Elsevier

Peptides

Volume 23, Issue 12, December 2002, Pages 2259-2264
Peptides

Structural requirements at the N-terminus of urotensin II octapeptides

https://doi.org/10.1016/S0196-9781(02)00266-8Get rights and content

Abstract

Urotensin II is the latest of a growing list of peptides exhibiting potent cardiovascular effects. It is an extremely potent vasoconstrictor in primates; its excretion is elevated in hypertensive patients thus suggesting therapeutic potential for urotensin II analogues, particularly receptor antagonists. In the present study, a number of interesting structural features pertaining to the N-terminus of urotensin II have been evaluated for binding to cloned human and rat urotensin II receptors and functional effects on rat upper thoracic aorta smooth muscle preparations. Shortened octapeptides retained full binding affinities and functional activities, did not require a free N-terminal amino group, and could tolerate an amidated C-terminus. The N-terminal Asp residue present in the octapeptides did not require a negatively charged side chain, merely one which contained a hydrogen bond acceptor CO group which could be present at a variety of positions on the side chain. The side chain could be constrained into a trans-olefinic configuration with full retention of potency, but potency was lost in the cis configuration. N-terminal aromatic amino substituted with polar groups such as OH and NO2 also resulted in high affinity analogues. Overall, the correlation between binding affinities for the human and rat receptors was quite good. These findings could be of value in the development of more potent urotensin II receptor antagonists based on the previously identified somatostatin antagonist octapeptides which we have recently found, function as relatively weak urotensin II antagonists.

Introduction

The precursor sequence of human urotensin II (hUII) was cloned in 1998 [3] and is composed of 11 amino acid residues retaining a conserved cyclic hexapeptide, c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-NH2, and an N-terminal region consisting of four amino acid residues (Glu-Thr-Pro-Asp) which is highly variable across animal species [6]. More recently, an high affinity rat receptor for urotensin II was identified as the known orphan G-protein-coupled receptor, GPR14, which has much sequence similarity to the somatostatin and opioid receptor family and has been designated as UII-Rla [10]. Simultaneously, a human G-protein-coupled receptor (GPCR) that has 75% sequence similarity to the orphan rat receptor GPR14 and exhibits the pharmacological characteristics of a human urotensin II receptor was also cloned [1]. Human UII binds with high affinity to this receptor, and the binding is functionally coupled to intracellular calcium mobilization. Human urotensin II and its prepro-mRNA have been localized primarily in the central nervous system and cardiovascular system and in smaller amounts in other organs [1], [3], [4], [11].

Subsequently, UII was found to be the most potent of the known vasoactive peptides when administered to primates, although its activity was generally restricted to the arterial part of the vasculature and varied depending on anatomical localization of these arteries [1]. In isolated rat thoracic aorta tissues, urotensin II-induced contraction consisted of two distinct tonic and phasic components and this system proved to be a useful functional assay system for urotensin II analogs [14].

The precise physiological functions of UII in cardiovascular regulatory systems remain to be evaluated, however, a recent report [12] described significant excretion of UII peptide in normal people and higher concentrations in those with essential hypertension and renal tubule abnormalities and a renal site of production was indicated. Furthermore, hUII mRNAs were highly expressed in kidney and right atrium and less so in the vasculature where GPR14 mRNAs were abundantly expressed in both cardiovascular and renal tissues. In terms of drug development it thus appears that an UII antagonist could be of therapeutic value in a number of cardiovascular disorders characterized by increased vasoconstriction, myocardial dysfunction and even artherosclerosis.

Although Liu et al. [10] have pointed out that GPR14 has much sequence homology to members of the somatostatin receptor family, it could only be activated by somatostatin and cortistatin at micromolar concentrations so that endogenous somatostatin would seem unlikely to exert any direct effect on the UII system. Conversely, recent studies [14] on the shortest, fully potent fragments of UII revealed that the octapeptide sequence, Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-NH2 was fully potent at the rat and human UII receptor and has great structural similarity (Fig. 1) to octapeptide somatostatin analogs of the octreotide series [2]. The existence of many potent somatostatin receptor antagonists [7], [8], [13] suggested to us that somatostatin SARs might be useful for UII antagonist development and several somatostatin antagonists were found to function as weak UII receptor antagonists [14]. Since the N-terminal amino acid residue is extremely important to designing somatostatin octapeptide antagonists, the present paper, we have examined the role of the N-terminal Asp residue in UII(4–11)NH2 since this acidic amino acid embodies one of the main structural differences between the UII(4–11) and somatostatin octapeptides [2] which results in little somatostatin affinity for the UII receptor.

Section snippets

Peptide synthesis

4-Methylbenzhydrylamine hydrochloride resin was obtained from Advanced ChemTech Inc., Louisville, KY. Na-tert-Butyloxycarbonyl (Boc) protected amino acids were purchased from Bachem Inc., Torrance, CA, Advanced ChemTech Inc., and Synthetech Inc., Albany, OR. The reactive side-chains of the amino acids were masked with one of the following groups: Cys, 4-methylbenzyl; Lys, 2-chlorobenzyloxycarbonyl; Thr, O-benzyl; Tyr, O-2,6-dichlorobenzyl. All reagents and solvents were ACS grade or better and

Results

It was found (Table 1) that the amidated version of hUII(4-1i) retained high potency in all three assay systems employed thus allowing this to be used as a basis for the synthesis of model amide containing analogs on amide-forming benzyhydrylamine resin. All peptides were synthesized in high yields using synthetic techniques also employed in the synthesis of somatostatin octapeptide analogs. Three N-terminal residues could be removed from the human sequence with retention, or even a slight

Discussion

Although the biological data generated with the shortened hUII sequences appear to agree well with limited previous reports, our data with the two fragmentary analogs UII(5–10)NH2 and UII(4–10)NH2 differs markedly from a recent report by Flohr et al. [5] in which the 5–11 and 4–10 sequences appeared to have full potencies employing cells expressing the cloned hGPR14 receptor and employing a FLIPR assay. No binding data was presented and since our rat aorta assay employs natural tissue and

Acknowledgements

We would like to thank Ms. Ethel Yauger for her expert technical assistance.

References (14)

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