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In Vivo and in Vitro Structure-Activity Relationships and Structural Conformation of Kisspeptin-10-Related Peptides

Department of Cell Biology, Physiology and Immunology, University of Córdoba, and CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn 06/03), Córdoba, Spain (E.G.-P., A.J.M.-F., R.P., J.R., M.D.-P., L.P., M.M.M., M.T.-S., J.P.C.); Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U413, Unité Affiliée au Centre National de la Recherche Scientifique, Equipe d'Accueil 4310, International Associated Laboratory Samuel de Champlain, European Institute for Peptide Research (l'Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23), University of Rouen, the Plate-forme Régionale de Recherche en Imagerie Cellulaire de Haute-Normandie (PRIMACEN), Mont-Saint-Aignan, France (J.L., M.-C.T., H.V.); and Laboratory of Nuclear Magnetic Resonance, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6014, l'Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23, University of Rouen, Mont-Saint-Aignan, France (I.S.-M., L.G., E.D., A.L.)

Kisspeptins, the natural ligands of the G protein-coupled receptor KISS1R, comprise a family of related peptides derived from the proteolytic processing of a common precursor encoded by the KISS1 gene. Among those, Kisspeptin-10 (Kp-10) contains the basic residues to retain full functional activity and exhibits higher receptor affinity and biopotency than longer forms of the peptide. Although kisspeptins were first characterized by their ability to inhibit tumor metastasis, recent studies have revealed that the KISS1/KISS1R system plays an essential role in the neuroendocrine control of the reproductive axis. In this context, development and functional analysis of Kp-10 analogs may help in the search for new agonists and antagonists as valuable tools to manipulate the KISS1/KISS1R system and hence fertility. We report herein functional and structural analyses of a series of Ala-substituted rat kp-10 analogs, involving [Ca²⁺]_i responses in rat kiss1r-transfected Chinese hamster ovary cells, dynamic luteinizing hormone (LH) responses in vivo, and NMR structural studies. In vitro assays revealed that Ala substitutions in positions 6 or 10 of kp-10 resulted in a significant increase in EC₅₀ values (>6.46 x 10^-6 M versus 1.54 to 2.6 x 10^-8 M for rat and human Kp-10, respectively) and a substantial decrease in the proportion of responsive cells coupled to a marked increase in the time required to reach maximal response. In vivo assays showed that Ala⁶ substitution diminished and Ala¹⁰ substitution eliminated LH secretory responses, whereas coadministration of each analog failed to affect the LH-releasing ability of kp-10. Molecular modeling under NMR restraints revealed that kp-10 exhibits a helicoidal structure between the Asn⁴ and Tyr¹⁰ residues, with mixed - and 3₁₀-helix characteristics. Ala⁶ substitution induced limited destabilization of the helix around the position of the substitution. Ala¹⁰ substitution was found to totally disrupt the helical structure in the C-terminal region of the molecule. Taken together, our results indicate that positions 6 and 10 are critical for kp-10 action at kiss1r and suggest that modifications in these positions could lead to the generation of new kisspeptin agonists and/or antagonists with altered functional and perhaps binding properties. Furthermore, they emphasize the importance of using combined, multidisciplinary approaches, including in vivo studies, to reliably evaluate structure function properties of novel kisspeptin analogs.

Received for publication November 30, 2008.

Accepted for publication April 23, 2009.

Address correspondence to: Dr. Justo P. Castaño, Department of Cell Biology, Physiology and Immunology, Edificio Severo Ochoa. Planta 3. Campus de Rabanales. University of Córdoba, E-14014 Córdoba, Spain. E-mail: justo{at}uco.es

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