Somsen, G. | Search bioRxiv

bioRxiv2020

Neutralizing effects of small molecule inhibitors and metal chelators on coagulopathic Viperinae snake venom toxins

Animal-derived antivenoms are the only specific therapies currently available for the treatment of snake envenoming, but these products have a number of limitations associated with their efficacy, safety and affordability for use in tropical snakebite victims. Small molecule drugs and drug candidates are regarded as promising alternatives for filling the critical therapeutic gap between snake envenoming and effective treatment. In this study, by using an advanced analytical technique that combines chromatography, mass spectrometry and bioassaying, we investigated the effect of several small molecule inhibitors that target phospholipase A2 (varespladib) and snake venom metalloproteinase (marimastat, dimercaprol and DMPS) toxin families on inhibiting the activities of coagulopathic toxins found in Viperinae snake venoms. The venoms of Echis carinatus, Echis ocellatus, Daboia russelii and Bitis arietans, which are known for their potent coagulopathic toxicities, were fractionated in high resolution onto 384-well plates using liquid chromatography followed by coagulopathic bioassaying of the obtained fractions. Bioassay activities were correlated to parallel recorded mass spectrometric and proteomics data to assign the venom toxins responsible for coagulopathic activity and assess which of these toxins could be neutralized by the inhibitors under investigation. Our results showed that the phospholipase A2-inhibitor varespladib neutralized the vast majority of anticoagulation activities found across all of the tested snake venoms. Of the snake venom metalloproteinase inhibitors, marimastat demonstrated impressive neutralization of the procoagulation activities detected in all of the tested venoms, whereas dimercaprol and DMPS could only partially neutralize these activities at the doses tested. Our results provide additional support for the concept that combination of small molecules, particularly the combination of varespladib with marimastat, serve as a drug-repurposing opportunity to develop new broad-spectrum inhibitor-based therapies for snakebite envenoming.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2019

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates before direct analysis using a plasma coagulation assay. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors.\n\nAuthor summarySnakebite is a neglected tropical disease that results in more than 100,000 deaths every year. Haemotoxicity is one of the most common signs of systemic envenoming observed after snakebite, and many snake venoms cause severe impairment of the blood coagulation that makes victims vulnerable to suffering life-threating hemorrhage. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that interfere with blood coagulation. Twenty snake venoms were screened for their effects on the blood coagulation cascade and based on the initial results and the medical relevance of the species, seven venoms were selected for in-depth analysis of the responsible toxins using advanced identification techniques. Our findings reveal a number of anticoagulant toxins that have not yet been reported before as such. The methodology described herein not only enables the identification of both known and unknown toxins that cause impairment of the blood coagulation, but offers a throughput platform to effectively screen for inhibitory molecules relevant for the development of next generation snakebite treatments.

pharmacology and toxicology↗