4- DISCUSSION
In this work, we enlarge the Kunitz-type toxin family active on the V2
vasopressin receptor through identification and characterization of
novel mamba and cobra V2R antagonist ligands. Thanks to screening
experiments and phylogenetic analysis, these structured ligands enable
us to understand a new molecular mechanism for their interaction with
the V2R.
Mamba (African) and cobra (here Asian species) are two evolutionary- and
geographically-distant snakes. The presence of a cobra toxin group also
active on V2R suggests either 1) independently derived V2R-activity of
Kunitz toxins from cobras and mambas, or 2) conserved V2R-activity due
to shared ancestry (ion-channel blockade of dendrotoxins would be the
derived activity then). The presence of toxins active on V2R in various
Elapid snake venoms suggests an important role of these toxins for
snakes’ survival. However, whether or not the V2R activity is their
selected function in the ecological context of venom use remains to be
established. We note that as long as victim has access to water,
blockage of V2R shouldn’t be toxic at all. In any case, the screening of
new targets, like GPCRs, allowed us the discovery of new group of toxins
with a strong activity on a pharmaceutically relevant receptor,
highlighting the extraordinary diversity of animal toxins present in
venoms that remains largely underexplored.
The identification of a new monophylogenic group in the Kunitz peptide
family constituted a great opportunity to better understand the
molecular mechanisms supporting the pharmacological property of these
toxins. The natural SAR highlighted the importance of the 2 MQ1 major
loops in V2R binding, which was confirmed by an extended number of
characterized MQ1 variants. MQ1 shows a new strategy to bind to its
target compared to α-DTX (Fig. 1, Gasparini et al., 1998) and BPTI (Fig.
1, Kawamura et al., 2011). Indeed, whereas α-DTX uses its structured
part to block Kv1.1 and BPTI its loop 1 to inhibit serine-proteases, MQ1
exploited its 2 major loops and engages more positions in its
interaction with V2R. The pharmacophore defined by numerus amino acids
positioned in loop 1 (9 to 18) and loop 2 (34, 39 and 44) may be at the
origin of the absolute selectivity of MQ1 for the V2R.
MQ1 displays the same nanomolar affinity for rat and human V2R (Droctové
et al., 2020) but no interaction with V1aR, V1bR or OTR (Ciolek et al.,
2017). The figure 6 shows a sequence alignment between the 3 external
loops (ELs) of the vasopressin-sensitive receptors. When comparing these
5 receptor sequences, 9 positions appeared as V2R specific versusV1Rs and OTR. Three are acidic residues: D103 (numbering according to
the hV2R) in ECL1, E198 in ECL2, E299 in ECL3 and 6 are non-polar
residues, all in ECL3. The 3 MQ1 basic residues implied in V2R binding
(K10, K39, R44) and the 3 V2R specific acidic residues just described,
point to a charge complementarity between the two partners. In addition
to these 9 specific positions, differences in loop length may play a
role in the MQ1 selectivity. OTR ECL2 is shorter by 2 residues compared
to V2R while the V1Rs ECL3 ones are longer by 3 residues. Even if
extensive structure-activity-relationships should be done to validate
these hypotheses, these data describe the implication of important
residues covering a large surface of contact for MQ1, which appear to be
in coherence with a possible implication of the 3 V2R ECLs in the
complex MQ1/V2R formation.
MQ1-K39A showed an interesting 14-fold higher specificity for hV2Rversus rV2R. Too many mutations exist between rat and human V2R
sequences to propose any hypothesis but MQ1-K39A represent a new tool to
gain insights into its mode of action. Blocking the V2R is a validated
therapeutic line for several pathologies like the autosomal dominant
polycystic kidney disease (Juul et al., 2014). Improving the in
vivo activity of MQ1 is important to develop a new therapeutic option
for V2R-related diseases. This work presents an upgraded version of MQ1,
which can not be validated on rats, showing the limits of rat animal
model.