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.