studied venomous animals with a number of unique proteins not previously found in animal venoms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Conorfamides are a poorly studied family of cone snail venom peptides with broad biological activities, including inhibition of glutamate receptors, acid-sensing ion channels, and voltage-gated potassium channels. The aim of this study was to characterize the pharmacological activity of two novel linear conorfamides (conorfamide&#95;As1a and conorfamide&#95;As2a) and their non-amidated counterparts (conopeptide&#95;As1b and conopeptide&#95;As2b) that were isolated from the venom of the Mexican cone snail Conus austini. Although As1a, As2a, As1b and As2b were identified by activity-guided fractionation using a high-throughput fluorescence imaging plate reader (FLIPR) assay assessing α7 nAChR activity, sequence determination revealed activity associated with four linear peptides of the conorfamide rather than the anticipated α-conotoxin family. Pharmacological testing revealed that the amidated peptide variants altered desensitization of acid-sensing ion channels (ASICs) 1a and 3, and the native lysine to arginine mutation differentiating As1a and As1b from As2a and As2b introduced ASIC1a peak current potentiation. Surprisingly, these conorfamides also inhibited α7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. This is the first report of conorfamides with dual activity, with the nAChR activity being the most potent molecular target of any conorfamide discovered to date., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Individual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between individual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis . Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly diverse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to diversifying selection.

Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus , the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that "messy" processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.

The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus , and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between individuals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, μ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.