Design of potent and selective agonists for the human vasopressin V1b receptor based on modifications of [deamino-cys1]arginine vasopressin at position 4.
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 structure and the function of the human V(1b) receptor.
Cheng
Ling Ling
LL
Department of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, Ohio 43614-5804, USA.
Stoev
Stoytcho
S
Manning
Maurice
M
Derick
Sylvain
S
Pena
Ana
A
Mimoun
Mohamed Ben
MB
Guillon
Gilles
G
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D003894:Deamino Arginine Vasopressin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000494:pharmacology; D018045:Receptors, Oxytocin / Q000378:metabolism; D017483:Receptors, Vasopressin / Q000819:agonists*; D013329:Structure-Activity Relationship
D007295:Inositol Phosphates; D010456:Peptides, Cyclic; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D000242:Cyclic AMP; D003894:Deamino Arginine Vasopressin