Structure–activity relationship study on Tyr9 of urotensin-II(4–11): Identification of a partial agonist of the UT receptor
Introduction
Urotensin-II (U-II) is a cyclic dodecapeptide originally identified and isolated from the teleost fish Gillichthys mirabilis [29]. Human U-II is comprised of 11 amino acids and shares some sequence homology with somatostatin-14 [10]. U-II binds to the previously orphan receptor GPR14 [1], which is now referred to as the U-II receptor UT [16]. Later, a novel gene coding for a peptide showing high potency at the UT receptor was cloned in rat, mouse and human and this peptide was named U-II related peptide (URP) [31].
There are numerous studies demonstrating the effects of U-II in the cardiovascular system in health and disease with the general consensus indicating elevated plasma U-II levels in heart failure, hypertension, diabetes and renal failure [14]. U-II causes vasoconstriction of the microvasculature in patients with heart failure and vasodilatation in healthy subjects. Furthermore, U-II also causes an increase in forearm blood flow in healthy individuals while reducing blood flow in hypertensive patients (for a review see [27]). U-II and the UT receptor are widely distributed in the nervous system and central administration of U-II stimulates locomotion, provokes anxiety- and depressive-like states, enhances feeding activity and increases the duration of paradoxical sleep episodes; thus it is likely that U-II might have a role to play as a neuromodulator [12], [13]. The N-terminus of U-II varies from species to species while the cyclic hexapeptide core is fully conserved throughout mammalian, amphibian and piscean species [15] and it is considered the bioactive part of U-II [18]. It is worthy of mention that the cyclic hexapeptide is identical in U-II and URP sequences [31]. In particular, the sequence Trp-Lys-Tyr is of paramount importance for biological activity [18], [25]. An important reduction of peptide efficacy has been obtained substituting Lys8 with ornithine [5] or diaminobutyric acid [20] while an increase of affinity for the UT receptor has been achieved replacing Cys5 with Pen [19]. Moreover, a similar increase of affinity has been obtained substituting Trp7 with its enantiomer, however this applies only for antagonist templates, i.e. urantide [28] and UFP-803 [6]. More recently, it has been reported that the replacement of Phe6 with Cha or Lys8 with (pNH2)Phe produced low potency UT receptor antagonists [8].
Previous structure–activity relationship studies of position 9 indicated that the phenol moiety of Tyr9 can be replaced with different aromatic moieties without loss of affinity [20], [22]. This position can be useful for increasing U-II bioactivity as demonstrated by the threefold increase of potency reported with the [2-Nal] substituted peptide [22]. Molecular modeling investigations suggested that the side chain of Tyr9 interacts with a large hydrophobic pocket of the UT receptor in which the receptor residues His208 (ELII), Leu212 (TMV), Trp277 (TMVI), Ala281 (TMVI), Gln285 (ELIII) and Val296 (ELIII) are involved [22], [24]. Binding experiments performed using surface plasmon resonance technology, established that U-II binds with synthetic sequences of the UT receptor, corresponding to ELII and ELIII, at micromolar concentrations [3], [4].
On this basis novel UT receptor ligands were synthesized by replacing Tyr9 with non-natural analogs characterized by the presence of the phenol ring potentially able to interact via hydrogen bonding with UT receptor residues. In particular, (i) the Tyr9 OH group was shifted from the para to meta and ortho positions, (ii) Tyr9 was replaced with (3,5-diiodo)-Tyr, (N-CH3)-Tyr and with the shorter analog (4OH)-Phg, (iii) Tyr9 was substituted with a series of conformationally constrained analogs obtained by cyclization of the side chain on the nitrogen or the C-alpha chiral carbon. The chemical structures of the Tyr analogs employed in this study are displayed in Fig. 1. This structure–activity relationship study was performed using human U-II(4–11) (H-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) as a peptide template. This peptide represents the minimum sequence maintaining the same affinity and potency as the native peptide [19] and has been used in previous studies which have successfully led to the identification of novel UT receptor ligands such as the agonist P5U ([Pen5]U-II(4–11) [19]), and the antagonists urantide ([Pen5,d-Trp7,Orn8]U-II(4-11) [28]) and UFP-803 ([Pen5,d-Trp7,Dab8]U-II(4-11) [6]) (see for a recent review [25]).
All peptides have been evaluated in vitro using a calcium mobilization assay in HEK293 cells stably expressing the rat UT receptor (HEK293rUT) with the aid of the FlexStation-II benchtop scanning fluorometer. The most interesting peptide, i.e. [(3,5-diiodo)-Tyr9]U-II(4–11) was then further characterized using a bioassay technique at native UT receptors expressed in the rat thoracic aorta.
Section snippets
Materials
Amino acids, protected amino acids, Fmoc-Val-Wang resin and chemicals were purchased from Bachem, Novabiochem or Fluka (Switzerland). The product 1′,3′-dihydro-spiro(imidazolidine-4,2′-(2H)indene)-2,5-dione for the synthesis of (5-OH)-Aic was purchased from Eburon Organics N.V. (Belgium) while the product 6-methoxy-2-tetralone for the synthesis of Hat was purchased from Magical Scientific (Oklahoma City, USA). (7-OH)-Tic and (6,8-diiodo-7-OH)-Tic were synthesized following literature methods
Results
Peptides were prepared by solid phase methods using a Wang resin as a solid support and standard Fmoc/HATU chemical protocol [2].
Racemic m-Tyr, o-Tyr, (5-OH)-Aic [30] and Hat [11] have been employed for the synthesis of compounds 1a–2b and 5a–6b. The corresponding diastereomeric U-II(4–11) analogs were fully separated by HPLC and compounds a and b correspond to the early and later HPLC elution time, respectively. The Tyr analog in which the OH function was shifted in position ortho was obtained
Discussion
In the present study the structure–activity relationships of Tyr9 of U-II(4–11) were investigated. Results obtained in this investigation, on one hand confirmed the important role of this position for UT receptor occupation and, on the other hand, demonstrated that this position is also important for UT receptor activation. This was suggested by the reduction of ligand efficacy displayed by compound 9 at both recombinant and native rat UT receptors.
Compounds 1a and 2a behaved as potent UT
Conclusions
Collectively the results of the present SAR study on position 9 of U-II(4–11) suggest that the position of the OH group of the Tyr side chain is not important for biological activity, and the distance of the phenol moiety from the peptide backbone and its conformational freedom are crucial for UT receptor recognition. Moreover this study demonstrated that this position is important not only for receptor binding but also for activation since the (3,5-diiodo)-Tyr9 chemical modification generated
Acknowledgements
This work was supported by funds from the University of Ferrara (FAR grant to SS), and the University of Leicester. MB holds a Leicester–Ferrara International PhD studentship.
References (32)
- et al.
Solution structure of urotensin-II receptor extracellular loop III and characterization of its interaction with urotensin-II
Peptides
(2008) - et al.
Structure–activity relationships and structural conformation of a novel urotensin II-related peptide
Peptides
(2004) - et al.
Neuropeptide interactions and REM sleep: a role for urotensin II?
Peptides
(2008) - et al.
Behavioral actions of urotensin-II
Peptides
(2008) - et al.
From ‘gills to pills’: urotensin-II as a regulator of mammalian cardiorenal function
Trends Pharmacol Sci
(2004) - et al.
Human urotensin-II, the most potent mammalian vasoconstrictor identified to date, as a therapeutic target for the management of cardiovascular disease
Trends Cardiovasc Med
(2000) Tissue sulfhydryl groups
Arch Biochem Biophys
(1959)- et al.
Structure–activity relationships of urotensin II and URP
Peptides
(2008) - et al.
Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain
Biochem Biophys Res Commun
(2003) - et al.
Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14
Nature
(1999)
Solid-phase synthesis
Chemistry of peptide synthesis
Characterization of urotensin-II receptor structural domains involved in the recognition of U-II, URP, and urantide
Biochemistry
A new ligand for the urotensin II receptor
Br J Pharmacol
In vitro and in vivo pharmacological characterization of the novel UT receptor ligand [Pen(5),DTrp(7),Dab(8)]urotensin II(4-11) (UFP-803)
Br J Pharmacol
The 9-fluorenylmethoxycarbonyl amino-protecting group
J Org Chem
Structure–activity relationships of a novel series of urotensin II analogues: identification of a urotensin II antagonist
J Med Chem
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