Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins.

Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody-based universal antivenom to treat snakebite envenoming.

Machine-learning guided Venom Induced Dermonecrosis Analysis tooL: VIDAL.

Snakebite envenoming is a global public health issue that causes significant morbidity and mortality, particularly in low-income regions of the world. The clinical manifestations of envenomings vary depending on the snake's venom, with paralysis, haemorrhage, and necrosis being the most common and medically relevant effects. To assess the efficacy of antivenoms against dermonecrosis, a preclinical testing approach involves in vivo mouse models that mimic local tissue effects of cytotoxic snakebites in humans. However, current methods for assessing necrosis severity are time-consuming and susceptible to human error. To address this, we present the Venom Induced Dermonecrosis Analysis tooL (VIDAL), a machine-learning-guided image-based solution that can automatically identify dermonecrotic lesions in mice, adjust for lighting biases, scale the image, extract lesion area and discolouration, and calculate the severity of dermonecrosis. We also introduce a new unit, the dermonecrotic unit (DnU), to better capture the complexity of dermonecrosis severity. Our tool is comparable to the performance of state-of-the-art histopathological analysis, making it an accessible, accurate, and reproducible method for assessing dermonecrosis in mice. Given the urgent need to address the neglected tropical disease that is snakebite, high-throughput technologies such as VIDAL are crucial in developing and validating new and existing therapeutics for this debilitating disease.

An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules.

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

(-)-Adaline from the Adalia Genus of Ladybirds Is a Potent Antagonist of Insect and Specific Mammalian Nicotinic Acetylcholine Receptors.

Ladybird beetles (Coleoptera: Coccinellidae) possess strong chemical defences that are secreted in response to stress and are also found on the coating of eggs, which are rich in alkaloids that are responsible for their toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists. Here, we have explored the actions of (-)-adaline, found in the 2-spot (Adalia bipunctata) and 10-spot (Adalia decempunctata) ladybirds, on both mammalian (α1ß1γδ, α7, α4ß2, α3ß4) and insect nAChRs using patch-clamp of TE671 cells and locust brain neurons natively expressing nAChRs, as well as two-electrode voltage clamp of Xenopus laevis oocytes recombinantly expressing nAChRs. All nAChR subtypes were antagonised by (-)-adaline in a time-dependent, voltage-dependent and non-competitive manner with the lowest IC50s at rat α3ß4 (0.10 µM) and locust neuron (1.28 µM) nAChRs, at a holding potential of -75 mV. The data imply that (-)-adaline acts as an open channel blocker of nAChRs.

Small Molecule Drug Discovery for Neglected Tropical Snakebite.

Snakebite envenoming is responsible for as many as 138 000 deaths annually, making it the world's most lethal neglected tropical disease (NTD). There is an urgent need to improve snakebite treatment, which currently relies on outdated and poorly tolerated biologic antivenoms that are often weakly efficacious, must be given intravenously in a healthcare setting, and are expensive to those who need them the most. Herein we describe the challenges associated with the discovery and development of new snakebite treatments and detail the great potential of venom toxin-inhibiting small molecule drugs. We finish by highlighting successful enabling strategies applied to other NTDs that could be exploited to facilitate the development of next-generation small molecule-based snakebite treatments.

Actions on mammalian and insect nicotinic acetylcholine receptors of harmonine-containing alkaloid extracts from the harlequin ladybird Harmonia axyridis.

The harlequin ladybird, Harmonia axyridis (H. axyridis), possesses a strong chemical defence that has contributed to its invasive success. Ladybird beetle defensive chemicals, secreted in response to stress and also found on the coating of laid eggs, are rich in alkaloids that are thought to be responsible for this beetle's toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists, hence we have explored the actions of alkaloids of the ladybird H. axyridis on both mammalian and insect nAChRs. Electrophysiological studies on native and functionally expressed recombinant nAChRs were used to establish whether an alkaloid extract from H. axyridis (HAE) targeted nAChRs and whether any selectivity exists for insect over mammalian receptors of this type. HAE was found to be an inhibitor of all nAChRs tested with the voltage-dependence of inhibition and the effect on ACh EC50 differing between nAChR subtypes. Our finding that an HAE fraction consisting almost entirely of harmonine had a strong inhibitory effect points to this alkaloid as a key component of nAChR inhibitory actions. Comparison of HAE inhibition between the mammalian and insect nAChRs investigated indicates some preference for the insect nAChR supporting the view that investigation of ladybird alkaloids shows promise as a method for identifying natural product leads for future insecticide development.

Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition.

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3ß4 nAChRs were most sensitive to PhTX-343 (IC50 = 12 nM at -80 mV) with α4ß4, α4ß2, α3ß2, α7 and α1ß1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1ß1γδ was most sensitive to PhTX-12 along with α3ß4 (IC50 values of 100 nM) with α4ß4, α4ß2, α3ß2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity.