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.