Neutralizing Effects of Small Molecule Inhibitors and Metal Chelators on Coagulopathic Viperinae Snake Venom Toxins

Abstract

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 haemotoxicities, 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 combinations 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.

Venom-Induced Blood Disturbances by Palearctic Viperid Snakes, and Their Relative Neutralization by Antivenoms and Enzyme-Inhibitors

“…The comparative testing of the enzyme inhibitors prinomastat and DMPS revealed highly contrasting differences in their specific abilities to neutralize the P-IIId SVMP responsible for FX activation by the venoms in this study. This is congruent with recent data published for FX activation of neonate Crotalus culminatus venom, which prinomastat neutralised but DMPS at the same concentration did not (59) and another report of DMPS requiring long incubation times to exert a discernable effect (60).…”

Echis carinatus snake venom metalloprotease-induced toxicities in mice: Therapeutic intervention by a repurposed drug, Tetraethyl thiuram disulfide (Disulfiram)

“…Some of these inhibitors were less effective as therapeutic regimen where the time lapse between venom and administration of inhibitors is prolonged [72]. Moreover, many small molecules and chelators were focused on interference in Viperinae snake venom-induced coagulopathy and local toxicities [69]. However, our findings highlight the efficacy of TTD in ECV-induced both local and systemic toxicities and, would be better repurposing to complement snakebite management.…”

“…Besides, TTD has been shown to inhibit MMP-2 and MMP-9 activity by directly interacting with them via a Zn ++ chelating mechanism [66]. Several scientific reports suggested that, many small inhibitors or chelators of SVMPs such as batimastat, marimastat, N,N,N 0 ,N 0 -tetrakis (2-pyridylmethyl) ethane-1,2-diamine and dimercaprol which targets the different classes of SVMPs-induced toxicities [67][68][69][70][71]. Some of these inhibitors were less effective as therapeutic regimen where the time lapse between venom and administration of inhibitors is prolonged [72].…”

Animal Venoms—Curse or Cure?

“…Two contributions from Jeroen Kool’s group examine the usefulness of the small-molecule PLA2 inhibitor Varespladib as a potential drug for the treatment of snake bites. In these studies, cutting-edge nanofractionation analytics are employed to determine the effects of Varespladib and other small molecules on the coagulopathic effects of various crotalid and viperid snake venoms [ 61 , 62 ]. The Hodgson lab examined the neutralising abilities of different antivenoms against the effects of venom from the Chinese cobra by using the chick biventer nerve muscle preparation [ 63 ].…”