1. INTRODUCTION
Animal venoms are rich in biologically active molecules, named toxins, displaying a wide variety of biological properties, being enzymes (protease, phospholipases, nuclease, etc…), enzyme inhibitors (as serine protease inhibitor) or ligands for membrane proteins like ion channels or G protein-coupled receptors (GPCRs). In addition to their high affinity and selectivity for their various targets, animal toxins are also highly stable thanks to a reticulated and rigid structure. Mamba snake venoms are mostly composed of 2 toxins scaffolds, the 3 finger-fold toxins (3FT) and the Kunitz-type toxins (Ainsworth et al., 2017). 3FT are active on ionic channels like calcium channels, ASIC channels and nicotinic receptors, on aminergic GPCRs, as well as on enzymes and integrins receptors (Maïga et al., 2012; Blanchet et al., 2014; Kessler et al., 2017). The Kunitz-type toxins are homologous to the bovine pancreatic trypsin inhibitor (BPTI) and have been initially described as inhibitors of various serine and cysteine proteases (Kunitz and Northrop, 1936). In the early 1980s, mamba Kunitz-type toxins known as dendrotoxins were identified with neurotoxic effects driven by to their potassium channel blockade activities (Harvey, 2001). Later on, other Kunitz-type toxins were also described to inhibit calcium channels (Schweitz et al., 1994), vanilloid receptor 1 (Andreev et al., 2008) and ASIC channels (Bohlen et al., 2011). We recently assigned a new function to this structural family. The mambaquaretin-1 (MQ1) isolated from the green mamba snake venom is the most selective antagonist of the type 2 arginine-vasopressin (AVP) human receptor (hV2R) having a nanomolar affinity for the hV2R and no effect on 156 other GPCRs (Ciolek et al., 2017). Up to now, it is the sole Kunitz peptide known to be active on a GPCR. We furthermore validated MQ1 as a therapeutic solution to hyponatremia and polycystic kidney diseases and as a diagnostic agent to detect V2R in vivo (Ciolek et al., 2017, Droctové et al., 2020).
Kunitz peptides are made of 56 to 60 residues in length and are characterized by a highly conserved α/β/α conformation stabilized by three disulfide bridges (C1-C6/C2-C4/C3-C5). They also exhibit a strong intramolecular hydrophobic network (Huber et al., 1970). Their N-terminal extremity forms an alpha helix followed by a loop 1, which links to the first part of the antiparallel β sheet. The loop 2 connects the β sheet with the short C-terminal α helix. BPTI-like peptides interact with serine proteases via only 4 residues of its loop 1 and more particularly a dyad (residues 15 and 16) made of a basic residue followed by a glycine or alanine residue (Harper and Berger, 1967; Otlewski et al., 2001). Dendrotoxins, on the other hand, block the potassium channel Kv1.1 predominantly with its lysine and an aliphatic residue positioned in the N-terminal extremity of the peptides (Gasparini et al., 1998). These two pharmacophores are diametrically opposed in the Kunitz structure (Fig. 1), and are not present in MQ1. Consequently, MQ1 barely blocks Kv1.1 conductivity and inhibits trypsin activity at 25 µM concentrations only (Ciolek et al., 2017). When restoring the pharmacophore of the dendrotoxin in MQ1 (MQ1-S3K, numbering according to the MQ1 sequence), potassium channel activity increases dramatically without affecting the V2R antagonism. When restoring the serine protease pharmacophore in MQ1 (MQ1-N15K+G16A), trypsin activity largely increases while the MQ1-V2R binding is disrupted (Ciolek et al., 2017).
In this study, we reassessed the diversity of mamba venoms by identifying 8 new mambaquaretins (MQs), which form a distinct functional monophyletic group among the Kunitz-type peptides.
These natural mambaquaretins altogether with synthetic variants brought new insights into MQ1 structure-activity relationships, highlighting the involvement of a large surface of the MQ1 structure in the V2R interaction, in sharp contrast to the way by which Kunitz-peptides block potassium channels or inhibit serine-proteases.