Probing the V_1a vasopressin receptor binding site with pyroglutamate-substituted linear antagonists

Summary We have synthesized eight analogues of the linear vasopressin antagonist DTyr(Et)²-Phe³-Gln⁴-Asn⁵-Arg ⁶-Pro⁷-Arg⁸-Tyr(NH₂)⁹ substituted with L-, or D-, pyroglutamate at position-1, Asn or Val at position-4 and Arg or Met at position 6. All of these peptides bound to the V_1a vasopressin receptor with affinities ranging 33.6-5, 470 nM. Of this series, only two peptides, [LpGlu¹Val⁴Arg⁶Tyr(NH₂) ⁹]AVP K_d = 48.4 nM and [DpGlu¹Val⁴Arg⁶Tyr(NH₂) ⁹]AVP K_d = 691 nM, bound to the V₂ vasopressin receptor. All of the neurohypophysial hormone receptors studied (V_1a VPR, V₂ VPR and OTR) were found to be stereoselective with respect to the N-terminal pGlu residue. The effect on binding characteristics of L-pGlu¹ and D-pGlu¹ analogues was dependent on both the sequence of the peptide and on the receptor subtype in question. From these data we found that peptide 5, which has the structure DpGlu-DTyr(Et)-Phe-Val-Asn-Arg-Pro-Arg-Tyr(NH₂), exhibited the highest V_1a/OTR selectivity reported to date (V_1aVPR K_d = 82 nM; OTR no binding at 10 μM). As such, peptide 5 will provide useful leads to the development of ligands with enhanced V_1a/OTR selectivity. The binding affinity and hydrophobicity of pyroglutamate-substituted peptides was compared with previously characterized V_1a receptor antagonists which contained a range of position-1 substitutions. The hydrophobicity of both cyclic and linear antagonists was markedly increased relative to the agonists AVP and [Phe²Om⁸]VT but increased hydrophobicity alone did not exclusively lead to high affinity antagonists. Data presented support the contention that in addition to a general increase in hydrophobicity/lipophilicity, position-1 influences the pharmacophore of vasopressin antagonists by providing molecular determinants for ligand/receptor interaction.