Unlocking nature's arsenal: Nanotechnology for targeted delivery of venom toxins in cancer therapy.

AIM: The aim of the present review is to shed light on the nanotechnological approaches adopted to overcome the shortcomings associated with the delivery of venom peptides which possess inherent anti-cancer properties. BACKGROUND: Venom peptides although have been reported to demonstrate anti-cancer effects, they suffer from several disadvantages such as in vivo instability, off-target adverse effects, limited drug loading and low bioavailability. This review presents a comprehensive compilation of different classes of nanocarriers while underscoring their advantages, disadvantages and potential to carry such peptide molecules for in vivo delivery. It also discusses various nanotechnological aspects such as methods of fabrication, analytical tools to assess these nanoparticulate formulations, modulation of nanocarrier polymer properties to enhance loading capacity, stability and improve their suitability to carry toxic peptide drugs. CONCLUSION: Nanotechnological approaches bear great potential in delivering venom peptide-based molecules as anticancer agents by enhancing their bioavailability, stability, efficacy as well as offering a spatiotemporal delivery approach. However, the challenges associated with toxicity and biocompatibility of nanocarriers must be duly addressed. PERSPECTIVES: The everlasting quest for new breakthroughs for safer delivery of venom peptides in human subjects is fuelled by unmet clinical needs in the current landscape of chemotherapy. In addition, exhaustive efforts are required in obtaining and purifying the venom peptides followed by designing and optimizing scale up technologies.

DeTox: a pipeline for the detection of toxins in venomous organisms.

Venomous organisms have independently evolved the ability to produce toxins 101 times during their evolutionary history, resulting in over 200 000 venomous species. Collectively, these species produce millions of toxins, making them a valuable resource for bioprospecting and understanding the evolutionary mechanisms underlying genetic diversification. RNA-seq is the preferred method for characterizing toxin repertoires, but the analysis of the resulting data remains challenging. While early approaches relied on similarity-based mapping to known toxin databases, recent studies have highlighted the importance of structural features for toxin detection. The few existing pipelines lack an integration between these complementary approaches, and tend to be difficult to run for non-experienced users. To address these issues, we developed DeTox, a comprehensive and user-friendly tool for toxin research. It combines fast execution, parallelization and customization of parameters. DeTox was tested on published transcriptomes from gastropod mollusks, cnidarians and snakes, retrieving most putative toxins from the original articles and identifying additional peptides as potential toxins to be confirmed through manual annotation and eventually proteomic analysis. By integrating a structure-based search with similarity-based approaches, DeTox allows the comprehensive characterization of toxin repertoire in poorly-known taxa. The effect of the taxonomic bias in existing databases is minimized in DeTox, as mirrored in the detection of unique and divergent toxins that would have been overlooked by similarity-based methods. DeTox streamlines toxin annotation, providing a valuable tool for efficient identification of venom components that will enhance venom research in neglected taxa.

Targeting Dysregulated Ion Channels in Liver Tumors with Venom Peptides.

The regulation of cellular processes by ion channels has become central to the study of cancer mechanisms. Designing molecules that can modify ion channels specific to tumor cells is a promising area of targeted drug delivery and therapy. Despite their potential in drug discovery, venom peptides-a group of natural products-have largely remained understudied and under-characterized. In general, venom peptides display high specificity and selectivity for their target ion channels. Therefore, they may represent an effective strategy for selectively targeting the dysregulation of ion channels in tumor cells. This review examines existing venom peptide therapies for different cancer types and focuses on the application of snail venom peptides in hepatocellular carcinoma (HCC), the most common form of primary liver cancer worldwide. We provide insights into the mode of action of venom peptides that have been shown to target tumors. We also explore the benefit of using new computational methods like de novo protein structure prediction to screen venom peptides and identify potential druggable candidates. Finally, we summarize the role of cell culture, animal, and organoid models in developing effective therapies against HCC and highlight the need for creating models that represent the most disproportionately affected ethnicities in HCC.

Uncovering CNS access of lipidated exendin-4 analogues by quantitative whole-brain 3D light sheet imaging.

Peptide-based drug development for CNS disorders is challenged by poor blood-brain barrier (BBB) penetrability of peptides. While acylation protractions (lipidation) have been successfully applied to increase circulating half-life of therapeutic peptides, little is known about the CNS accessibility of lipidated peptide drugs. Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method to visualize whole-brain 3D distribution of fluorescently labelled therapeutic peptides at single-cell resolution. Here, we applied LSFM to map CNS distribution of the clinically relevant GLP-1 receptor agonist (GLP-1RA) exendin-4 (Ex4) and lipidated analogues following peripheral administration. Mice received an intravenous dose (100 nmol/kg) of IR800 fluorophore-labelled Ex4 (Ex4), Ex4 acylated with a C16-monoacid (Ex4_C16MA) or C18-diacid (Ex4_C18DA). Other mice were administered C16MA-acylated exendin 9-39 (Ex9-39_C16MA), a selective GLP-1R antagonist, serving as negative control for GLP-1R mediated agonist internalization. Two hours post-dosing, brain distribution of Ex4 and analogues was predominantly restricted to the circumventricular organs, notably area postrema and nucleus of the solitary tract. However, Ex4_C16MA and Ex9-39_C16MA also distributed to the paraventricular hypothalamic nucleus and medial habenula. Notably, Ex4_C18DA was detected in deeper-lying brain structures such as dorsomedial/ventromedial hypothalamic nuclei and the dentate gyrus. Similar CNS distribution maps of Ex4_C16MA and Ex9-39_C16MA suggest that brain access of lipidated Ex4 analogues is independent on GLP-1 receptor internalization. The cerebrovasculature was devoid of specific labelling, hence not supporting a direct role of GLP-1 RAs in BBB function. In conclusion, peptide lipidation increases CNS accessibility of Ex4. Our fully automated LSFM pipeline is suitable for mapping whole-brain distribution of fluorescently labelled drugs.

Venom composition and bioactive RF-amide peptide toxins of the saddleback caterpillar, Acharia stimulea (Lepidoptera: Limacodidae).

Limacodidae is a family of lepidopteran insects comprising >1500 species. More than half of these species produce pain-inducing defensive venoms in the larval stage, but little is known about their venom toxins. Recently, we characterised proteinaceous toxins from the Australian limacodid caterpillar Doratifera vulnerans, but it is unknown if the venom of this species is typical of other Limacodidae. Here, we use single animal transcriptomics and venom proteomics to investigate the venom of an iconic limacodid, the North American saddleback caterpillar Acharia stimulea. We identified 65 venom polypeptides, grouped into 31 different families. Neurohormones, knottins, and homologues of the immune signaller Diedel make up the majority of A.stimulea venom, indicating strong similarities to D. vulnerans venom, despite the large geographic separation of these caterpillars. One notable difference is the presence of RF-amide peptide toxins in A. stimulea venom. Synthetic versions of one of these RF-amide toxins potently activated the human neuropeptide FF1 receptor, displayed insecticidal activity when injected into Drosophila melanogaster, and moderately inhibited larval development of the parasitic nematode Haemonchus contortus. This study provides insights into the evolution and activity of venom toxins in Limacodidae, and provides a platform for future structure-function characterisation of A.stimulea peptide toxins.

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39).

PURPOSE: Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. METHODS: We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. RESULTS: Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). CONCLUSION: We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

Unraveling the venom chemistry with evidence for histamine as key regulator in the envenomation by caterpillar Automeris zaruma.

Over the past decades, envenomation by caterpillars of Automeris spp. became an increasing health problem in Latin America. Accidental contact with the stinging spines of these caterpillars cause acute local pain, itching, inflammation and skin rashes that persists for days. Even when the cause is obvious, the exact molecular mechanisms responsible for the observed symptoms are yet to be elucidated. Here, we describe for the first time, an active compound in the venom and the study of the bioactivity of the venom extracted from the spines of the caterpillar Automeris zaruma. Electrophysiological screening of a library of membrane proteins important for pain and itch enabled us to investigate and reveal the mode of action of the venom of A. zaruma. Further mass spectrometric analysis (Q-TOF-MS) made it possible to establish a link between the bioactivity and the components found in the venom. We show that the spine extract of A. zaruma contains histamine that potently activates the four types of the human histamine receptors (H1R, H2R, H3R and H4R) with a selectivity preference towards H3R and H4R. Furthermore, a modulation of the target MRGPRX2 was found. Together, these findings are the first to explain the symptomology of A. zaruma envenomation, enabling us a better understanding of caterpillar envenomation and predict that the hurdle of the scarce efficacy of the currently used antihistaminic drugs can be overcome by including H3R and H4R blockers in the clinical used medication. Such an approach might be used for other caterpillar envenomation in the world and represent a significant improvement for the well-being of the patient.

Somatostatin venom analogs evolved by fish-hunting cone snails: From prey capture behavior to identifying drug leads.

Somatostatin (SS) is a peptide hormone with diverse physiological roles. By investigating a deep-water clade of fish-hunting cone snails, we show that predator-prey evolution has generated a diverse set of SS analogs, each optimized to elicit specific systemic physiological effects in prey. The increased metabolic stability, distinct SS receptor activation profiles, and chemical diversity of the venom analogs make them suitable leads for therapeutic application, including pain, cancer, and endocrine disorders. Our findings not only establish the existence of SS-like peptides in animal venoms but also serve as a model for the synergy gained from combining molecular phylogenetics and behavioral observations to optimize the discovery of natural products with biomedical potential.

Proteogenomic Assessment of Intraspecific Venom Variability: Molecular Adaptations in the Venom Arsenal of Conus purpurascens.

Cone snails produce venom that contains diverse groups of peptides (conopeptides/conotoxins) and display a wide mass range, high rate of posttranslational modifications, and many potential pharmacological targets. Here we employ a proteogenomic approach to maximize conopeptide identification from the injected venom of Conus purpurascens. mRNA sequences from C. purpurascens venom ducts were assembled into a search database and complemented with known sequences and de novo approaches. We used a top-down peptidomic approach and tandem mass spectrometry identification to compare injected venom samples of 27 specimens. This intraspecific analysis yielded 543 unique conopeptide identifications, which included 33 base conopeptides and their toxiforms, 21 of which are novel. The results reveal two distinct venom profiles with different synergistic interactions to effectively target neural pathways aimed to immobilize prey. These venom expression patterns will aid target prediction, a significant step toward developing conotoxins into valuable drugs or neural probes.

Advances in venom peptide drug discovery: where are we at and where are we heading?

Introduction: Animal venoms are a complex mixture of bioactive molecules that have evolved over millions of years for prey capture and defense from predators. Venom consists of many different types of molecules, with disulfide-rich peptides being a major component in most venoms. The study of these potent and highly selective molecules has led to the development of venom-derived drugs for diseases such as type 2 diabetes mellitus and chronic pain. As technologies have improved, more bioactive peptides have been discovered from venomous animals. Many of these molecules may have applications as tools for understanding normal and disease physiology, therapeutics, cosmetics or in agriculture.Areas covered: This article reviews venom-derived drugs approved by the FDA and venom-derived peptides currently in development. It discusses the challenges faced by venom-derived peptide drugs during drug development and the future for venom-derived peptides.Expert opinion: New techniques such as toxin driven discovery are expanding the pipeline of venom-derived peptides. There are many venom-derived peptides currently in preclinical and clinical trials that would have remained undiscovered using traditional approaches. A renewed focus on venoms, with advances in technology, will broaden the diversity of venom-derived peptide therapeutics and expand our knowledge of their molecular targets.

Interactions of Sea Anemone Toxins with Insect Sodium Channel-Insights from Electrophysiology and Molecular Docking Studies.

Animal venoms are considered as a promising source of new drugs. Sea anemones release polypeptides that affect electrical activity of neurons of their prey. Voltage dependent sodium (Nav) channels are the common targets of Av1, Av2, and Av3 toxins from Anemonia viridis and CgNa from Condylactis gigantea. The toxins bind to the extracellular side of a channel and slow its fast inactivation, but molecular details of the binding modes are not known. Electrophysiological measurements on Periplaneta americana neuronal preparation revealed differences in potency of these toxins to increase nerve activity. Av1 and CgNa exhibit the strongest effects, while Av2 the weakest effect. Extensive molecular docking using a modern SMINA computer method revealed only partial overlap among the sets of toxins' and channel's amino acid residues responsible for the selectivity and binding modes. Docking positions support earlier supposition that the higher neuronal activity observed in electrophysiology should be attributed to hampering the fast inactivation gate by interactions of an anemone toxin with the voltage driven S4 helix from domain IV of cockroach Nav channel (NavPaS). Our modelling provides new data linking activity of toxins with their mode of binding in site 3 of NavPaS channel.

Venom peptides in cancer therapy: An updated review on cellular and molecular aspects.

Based on the high incidence and mortality rates of cancer, its therapy remains one of the most vital challenges in the field of medicine. Consequently, enhancing the efficacy of currently applied treatments and finding novel strategies are of great importance for cancer treatment. Venoms are important sources of a variety of bioactive compounds including salts, small molecules, macromolecules, proteins, and peptides that are defined as toxins. They can exhibit different pharmacological effects, and in recent years, their anti-tumor activities have gained significant attention. Several different compounds are responsible for the anti-tumor activity of venoms, and peptides are one of them. In the present review, we discuss the possible anti-tumor activities of venom peptides by highlighting molecular pathways and mechanisms through which these molecules can act effectively. Venom peptides can induce cell death in cancer cells and can substantially enhance the efficacy of chemotherapy and radiotherapy. Also, the venom peptides can mitigate the migration of cancer cells via suppression of angiogenesis and epithelial-to-mesenchymal transition. Notably, nanoparticles have been applied in enhancing the bioavailability of venom peptides and providing targeted delivery, thereby leading to their elevated anti-tumor activity and potential application for cancer therapy.

Induced Disassembly of a Virus-like Particle under Physiological Conditions for Venom Peptide Delivery.

Virus-like particles (VLPs) show considerable promise for the in vivo delivery of therapeutic compounds such as bioactive venom peptides. While loading and targeting protocols have been developed for numerous VLP prototypes, induced disassembly under physiological conditions of neutral pH, moderate temperature, and aqueous medium remain a challenge. Here, we implement and evaluate a general mechanism, based on ring-opening metathesis polymerization (ROMP), for controllable VLP disassembly. This mechanism is independent of cell-specific factors or the manipulation of environmental conditions such as pH and temperature that cannot be readily controlled in vivo. The ROMP substrate norbornene is covalently conjugated to surface-exposed lysine residues of a P22 bacteriophage-derived VLP, and ROMP is induced by treatment with the water-soluble ruthenium catalyst AquaMet. Disruption of the P22 shell and release of a GFP reporter is confirmed via native agarose electrophoresis, TEM, and dynamic light scattering (DLS) analyses. Our ROMP disassembly strategy does not depend on the particular structure or morphology of the P22 nanocontainer and is adaptable to other VLP prototypes for the potential delivery of venom peptides for pharmacological applications.

The voltage-gated potassium channel KV1.3 as a therapeutic target for venom-derived peptides.

The voltage-gated potassium channel KV1.3 is a well-established therapeutic target for a range of autoimmune diseases, in addition to being the site of action of many venom-derived peptides. Numerous studies have documented the efficacy of venom peptides that target KV1.3, in particular from sea anemones and scorpions, in animal models of autoimmune diseases such as rheumatoid arthritis, psoriasis and multiple sclerosis. Moreover, an analogue of the sea anemone peptide ShK (known as dalazatide) has successfully completed Phase 1 clinical trials in mild-to-moderate plaque psoriasis. In this article we consider other potential therapeutic applications of inhibitors of KV1.3, including in inflammatory bowel disease and neuroinflammatory conditions such as Alzheimer's and Parkinson's diseases, as well as fibrotic diseases. We also summarise strategies for facilitating the entry of peptides to the central nervous system, given that this will be a pre-requisite for the treatment of most neuroinflammatory diseases. Venom-derived peptides that have been reported recently to target KV1.3 are also described. The increasing number of autoimmune and other conditions in which KV1.3 is upregulated and is therefore a potential therapeutic target, combined with the fact that many venom-derived peptides are potent inhibitors of KV1.3, suggests that venoms are likely to continue to serve as a rich source of new pharmacological tools and therapeutic leads targeting this channel.

Neuropeptide signalling systems - An underexplored target for venom drug discovery.

Neuropeptides are signalling molecules mainly secreted from neurons that act as neurotransmitters or peptide hormones to affect physiological processes and modulate behaviours. In humans, neuropeptides are implicated in numerous diseases and understanding their role in physiological processes and pathologies is important for therapeutic development. Teasing apart the (patho)physiology of neuropeptides remains difficult due to ligand and receptor promiscuity and the complexity of the signalling pathways. The current approach relies on a pharmacological toolbox of agonists and antagonists displaying high selectivity for independent receptor subtypes, with the caveat that only few selective ligands have been discovered or developed. Animal venoms represent an underexplored source for novel receptor subtype-selective ligands that could aid in dissecting human neuropeptide signalling systems. Multiple endogenous-like neuropeptides as well as peptides acting on neuropeptide receptors are present in venoms. In this review, we summarise current knowledge on neuropeptides and discuss venoms as a source for ligands targeting neuropeptide signalling systems.

Multi-targeting sodium and calcium channels using venom peptides for the treatment of complex ion channels-related diseases.

Venom peptides are amongst the most exquisite group of bioactive molecules able to alter the normal physiology of organisms. These bioactive peptides penetrate tissues and blood vessels to encounter a number of receptors and ion channels to which they bind with high affinity and execute modulatory activities. Arachnid is the most diverse class of venomous animals often rich in peptides modulating voltage-gated sodium (NaV), calcium (CaV), and potassium (KV) channels. Spider venoms, in particular, contain potent and selective peptides targeting these channels, with a few displaying interesting multi-target properties for NaV and CaV channels underlying disease mechanisms such as in neuropathic pain, motor neuron disease and cancer. The elucidation of the pharmacology and structure-function properties of these venom peptides are invaluable for the development of effective drugs targeting NaV and CaV channels. This perspective discusses spider venom peptides displaying multi-target properties to modulate NaV and CaV channels in regard to their pharmacological features, structure-function relationships and potential to become the next generation of effective drugs to treat neurological disorders and other multi-ion channels related diseases.

Animal toxins - Nature's evolutionary-refined toolkit for basic research and drug discovery.

Venomous animals have evolved toxins that interfere with specific components of their victim's core physiological systems, thereby causing biological dysfunction that aids in prey capture, defense against predators, or other roles such as intraspecific competition. Many animal lineages evolved venom systems independently, highlighting the success of this strategy. Over the course of evolution, toxins with exceptional specificity and high potency for their intended molecular targets have prevailed, making venoms an invaluable and almost inexhaustible source of bioactive molecules, some of which have found use as pharmacological tools, human therapeutics, and bioinsecticides. Current biomedically-focused research on venoms is directed towards their use in delineating the physiological role of toxin molecular targets such as ion channels and receptors, studying or treating human diseases, targeting vectors of human diseases, and treating microbial and parasitic infections. We provide examples of each of these areas of venom research, highlighting the potential that venom molecules hold for basic research and drug development.

Antimicrobials from Venomous Animals: An Overview.

The inappropriate or excessive use of antimicrobial agents caused an emerging public health problem due to the resulting resistance developed by microbes. Therefore, there is an urgent need to develop effective antimicrobial strategies relying on natural agents with different mechanisms of action. Nature has been known to offer many bioactive compounds, in the form of animal venoms, algae, and plant extracts that were used for decades in traditional medicine. Animal venoms and secretions have been deeply studied for their wealth in pharmaceutically promising molecules. As such, they were reported to exhibit many biological activities of interest, such as antibacterial, antiviral, anticancer, and anti-inflammatory activities. In this review, we summarize recent findings on the antimicrobial activities of crude animal venoms/secretions, and describe the peptides that are responsible of these activities.

Nanoparticles Functionalized with Venom-Derived Peptides and Toxins for Pharmaceutical Applications.

Venom-derived peptides display diverse biological and pharmacological activities, making them useful in drug discovery platforms and for a wide range of applications in medicine and pharmaceutical biotechnology. Due to their target specificities, venom peptides have the potential to be developed into biopharmaceuticals to treat various health conditions such as diabetes mellitus, hypertension, and chronic pain. Despite the high potential for drug development, several limitations preclude the direct use of peptides as therapeutics and hamper the process of converting venom peptides into pharmaceuticals. These limitations include, for instance, chemical instability, poor oral absorption, short halflife, and off-target cytotoxicity. One strategy to overcome these disadvantages relies on the formulation of bioactive peptides with nanocarriers. A range of biocompatible materials are now available that can serve as nanocarriers and can improve the bioavailability of therapeutic and venom-derived peptides for clinical and diagnostic application. Examples of isolated venom peptides and crude animal venoms that have been encapsulated and formulated with different types of nanomaterials with promising results are increasingly reported. Based on the current data, a wealth of information can be collected regarding the utilization of nanocarriers to encapsulate venom peptides and render them bioavailable for pharmaceutical use. Overall, nanomaterials arise as essential components in the preparation of biopharmaceuticals that are based on biological and pharmacological active venom-derived peptides.