RP-HPLC/ESI-MS analyses of the synthesized conopressins and alignment of conopressin-related sequences. (A) Alignment of conopressin-related sequences. The asterisks * indicate an amidated C-terminal. Conopressin-M1 and M2 with γ-conopressin-vil are the only sequences that display a negatively charged amino acid at position 8. Interestingly, conopressin-M1 also displays an unusual proline residue at position 3. The highly conserved glycine residue at position 9 is replaced by a serine residue in conopressin-M1 and M2. (B) RP-HPLC/ESI-MS analyses of the synthesized conopressins. Acetonitrile (ACN) gradient from 0% to 30% over 30 min. For Con-M1 the two peaks display the same mass, possibly caused by the two proline residues inducing cis-trans isomerization causing dynamic conformational exchange leading to the splitting of the UV chromatogram peak [16,17,18]. The asterisk (*) on ESI-MS insets indicate an ion resulting from in source fragmentation of the proline residue [19].

Three-dimensional structures of Con-G, Con-M1, Con-M2 and Con-T. The 20 lowest NMR structures are superimposed over the backbone atoms. The backbone is shown in ribbon format and the side-chains as sticks. Proline residues bringing constraints to the structures are highlighted in yellow.

Representative concentration-response curves measuring increasing concentrations of intracellular calcium using a FLIPR assay for the hOTR, hV1aR and hV1bR, and representative concentration-response curves measuring accumulation of cAMP using a cAMP signaling assay for the hV2R of all tested compounds. Each point represents the mean of measurements from one experiment performed in triplicate. Error bars represent S.E.M.

Representative concentration-response curves measuring increasing concentrations of intracellular calcium using a FLIPR assay of all tested compounds against Danio rerio (zebrafish) oxytocin-vasopressin related receptors. Each point represents the mean of measurements from one experiment performed in triplicate. Error bars represent S.E.M.

Alignment between hV2R (uniprot entry P30518) and cloned ZF V2R. The substitution of D297 in hV2R with S275 in ZF V2R is bordered in black. Asterisks (*) indicate amino-acid residues that have been suggested to participate and to be important in receptor–ligand interaction. Arrows indicate the seven putative transmembrane domains (TM 1–7).