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PAC1 receptor is abundant in the CNS and plays an important role in neuronal survival. To identify the molecular determinants and the conformational components responsible for the activation of the PAC1 receptor, we performed a SAR study focusing on the N-terminal domain of its endogenous ligand, PACAP. This approach revealed that residues Asp(3) and Phe(6) are key elements of the pharmacophore of the PAC1 receptor. This study, supported by NMR structural analyses, suggests that the N-terminal tail of PACAP (residues 1 to 4) adopts a specific conformation similar to a turn when it activates the PAC1 receptor. Moreover, the integrity of the alpha-helix conformation observed at positions 5 to 7 appears crucial to allow the binding of PACAP. Characterization of analogues led to the identification of several superagonists, such as [Bip(6)]PACAP27, and of a new potent PAC1 receptor antagonist, [Sar(4)]PACAP38. The bioactive conformation inferred from this SAR study could constitute an appropriate molecular scaffold supporting the design of nonpeptidic PAC1 receptor agonists.

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26RFa, a novel RFamide neuropeptide, is the endogenous ligand of the former orphan receptor GPR103. Intracerebroventricular injection of 26RFa and its C-terminal heptapeptide, 26RFa((20-26)), stimulates food intake in rodents. To develop potent, stable ligands of GPR103 with low molecular weight, we have designed a series of aza-β(3)-containing 26RFa((20-26)) analogues for their propensity to establish intramolecular hydrogen bonds, and we have evaluated their ability to increase [Ca(2+)](i) in GPR103-transfected cells. We have identified a compound, [Cmpi(21),aza-β(3)-Hht(23)]26RFa((21-26)), which was 8-fold more potent than 26RFa((20-26)) in mobilizing [Ca(2+)](i). This pseudopeptide was more stable in serum than 26RFa((20-26)) and exerted a longer lasting orexigenic effect in mice. This study constitutes an important step toward the development of 26RFa analogues that could prove useful for the treatment of feeding disorders.

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 [Ca 2ϩ

Background and Purpose The neuropeptide 26RFa and its cognate receptor GPR103 are involved in the control of food intake and bone mineralization. Here, we have tested, experimentally, the predicted ligand‐receptor interactions by site‐directed mutagenesis of GPR103 and designed point‐substituted 26RFa analogues. Experimental Approach Using the X‐ray structure of the β2‐adrenoceptor, a 3‐D molecular model of GPR103 has been built. The bioactive C‐terminal octapeptide 26RFa(19–26), KGGFSFRF‐NH2, was docked in this GPR103 model and the ligand‐receptor complex was submitted to energy minimization. Key Results In the most stable complex, the Phe‐Arg‐Phe‐NH2 part was oriented inside the receptor cavity, whereas the N‐terminal Lys residue remained outside. A strong intermolecular interaction was predicted between the Arg25 residue of 26RFa and the Gln125 residue located in the third transmembrane helix of GPR103. To confirm this interaction experimentally, we tested the ability of 26RFa and Arg‐modified 26RFa analogues to activate the wild‐type and the Q125A mutant receptors transiently expressed in CHO cells. 26RFa (10−6 M) enhanced [Ca2+]i in wild‐type GPR103‐transfected cells, but failed to increase [Ca2+]i in Q125A mutant receptor‐expressing cells. Moreover, asymmetric dimethylation of the side chain of arginine led to a 26RFa analogue, [ADMA25]26RFa(20–26), that was unable to activate the wild‐type GPR103, but antagonized 26RFa‐evoked [Ca2+]i increase. Conclusion and Implications Altogether, these data provide strong evidence for a functional interaction between the Arg25 residue of 26RFa and the Gln125 residue of GPR103 upon ligand‐receptor activation, which can be exploited for the rational design of potent GPR103 agonists and antagonists.

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