Identification of New Snake Venom Metalloproteinase Inhibitors Using Compound Screening and Rational Peptide Design

Abstract

ABSTRACT:The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP. KEYWORDS: BaP1, metalloproteinase inhibitors, protein docking, snake venom metalloproteinases T he treatment of snakebite envenomations is based on the parenteral administration of animal-derived antivenoms, 1 which have proved highly effective in the neutralization of systemic effects induced by snake venoms; however, they are only partially effective in abrogating the local pathological alterations induced by viperid snake venoms. 2 This is in part due to the very rapid onset of these effects, associated with the delay in reaching health centers where antivenoms are available. 3 Local pathological alterations induced by viperid snake venoms are predominantly due to the action of hemorrhagic zinc-dependent metalloproteinases (SVMP) and myotoxic phospholipases A 2 (PLA 2 ). 2 Bothrops asper metalloproteinase P1 (BaP1) is a representative member of the SVMP family. In the high resolution structure of BaP1, as well as in matrix metalloproteinases (MMP), a Zn 2+ ion is coordinated by a tri(histidine) motif, which is critical for substrate binding and cleavage. 4−9 Most MMP inhibitors to date developed consist of a zinc-binding group (ZBG), which binds the catalytic metal ion, 5,8,10 and a peptidomimetic backbone, which interacts noncovalently with the active site of the enzyme. 7,11 The peptidomimetic Batimastat (BB-94) is a first generation MMP inhibitor that contains the most common ZBG, that is, a hydroxamate moiety.Because of the difficulty in neutralizing locally acting SVMPs by antivenoms, the possibility has been raised that specific enzyme inhibitors may represent a new alternative for the treatment of these envenomations. 12 At the experimental level, it has been shown that chelating agents, such as EDTA salts, as well as Batimastat, are effective at inhibiting both the isolated SVMPs and the hemorrhagic activity of crude viperid venoms in animal models, 13,14 underscoring the potential therapeutic value of such inhibitors in this pathology. Nevertheless, the public access to metalloproteinase inhibitors designed by the pharma...

Natural Inhibitors of Snake Venom Metallopeptidases

“…Synthetic peptide inhibitors of MMPs (batimastat, marimastat, CGS-270 23A, AG-3340, and Bay-12 9566) have been tested against SVMPs, showing a few encouraging results (Escalante et al 2000;Howes et al 2007). Novel SVMP inhibitors are also being pursued by a combination of chemical library screening and rational peptide design (Villalta-Romero et al 2012).…”

Discovery of small molecule inhibitors for the snake venom metalloprotease BaP1 using in silico and in vitro tests

“…dm -3 (BaP1-A2), together with the inhibition of BaP1 activity of almost 100% during in vitro experiments make the pyrimidine-2,4,6-trione structure moiety very interesting for further investigation aimed to improve its binding and affinity properties against BaP1. Also, when compared with the known peptidomimetic inhibitor 31 (Fig. 1.…”

“…Some synthetic substances like O-phenathroline, EDTA 8,9 and Batimastat, because of their ability to chelate metals (Zn 2+ ion in the case of SVMP) have been shown to inhibit local hemorrhage induced by the venom of Bothrops asper, were tested. It was demonstrated that these compounds were effective at inhibiting both the isolated SVMPs and the hemorrhagic activity of crude venoms in animal models 9,10 . Also, some hydroxamatecontaining tripeptides with sequence Phe-Leu-Phe showed good results in vitro 10,11 .…”

“…It was demonstrated that these compounds were effective at inhibiting both the isolated SVMPs and the hemorrhagic activity of crude venoms in animal models 9,10 . Also, some hydroxamatecontaining tripeptides with sequence Phe-Leu-Phe showed good results in vitro 10,11 . Although hydroxamates are often pointed out as very potent SVMPs inhibitors, several challenges need to be addressed in the context of producing new drugs for these enzymes.…”

“…Results for in vitro inhibition[30][31][32][33][34][35] of BaP1's proteolytic activity over azocasein at 1 mmol• dm -3 :…”