Natural Inhibitors of Snake Venom Metalloendopeptidases: History and Current Challenges

Abstract

The research on natural snake venom metalloendopeptidase inhibitors (SVMPIs) began in the 18th century with the pioneering work of Fontana on the resistance that vipers exhibited to their own venom. During the past 40 years, SVMPIs have been isolated mainly from the sera of resistant animals, and characterized to different extents. They are acidic oligomeric glycoproteins that remain biologically active over a wide range of pH and temperature values. Based on primary structure determination, mammalian plasmatic SVMPIs are classified as members of the immunoglobulin (Ig) supergene protein family, while the one isolated from muscle belongs to the ficolin/opsonin P35 family. On the other hand, SVMPIs from snake plasma have been placed in the cystatin superfamily. These natural antitoxins constitute the first line of defense against snake venoms, inhibiting the catalytic activities of snake venom metalloendopeptidases through the establishment of high-affinity, non-covalent interactions. This review presents a historical account of the field of natural resistance, summarizing its main discoveries and current challenges, which are mostly related to the limitations that preclude three-dimensional structural determinations of these inhibitors using "gold-standard" methods; perspectives on how to circumvent such limitations are presented. Potential applications of these SVMPIs in medicine are also highlighted.

Keywords: cross-linking; hydrogen/deuterium exchange; mass spectrometry; metalloendopeptidase inhibitor; modeling; natural immunity; natural resistance; snake venom; structure; therapeutic application.

Figure 1

Research milestones on natural inhibitors of metalloendopeptidases. The investigation on the natural resistance that some animals presented to snake venoms began in the eighteenth century. Since Fontana’s pioneering work, the field has grown considerably. Researchers have managed to purify several inhibitors from the sera of snakes and mammals and determined their relevant physicochemical properties. The challenges that lie ahead are the three-dimensional structure elucidation of these snake venom metalloendopeptidase inhibitors (SVMPIs) in their free and toxin-complexed forms in order to better understand the molecular dynamics of this interaction.

Figure 2

Strategies for a structural view of SVMPIs. (Left) The experimental methods for structure determination, NMR spectroscopy and XRD crystallography, are the “gold-standard” techniques in protein structure elucidation, providing atomic resolution of individual proteins and their complexes. The SVMPIs DM43 and BJ46a represent a challenge for these techniques. For NMR spectroscopy, due to the molecular size of both molecules, costly and time-consuming methods for sample labeling and analysis are required. For XRD crystallography, crystals of DM43 produced low-resolution diffraction pattern while BJ46a could not be crystallized, highlighting the limiting character of the crystallization step. Hence, modeling becomes an important tool for the structural studies of these molecules. (Right) In molecular modeling, the main step is the identification of a homologous protein, whose experimental structure has already been determined, to be used as a template structure. The identification in structure databases of sequences evolutionarily correlated with sequential identity greater than 40% is done by standard pairwise sequence search methods, allowing the generation of high accuracy models. However, below this sequence identity threshold the correlation between two structures is difficult to address. In this range, sequences are correlated directly with proteins of known structure (fold recognition). A drawback is that, due to the low evolutionary correlation and the low sensitivity in the sequence alignment building, the accuracy of the produced models is lower. On the other hand, the ensemble of models produced can be filtered according to their agreement with experimental data. In our proposed strategy, these data would come from XL-MS, HDX-MS and SAXS assays, leading to the selection of accurate models, and shedding some light on the three-dimensional structural characteristics of these SVMPIs. Consequently, the molecular basis of the interaction between the inhibitors and their target toxins could be established.