Computational and in vitro analyses to identify the anticoagulant regions of Echicetin, a snake venom anticoagulant C-type lectin (snaclec): possibility to develop anticoagulant peptide therapeutics?

Snake venom C-type lectins (Snaclecs) display anticoagulant and platelet-modulating activities; however, their interaction with the critical components of blood coagulation factors was unknown. Computational analysis revealed that Echicetin (Snaclec from Echis carinatus venom) interacted with heavy chain of thrombin, and heavy and light chains of factor Xa (FXa). Based on FXa and thrombin binding regions of Echicetin, the two synthetic peptides (1A and 1B) were designed. The in silico binding studies of the peptides with thrombin and FXa showed that peptide 1B interacted with both heavy and light chains of thrombin and, peptide 1A interacted with only heavy chain of thrombin. Similarly, peptide 1B interacted with both heavy and light chains of FXa; however, peptide 1A interacted only with heavy chain of FXa. Alanine screening predicted the hot-spots residues for peptide 1A (Aspartic acid6, Valine8, Valine9, and Tyrosine17 with FXa, and Isoleucine14, Lysine15 with thrombin) and peptide 1B (Valine16 with FXa). Spectrofluorometric interaction study showed a lower Kd value for peptide 1B binding with both FXa and thrombin than peptide 1A, indicating higher binding strength of the former peptide. The circular dichroism spectroscopy also established the interaction between thrombin and the custom peptides. The in vitro study demonstrated higher anticoagulant activity of peptide 1B than peptide 1A due to higher inhibition of thrombin and FXa. Inhibition of anticoagulant activity of the peptides by respective anti-peptide antibodies corroborates our hypothesis that peptides 1A and 1B represent the anticoagulant regions of Echicetin and may be developed as antithrombotic peptide drug prototypes.Communicated by Ramaswamy H. Sarma.

A Novel Therapeutic Formulation for the Improved Treatment of Indian Red Scorpion (Mesobuthus tamulus) Venom-Induced Toxicity-Tested in Caenorhabditis elegans and Rodent Models.

Indian Red Scorpion (Mesobuthus tamulus) stings are a neglected public health problem in tropical and sub-tropical countries, including India. The drawbacks of conventional therapies using commercial anti-scorpion antivenom (ASA) and α1-adrenoreceptor antagonists (AAA) have prompted us to search for an adequate formulation to improve treatment against M. tamulus stings. Novel therapeutic drug formulations (TDF) of low doses of commercial ASA, AAA, and ascorbic acid have remarkably improved in neutralising the in vivo toxic effects of M. tamulus venom (MTV) tested in Caenorhabditis elegans and Wistar strain albino rats in vivo models. The neutralisation of MTV-induced production of free radicals, alteration of the mitochondrial transmembrane potential, and upregulated expression of genes involved in apoptosis, detoxification, and stress response in C. elegans by TDF surpassed the same effect shown by individual components of the TDF. Further, TDF efficiently neutralized the MTV-induced increase in blood glucose level within 30 to 60 min post-treatment, organ tissue damage, necrosis, and pulmonary oedema in Wistar rats, indicating its clinical application for effecting treating M. tamulus envenomation. This study demonstrates for the first time that C. elegans can be a model organism for screening the neutralization potency of the drug molecules against a neurotoxic scorpion venom.

Pharmacological re-assessment of traditional medicinal plants-derived inhibitors as antidotes against snakebite envenoming: A critical review.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional healers have used medicinal plants to treat snakebite envenomation worldwide; however, mostly without scientific validation. There have been many studies on the therapeutic potential of the natural products against snake envenomation. AIM OF THE STUDY: This review has highlighted snake venom inhibitory activity of bioactive compounds and peptides from plants that have found a traditional use in treating snakebite envenomation. We have systematically reviewed the scenario of different phases of natural snake venom inhibitors characterization covering a period from 1994 until the present and critically analysed the lacuna of the studies if any, and further scope for their translation from bench to bedside. MATERIALS AND METHODS: The medicinal plant-derived compounds used against snakebite therapy were reviewed from the available literature in public databases (Scopus, MEDLINE) from 1994 till 2020. The search words used were 'natural inhibitors against snakebite,' 'natural products as therapeutics against snakebite,' 'natural products as antidote against snake envenomation,' ' snake venom toxin natural inhibitors,' 'snake venom herbal inhibitors'. However, the scope of this review does not include computational (in silico) predictions without any wet laboratory validation and snake venom inhibitory activity of the crude plant extracts. In addition, we have also predicted the ADMET properties of the identified snake venom inhibitors to highlight their valuable pharmacokinetics for future clinical studies. RESULTS: The therapeutic application of plant-derived natural inhibitors to treat snakebite envenomation as an auxiliary to antivenom therapy has been gaining significant momentum. Pharmacological reassessment of the natural compounds derived from traditional medicinal plants has demonstrated inhibition of the principal toxic enzymes of snake venoms at various extents to curb the lethal and/or deleterious effects of venomous snakebite. Nevertheless, such molecules are yet to be commercialized for clinical application in the treatment of snakebite. There are many obstacles in the marketability of the plant-derived natural products as snake envenomation antidote and strategies must be explored for the translation of these compounds from drug candidates to their clinical application. CONCLUSION: In order to minimize the adverse implications of snake envenomation, strategies must be developed for the smooth transition of these plant-derived small molecule inhibitors from bench to bedside. In this article we have presented an inclusive review and have critically analysed natural products for their therapeutic potential against snake envenomation, and have proposed a road map for use of natural products as antidote against snakebite.

In vitro laboratory analyses of commercial anti-scorpion (Mesobuthus tamulus) antivenoms reveal their quality and safety but the prevalence of a low proportion of venom-specific antibodies.

Mesobuthus tamulus (Indian Red Scorpion) sting is a severe but neglected health issue in India. The accomplishment of in-patient scorpion sting management is highly dependent on the safety, efficacy, and homogeneity of scorpion antivenom preparation. Therefore, in this study, the above qualities of commercial anti-scorpion antivenoms manufactured in India were assessed by in vitro laboratory analyses. Biophysical characterization of venom by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size exclusion chromatography, and proteomics analysis demonstrated that anti-scorpion antivenoms (ASAs) mostly contain F(ab')2 molecules with a trace amount of undigested immunoglobulin (Ig) G. The physicochemical characterization, electron microscopy, and dynamic light scattering studies revealed that ASAs were prepared according to the guidelines of World Health Organization (WHO), and were devoid of aggregate content and virus particles. ASAs did not show IgE contamination and bacterial endotoxin but demonstrated moderate complement activation properties, which may have adverse effects in treated patients. Spectrofluorometric and atomic force microscopy analyses showed poor binding of venom with commercial ASAs. The percent of antibodies raised against the venom toxins in commercial ASAs was determined at the range of 5.3-6.3%, which is a reason for their poor efficacy. This study advocates the importance of in vitro laboratory analyses for assessing commercial antivenom's quality and safety parameters before their pre-clinical research and clinical use to treat Indian red scorpion sting.

Advances in the Therapeutic Application of Small-Molecule Inhibitors and Repurposed Drugs against Snakebite.

The World Health Organization has declared snakebite as a neglected tropical disease. Antivenom administration is the sole therapy against venomous snakebite; however, several limitations of this therapy reinforce the dire need for an alternative and/or additional treatment against envenomation. Inhibitors against snake venoms have been explored from natural resources and are synthesized in the laboratory; however, repurposing of small-molecule therapeutics (SMTs) against the principal toxins of snake venoms to inhibit their lethality and/or obnoxious effect of envenomation has been garnering greater attention owing to their established pharmacokinetic properties, low-risk attributes, cost-effectiveness, ease of administration, and storage stability. Nevertheless, SMTs are yet to be approved and commercialized for snakebite treatment. Therefore, we have systematically reviewed and critically analyzed the scenario of small synthetic inhibitors and repurposed drugs against snake envenomation from 2005 to date and proposed novel approaches and commercialization strategies for the development of efficacious therapies against snake envenomation.

Recent developments in diagnostic tools and bioanalytical methods for analysis of snake venom: A critical review.

Snakebite is a neglected medical emergency causing fatalities and long-term disabilities throughout the world, especially in tropical countries. The effectiveness of therapy against snakebite is reliant on the unambiguous identification of bitten species of snake followed by immediate administration of venom-specific monovalent antivenom. However, this is a challenging task and therefore, over the several years scientists are constantly trying to address this issue by developing species-specific snake venom diagnostic kits as an alternative to classical methods of snake identification in clinics. Recently quite a few modern tools and techniques have been deployed for the development of simple, inexpensive, rapid, specific, and sensitive snake venom detection kits. However, despite these efforts a lone snakebite diagnostic kit is available until now which is a severe concern for efficacious snakebite therapy. In this article, we have reviewed the key issues pertaining to the rapid diagnosis of snake envenomation, tools and techniques developed and/or invented particularly over the past 40 years for the detection of snakebite as well as quantity of venom in the body fluids and/or tissues of victims. To overcome the practical constraints against the successful commercialization of these diagnostic kits, much more intensive studies for their improvement in terms of efficacy, affordability, storage stability, and usability, in addition to standardization of techniques for use in clinics are required to fulfil the objectives of the user-friendliness and commercial viability of snakebite diagnostic kits particularly in the rural and underdeveloped areas of tropical countries showing the maximum incidence of snakebite.