The Relative Efficacy of Chemically Diverse Small-Molecule Enzyme-Inhibitors Against Anticoagulant Activities of African Spitting Cobra (Naja Species) Venoms.

Abhinandan Chowdhury,Bryan G Fry,Matthew R Lewin,Rebecca Carter,Christina N Zdenek

doi:10.3389/fimmu.2021.752442

Abstract

African spitting cobras are unique among cobras for their potent anticoagulant venom activity arising from strong inhibition of Factor Xa. This anticoagulant effect is exerted by venom phospholipase A2 (Group I PLA2) toxins whose activity contributes to the lethality of these species. This anticoagulant toxicity is particularly problematic as it is not neutralized by current antivenoms. Previous work demonstrated this trait for Naja mossambica, N. nigricincta, N. nigricollis, and N. pallida. The present work builds upon previous research by testing across the full taxonomical range of African spitting cobras, demonstrating that N. ashei, N. katiensis, and N. nubiae are also potently anticoagulant through the inhibition of Factor Xa, and therefore the amplification of potent anticoagulant activity occurred at the base of the African spitting cobra radiation. Previous work demonstrated that the enzyme-inhibitor varespladib was able to neutralize this toxic action for N. mossambica, N. nigricincta, N. nigricollis, and N. pallida venoms. The current work demonstrates that varespladib was also able to neutralize N. ashei, N. katiensis, and N. nubiae. Thus varespladib is shown to have broad utility across the full range of African spitting cobras. In addition, we examined the cross-reactivity of the metalloprotease inhibitor prinomastat, which had been previously intriguingly indicated as being capable of neutralizing viperid venom PLA2 (Group II PLA2). In this study prinomastat inhibited the FXa-inhibiting PLA2 toxins of all the African spitting cobras at the same concentration at which it has been shown to inhibit metalloproteases, and thus was comparably effective in its cross-reactivity. In addition we showed that the metalloprotease-inhibitor marimastat was also able to cross-neutralize PLA2 but less effectively than prinomastat. Due to logistical (cold-chain requirement) and efficacy (cross-reactivity across snake species) limitations of traditional antivenoms, particularly in developing countries where snakebite is most common, these small molecule inhibitors (SMIs) might hold great promise as initial, field-based, treatments for snakebite envenoming as well as addressing fundamental limitations of antivenom in the clinical setting where certain toxin effects are unneutralized.

Highlights

In many tropical and subtropical nations, snake bite is a neglected public health issue, despite relatively cost-effective treatments available
African spitting cobras evolved around 15 million years ago, and represent one of the three lineages within the Hemachatus/ Naja clade that have convergently evolved the spitting of venom trait [9,10,11,12,13]
We ascertained the ability of the PLA2-inhibitor varespladib and the cross-reactivity of the metalloprotease-inhibitors prinomastat and marimastat with the FXa-blocking anticoagulant PLA2 of African spitting cobras

Summary

INTRODUCTION

In many tropical and subtropical nations, snake bite is a neglected public health issue, despite relatively cost-effective treatments available. Rather than inhibiting FXa active site directly, the toxins compete with Factor Va for binding to FXa at a site distinct from the enzymatic pocket [20, 26,27,28,29] These FXa-inhibiting toxins have been shown to unaffected by the SAVP polyvalent antivenom (which is made using the venoms of Bitis arietans, Bitis gabonica, Dendroaspis angusticeps, Dendroaspis jamesoni, Dendroaspis polylepis, Haemachatus haemachatus, Naja nivea, Naja melanoleuca, Naja annulifera, and Naja mossambica) but have been shown to be well neutralized by the PLA2 inhibitor varespladib [26]. Only included viperid venoms and the intriguing ability of prinomastat to neutralize anticoagulant activity of PLA2 in cobra venom was unexplored. To fill this gap, our study set out to examine the concentration-dependent effects of varespladib across the full taxonomical range of African spitting cobras. These approaches can be combined to expedite the development of improved treatments for snakebite and reduce dependence on animal models

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RESULTS AND DISCUSSION