Efficacy and Limitations of Chemically Diverse Small-Molecule Enzyme-Inhibitors against the Synergistic Coagulotoxic Activities of Bitis Viper Venoms.

Matthew R. Lewin,Rebecca Carter,Nicholas J. Youngman,Arno Naude,Bryan G. Fry

doi:10.3390/molecules27051733

Abstract

Snakebite remains a significant public health burden globally, disproportionately affecting low-income and impoverished regions of the world. Recently, researchers have begun to focus on the use of small-molecule inhibitors as potential candidates for the neutralisation of key snake venom toxins and as potential field therapies. Bitis vipers represent some of the most medically important as well as frequently encountered snake species in Africa, with a number of species possessing anticoagulant phospholipase A2 (PLA2) toxins that prevent the prothrombinase complex from inducing clot formation. Additionally, species within the genus are known to exert pseudo-procoagulant activity, whereby kallikrein enzymatic toxins cleave fibrinogen to form a weak fibrin clot that rapidly degrades, thereby depleting fibrinogen levels and contributing to the net anticoagulant state. Utilising well-validated coagulation assays measuring time until clot formation, this study addresses the in vitro efficacy of three small molecule enzyme inhibitors (marimastat, prinomastat and varespladib) in neutralising these aforementioned activities. The PLA2 inhibitor varespladib showed the greatest efficacy for the neutralisation of PLA2-driven anticoagulant venom activity, with the metalloproteinase inhibitors prinomastat and marimastat both showing low and highly variable degrees of cross-neutralisation with PLA2 anticoagulant toxicity. However, none of the inhibitors showed efficacy in neutralising the pseudo-procoagulant venom activity exerted by the venom of B. caudalis. Our results highlight the complex nature of snake venoms, for which single-compound treatments will not be universally effective, but combinations might prove highly effective. Despite the limitations of these inhibitors with regards to in vitro kallikrein enzyme pseudo-procoagulant venom activity, our results further support the growing body of literature indicating the potential use of small molecule inhibitors to enhance first-aid treatment of snakebite envenoming, particularly in cases where hospital and thus antivenom treatment is either unavailable or far away.

Highlights

Snakebite envenoming remains a neglected health issue, in tropical and subtropical countries around the world, with approximately 5.4 million snakebites and up to 140,000 deaths occurring annually [1–3]
B. atropos, B. caudalis and B. cornuta all possess snake venom phospholipase A2 toxins, which exert anticoagulant activity through the inhibition of the prothrombinase complex, impeding its ability to induce clot formation by converting prothrombin to thrombin [15]. This activity is highly variable across the allopatric populations of B. atropos and B. caudalis, across B. caudalis, as some populations possess venom with a greater proportion of snake venom kallikrein-type serine proteinases, which deplete fibrinogen levels by directly cleaving fibrinogen in a pseudo-procoagulant manner to induce the formation of weak fibrin clots with short half-lives [15]
Our results expand upon recent work regarding the cross-neutralising efficacy of metalloprotease inhibitors in neutralising the anticoagulant activity of snake venom phospholipase A2 (sPLA2) toxins [25]

Summary

Introduction

Snakebite envenoming remains a neglected health issue, in tropical and subtropical countries around the world, with approximately 5.4 million snakebites and up to 140,000 deaths occurring annually [1–3]. These numbers are well-recognised as gross underestimations due to the poor to non-existent record keeping in the most affected regions. B. atropos, B. caudalis and B. cornuta all possess snake venom phospholipase A2 (sPLA2) toxins, which exert anticoagulant activity through the inhibition of the prothrombinase complex, impeding its ability to induce clot formation by converting prothrombin to thrombin [15]. This activity is highly variable across the allopatric populations of B. atropos and B. caudalis, across B. caudalis, as some populations possess venom with a greater proportion of snake venom kallikrein-type serine proteinases, which deplete fibrinogen levels by directly cleaving fibrinogen in a pseudo-procoagulant manner to induce the formation of weak fibrin clots with short half-lives [15]

Objectives

Methods

Results