PT  - JOURNAL ARTICLE
AU  - Ester Gutiérrez-Pascual
AU  - Jérôme Leprince
AU  - Antonio J. Martínez-Fuentes
AU  - Isabelle Ségalas-Milazzo
AU  - Rafael Pineda
AU  - Juan Roa
AU  - Mario Duran-Prado
AU  - Laure Guilhaudis
AU  - Elia Desperrois
AU  - Aurélie Lebreton
AU  - Leonor Pinilla
AU  - Marie-Christine Tonon
AU  - María M. Malagón
AU  - Hubert Vaudry
AU  - Manuel Tena-Sempere
AU  - Justo P. Castaño
TI  - In Vivo and in Vitro Structure-Activity Relationships and Structural Conformation of Kisspeptin-10-Related Peptides
AID  - 10.1124/mol.108.053751
DP  - 2009 Jul 01
TA  - Molecular Pharmacology
PG  - 58--67
VI  - 76
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/76/1/58.short
4100  - http://molpharm.aspetjournals.org/content/76/1/58.full
SO  - Mol Pharmacol2009 Jul 01; 76
AB  - 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 × 10-6 M versus 1.54 to 2.6 × 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 α- 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.