Design of Potent and Selective Agonists for the Human Vasopressin V_1bReceptor Based on Modifications of [Deamino-Cys]arginine Vasopressin at Position 4

Abstract

The glutamine(4) residue in [deamino-Cys(1)]arginine vasopressin (dAVP) was replaced by a broad series of aliphatic, aromatic, polar, and charged amino acids to give the following peptides: d[Gly(4)]AVP (1), d[Ala(4)]AVP (2), d[Abu(4)]AVP (3), d[Nva(4)]AVP (4), d[Nle(4)]AVP (5), d[Leu(4)]AVP (6), d[Ile(4)]AVP (7), d[Thi(4)]AVP (8), d[Phe(4)]AVP (9), d[Tyr(4)]AVP (10), d[Trp(4)]AVP (11), d[Asn(4)]AVP (12), d[Ser(4)]AVP (13), d[Thr(4)]AVP (14), d[Dap(4)]AVP (15), d[Dab(4)]AVP (16), d[Orn(4)]AVP (17), d[Lys(4)]AVP (18), d[Arg(4)]AVP (19), d[Har(4)]AVP (20), and d[Glu(4)]AVP (21). All peptides were synthesized by solid-phase methods using BOC chemistry for all but one peptide (8), which required the use of Fmoc chemistry. The binding and functional properties of these position 4 substituted analogues of dAVP (d[X(4)]AVP) and the previously reported d[Cha(4)]AVP (Derick et al. Endocrinology 2002, 143, 4655-4664) were evaluated on human arginine vasopressin (AVP) V(1a), V(1b), and V(2) receptors and on the human oxytocin (OT) receptor expressed in living Chinese hamster ovary (CHO) cells. Binding studies revealed that broad modifications of the fourth residue of dAVP do not significantly alter affinity for the human V(1b) receptor. Only aromatic (Phe, Tyr, Trp) or negatively charged (Glu) residues reduce V(1b) affinity. By contrast, the human V(1a) and more particularly the human V(2) and the OT receptors are more sensitive to many of these modifications. Thus, the replacement of the Gln(4) residue of dAVP by aliphatic (Leu, Cha) or positively charged (Orn, Lys, Arg, Har) amino acids led to analogues exhibiting drastic reductions of their affinity for the human V(1a), V(2), and OT receptors. Consequently, in addition to the previously reported d[Cha(4)]AVP, peptides 6 and 17-20 display excellent selectivities for the human V(1b) receptor. The key structural requirement responsible for optimal V(1b) selectivity appears to be the length and branching of the aliphatic side chain of the fourth residue of dAVP. Functional studies performed on CHO cells expressing the different human AVP/OT receptors confirm the V(1b) selectivity of peptides 6, 17, 18, 20, and d[Cha(4)]AVP. However, d[Arg(4)]AVP (19), which triggers an excellent coupling between the human V(2) receptor and adenylyl cyclase, was found to exhibit both V(1b) and V(2) agonism in functional tests. More interestingly, these functional experiments revealed that, depending on the AVP/OT receptor, a given d[X(4)]AVP analogue may behave as a full agonist or as a partial agonist. This strongly suggests that the fourth residue of dAVP plays an important role in the coupling between the hormone-receptor complex, the heterotrimeric G protein, and the effectors. In conclusion, the synthesis of these d[X(4)]AVP analogues led to the discovery of new V(1b) agonists with high affinity and greatly enhanced selectivities. Thus, in addition to d[Cha(4)]AVP, d[Leu(4)]AVP (6), d[Orn(4)]AVP (17), d[Lys(4)]AVP (18), and d[Har(4)]AVP (20) are useful new tools for studying the structur...

Mapping the Binding Site of Arginine Vasopressin to V1aand V1bVasopressin Receptors

“…In contrast to AVP, which does not distinguish V 1a from V 1b subtype with regard to binding affinity, d[Cha 4 ]AVP exhibits a much stronger affinity for the human V 1b receptor than for the human V 1a subtype (Table 3). A previous report demonstrated that a hydrophobic side chain at position 4 is clearly responsible for the favored binding to the human V 1b subtype (20). Modeling the complex between the latter AVP analog and V 1a /V 1b receptor subtypes did not reveal major changes in the binding mode with respect to the previously described recognition model of endogenous AVP.…”

“…To furthermore challenge the herein described 3-D models, we took advantage of the fine selectivity profile of a recently described V 1b peptide agonist (d[Cha 4 ]AVP), an analog of dAVP ([deaminocys1]arginine vasopressin) (20) for which glutamine 4 has been changed to cyclohexylalanine (Cha) and which exhibits a 70-fold higher affinity for the V 1a subtype than for the V 1b subtype (Table 3). Our 3-D models suggest that the fourth residue of the peptide ligand is accommodated by a pocket delineated both by four conserved residues among the AVP receptor family (K 3.29 , V 3.33 , Q 4.60 , and Y 5.38 ; Fig.…”

“…Our 3-D models were then used to find a plausible explanation to the previously described nice selectivity profile of d[Cha 4 ]AVP, a desamino-cys-1 analog of AVP bearing a cyclohexylalanine side chain at position 4 (20). In contrast to AVP, which does not distinguish V 1a from V 1b subtype with regard to binding affinity, d[Cha 4 ]AVP exhibits a much stronger affinity for the human V 1b receptor than for the human V 1a subtype (Table 3).…”

“…Proposed 3-D models, compatible with most known experimental data, have been successfully challenged by site-directed mutagenesis focusing on yet undisclosed peptide-receptor interactions. Binding and functional properties of six mutants of the V 1a or the V 1b human receptor were investigated using either AVP or the recently described d[Cha 4 ]AVP analog exhibiting a nice V 1b selectivity profile for human vasopressin receptor subtypes (20).…”

Vasopressin modulates lateral septal network activity via two distinct electrophysiological mechanisms

“…[1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha 4 ]AVP; Derick et al, 2002;Cheng et al, 2004;Guillon et al, 2004) is a recently synthesized selective V 1B receptor agonist and (HO)Phaa-d-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH 2 is a selective V 1A receptor antagonist (Manning et al, 1992). Both were kindly donated by Dr M. Manning (Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH, USA).…”