Stingray Venom Proteins: Mechanisms of Action Revealed Using a Novel Network Pharmacology Approach

Animal venoms offer a valuable source of potent new drug leads, but their mechanisms of action are largely unknown. We therefore developed a novel network pharmacology approach based on multi-omics functional data integration to predict how stingray venom disrupts the physiological systems of target animals. We integrated 10 million transcripts from five stingray venom transcriptomes and 848,640 records from three high-content venom bioactivity datasets into a large functional data network. The network featured 216 signaling pathways, 29 of which were shared and targeted by 70 transcripts and 70 bioactivity hits. The network revealed clusters for single envenomation outcomes, such as pain, cardiotoxicity and hemorrhage. We carried out a detailed analysis of the pain cluster representing a primary envenomation symptom, revealing bibrotoxin and cholecystotoxin-like transcripts encoding pain-inducing candidate proteins in stingray venom. The cluster also suggested that such pain-inducing toxins primarily activate the inositol-3-phosphate receptor cascade, inducing intracellular calcium release. We also found strong evidence for synergistic activity among these candidates, with nerve growth factors cooperating with the most abundant translationally-controlled tumor proteins to activate pain signaling pathways. Our network pharmacology approach, here applied to stingray venom, can be used as a template for drug discovery in neglected venomous species.

Interaction of α9α10 Nicotinic Receptors With Peptides and Proteins From Animal Venoms

Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any β subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.

Animal Venoms and Their Components: Molecular Mechanisms of Action

Animal venoms comprise numerous toxin families, consisting mainly of peptides and proteins [...]

Variation of Two S3b Residues in KV4.1–4.3 Channels Underlies Their Different Modulations by Spider Toxin κ-LhTx-1

The naturally occurred peptide toxins from animal venoms are valuable pharmacological tools in exploring the structure-function relationships of ion channels. Herein we have identified the peptide toxin κ-LhTx-1 from the venom of spider Pandercetes sp (the Lichen huntsman spider) as a novel selective antagonist of the KV4 family potassium channels. κ-LhTx-1 is a gating-modifier toxin impeded KV4 channels’ voltage sensor activation, and mutation analysis has confirmed its binding site on channels’ S3b region. Interestingly, κ-LhTx-1 differently modulated the gating of KV4 channels, as revealed by toxin inhibiting KV4.2/4.3 with much more stronger voltage-dependence than that for KV4.1. We proposed that κ-LhTx-1 trapped the voltage sensor of KV4.1 in a much more stable resting state than that for KV4.2/4.3 and further explored the underlying mechanism. Swapping the non-conserved S3b segments between KV4.1(280FVPK283) and KV4.3(275VMTN278) fully reversed their voltage-dependence phenotypes in inhibition by κ-LhTx-1, and intensive mutation analysis has identified P282 in KV4.1, D281 in KV4.2 and N278 in KV4.3 being the key residues. Furthermore, the last two residues in this segment of each KV4 channel (P282/K283 in KV4.1, T280/D281 in KV4.2 and T277/N278 in KV4.3) likely worked synergistically as revealed by our combinatorial mutations analysis. The present study has clarified the molecular basis in KV4 channels for their different modulations by κ-LhTx-1, which have advanced our understanding on KV4 channels’ structure features. Moreover, κ-LhTx-1 might be useful in developing anti-arrhythmic drugs given its high affinity, high selectivity and unique action mode in interacting with the KV4.2/4.3 channels.

A Venomics Approach to Identify and Characterize Biocactive Peptides (BAP) with Anti-Cancer Activity against Colorectal Cancer Cells from Animal Venoms (Preprint)

BACKGROUND Cancer is the third leading cause of death in the United Arab Emirates (UAE) after cardiovascular diseases and accidents. In UAE, colorectal cancer (CRC) is the first and fourth most common cancer in males and females respectively. Several treatment modalities have been employed for cancer treatment such as surgery, radiotherapy, chemotherapy, hormone replacement therapy, and immunotherapy. These treatment modalities often elicit adverse effects on normal cells, causing toxic side effects. To circumvent these toxicities, there has been an increased impetus towards the identification of alternate treatment strategies. Animal venoms are veritable gold mines of pharmacologically active polypeptides and proteins. OBJECTIVE In this proof-of-concept study, we avail a high throughput “Venomics” strategy to identify and characterize anticancer bioactive peptides (BAP) from 20 different animal venoms specifically targeting CRC. We chose to focus on CRC as it is one of the foremost health issues in the UAE. METHODS In initial study, we will screen 2500 different peptides derived from 20 different animal venoms for anticancer activity specifically directed against three CRC cell lines and two control cell lines employing the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay for cytotoxicity. Three venoms of the 20, which exhibited specific and potent anticancer activity directed against the three CRC cell lines will be selected; and from these three venoms the specific peptide(s) with anti-CRC activity will be isolated and characterized. RESULTS This study is at the protocol development stage only, and as such, no results are available. CONCLUSIONS In summary, the proposed study will not only generate therapeutic leads to manage/treat one of the leading health issues in the UAE i.e., CRC, but is also of commercial interest as the identified BAP with specific anti-cancer activity against CRC can be patented for commercialization.

Animal Venoms—Curse or Cure?

An estimated 15% of animals are venomous, with representatives spread across the majority of animal lineages. Animals use venoms for various purposes, such as prey capture and predator deterrence. Humans have always been fascinated by venomous animals in a Janus-faced way. On the one hand, humans have a deeply rooted fear of venomous animals. This is boosted by their largely negative image in public media and the fact that snakes alone cause an annual global death toll in the hundreds of thousands, with even more people being left disabled or disfigured. Consequently, snake envenomation has recently been reclassified by the World Health Organization as a neglected tropical disease. On the other hand, there has been a growth in recent decades in the global scene of enthusiasts keeping venomous snakes, spiders, scorpions, and centipedes in captivity as pets. Recent scientific research has focussed on utilising animal venoms and toxins for the benefit of humanity in the form of molecular research tools, novel diagnostics and therapeutics, biopesticides, or anti-parasitic treatments. Continued research into developing efficient and safe antivenoms and promising discoveries of beneficial effects of animal toxins is further tipping the scales in favour of the “cure” rather than the “curse” prospect of venoms.

Cell-Penetrating Peptides Derived from Animal Venoms and Toxins

Cell-penetrating peptides (CPPs) comprise a class of short polypeptides that possess the ability to selectively interact with the cytoplasmic membrane of certain cell types, translocate across plasma membranes and accumulate in the cell cytoplasm, organelles (e.g., the nucleus and mitochondria) and other subcellular compartments. CPPs are either of natural origin or de novo designed and synthesized from segments and patches of larger proteins or designed by algorithms. With such intrinsic properties, along with membrane permeation, translocation and cellular uptake properties, CPPs can intracellularly convey diverse substances and nanomaterials, such as hydrophilic organic compounds and drugs, macromolecules (nucleic acids and proteins), nanoparticles (nanocrystals and polyplexes), metals and radionuclides, which can be covalently attached via CPP N- and C-terminals or through preparation of CPP complexes. A cumulative number of studies on animal toxins, primarily isolated from the venom of arthropods and snakes, have revealed the cell-penetrating activities of venom peptides and toxins, which can be harnessed for application in biomedicine and pharmaceutical biotechnology. In this review, I aimed to collate examples of peptides from animal venoms and toxic secretions that possess the ability to penetrate diverse types of cells. These venom CPPs have been chemically or structurally modified to enhance cell selectivity, bioavailability and a range of target applications. Herein, examples are listed and discussed, including cysteine-stabilized and linear, α-helical peptides, with cationic and amphipathic character, from the venom of insects (e.g., melittin, anoplin, mastoparans), arachnids (latarcin, lycosin, chlorotoxin, maurocalcine/imperatoxin homologs and wasabi receptor toxin), fish (pardaxins), amphibian (bombesin) and snakes (crotamine and cathelicidins).