Alfonso Carotenuto

Design, Synthesis, Conformational Analysis, and Biological Studies of Urotensin-II Lactam Analogues

Human urotensin II (hU-II; H-Glu-Thr-Pro-Asp-cyclo[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) is a disulfide bridged undecapeptide recently identified as the ligand of an orphan G protein-coupled receptor. hU-II has been described as the most potent vasoconstrictor compound identified to date. With the aim of replacing the disulfide bridge by a chemically more stable moiety, we have synthesized and tested a series of lactam analogues of hU-II minimum active fragment, that is hU-II(4-11). The contractile activity of the synthetic analogues on the rat isolated thoracic aorta was found to be dependent upon the dimension of the lactam bridge. The most active peptide, H-Asp-cyclo[Orn-Phe-Trp-Lys-Tyr-Asp]-Val-OH (3), is approximately 2 logs less potent than hU-II (pD(2)=6.3 vs 8.4). A conformational analysis in solution of the active peptide 3, one of the inactive analogues, and hU-II was performed, using NMR and molecular modelling techniques. A superposition of the calculated structures of hU-II and 3 clearly shows that three out of four key residues (i.e., Phe(6), Lys(8) and Tyr(9)) maintain the same side- chain orientation, while the fourth one, Trp(7), cannot be superimposed. This observation could explain the reduced biological activity of the synthetic analogue.

Design, Synthesis and Conformational Analysis of Human Urotensin II (U-II) Analogues with Lactam Bridge

Urotensin II (U-II), a cyclic neuropeptide with potent vasoconstrictor activity, has been originally isolated from the urophysis of the fish goby. Several structural forms of U-II, showing a disulfide bridge between cysteines, have been reported in different species of fish and amphibian, with variation occurring in the first five to seven N-terminal amino acids, while the C-terminal cyclic hexapeptide sequence is conserved in all species [1,2]. The recent cloning of the U-II precursor in frog and human has demonstrated that the peptide is not restricted to the fish urophysis, but is also expressed in the central nervous system of tetrapods. The identification of a human G protein-coupled receptor homologous to rat GPR14, expressed predominantly in cardiovascular tissue, has demonstrated that U-II is an endogenous ligand of this orphan receptor. Human U-II is an 11 amino acids peptide that retains the cyclohexapeptide sequence typical of this family (GluThrProAspCysPheTrpLysTyrCysVal). The cyclic sequence of U-II is the biologically active fragment, and it has been suggested that the conformation of the disulfide loop might modulate the activity of the molecule [3,4]. U-II is partially homologous and analogous to somatostatin, a peptide whose disulfide bridge is known to be important for activity.

Conformational Studies of a Glycopeptide Recognized with High Affinity by Autoantibodies in Multiple Sclerosis

Myelin oligodendrocyte glycoprotein (MOG), a minor myelin component, is an important central-nervous system-specific target autoantigen for primary demyelination in autoimmune diseases like multiple sclerosis (MS). It has been described that glycosylation of a MOG peptide epitope improved the detection of specific autoantibodies in sera of MS patients [1]. We have recently found that the specific antibody recognition of this peptide is most likely driven by direct interactions of the antibody binding site with the Asn-linked sugar moiety and then stabilized by putative specific peptide-antibody interactions [2]. We have subsequently prepared and characterized a new 21-mer MOG derived glycopeptide N7 (H-Thr-Pro-Arg-Val-Glu-Arg-Asn(Glc)-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-OH — 1) showing enhanced affinity for MS autoantibodies in ELISA experiments compared to the native N-glycosylated MOG peptide epitope Asn31(Glc)hMOG(30–50). Thus, the peptide (1) is a synthetic antigen able to detect pathogenic demyelinating autoantibodies in MS patients and, most importantly, it can be used as a template for the design of a new generation of drugs capable of specific blockage of circulating autoantibodies in patients affected by MS. In view of this goal, we describe here a conformational analysis of this peptide, and of its unglycosylated and inactive counterpart (H-Thr-Pro-Arg-Val-Glu-Arg-Asn-Gly-His-Ser-Val-Phe-Leu-Ala-Pro-Tyr-Gly-Trp-Met-Val-Lys-OH — 2).