Structure−Activity Studies of Orexin A and Orexin B at the Human Orexin 1 and Orexin 2 Receptors Led to Orexin 2 Receptor Selective and Orexin 1 Receptor Preferring Ligands

Abstract

The neuropeptides orexin A and B (also known as hypocretins) play an important role in many physiological and behavioral activities. Orexins are ligands of two closely related G-protein-coupled receptors, that are the named orexin 1 and orexin 2 receptors. To clearly identify the minimal ligand sequences required for receptor activation, we synthesized and analyzed different centrally, C- and N-terminally truncated analogues of orexins A and B. Furthermore, we used the shortest active analogue to screen for important amino acid residues by l-alanine and l-proline replacement scans. For orexin A, only full-length peptides were able to show the same activity as orexin A, but interestingly, reduced orexin A and natural orexin A, which contains the two disulfide bonds, had the same activity. The shortest highly active orexin B analogue was orexin B 6-28. In addition, we identified orexin A 2-33 as the first analogue with orexin 1 receptor preference and orexin B 10-28, [A27]orexin B 6-28, and [P11]orexin B 6-28 as being highly potent orexin 2 receptor selective (>1000-fold) peptides.

Mapping the Binding Pocket of Dual Antagonist Almorexant to Human Orexin 1 and Orexin 2 Receptors: Comparison with the Selective OX1 Antagonist SB-674042 and the Selective OX2 Antagonist N-Ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA)

“…Blue, nitrogen atoms; red, oxygen; yellow, sulfur; and green, fluorine. from orexin-A for activation of the receptor was similar between OX 1 and OX 2 (Ammoun et al, 2003;Lang et al, 2004 (Fig. 6B).…”

“…Several researchers, who have investigated the determinants of orexin-A required to activate OX 1 and OX 2 using truncated peptides and alanine-scanned peptides (systematic replacement of the natural amino acids with L-alanine) (Ammoun et al, 2003;Lang et al, 2004Lang et al, , 2006Takai et al, 2006) have indicated that: 1) a minimal 19 amino acids of C-terminal segment of orexin-A (Arg15-Leu33) is required for OX activation, though functional activity of this peptide is reduced; 2) the replacement of orexin-A (Arg15-Leu33) truncated peptide residues, Leu16, Leu19, Leu20, His26, Gly29, Ile30, Leu31, Thr32, and Leu33 with alanine led to a significant reduction in the functional potency at the OX 1 (Darker et al, 2001); and 3) orexin-A distinctly recognized OX 1 from OX 2 and its binding to OX 1 required more molecular determinants than binding to OX 2 . Thus far, little is known about the OX ligand-binding pocket.…”

Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist

“…Peptide Synthesis-The peptides were synthesized by solidphase technique on an automated multiple peptide synthesizer (Syro; MultiSynTech, Bochum, Germany) by using Rink amide resin (30 mg, resin loading 0.6 mmol/g) as described recently (23). The non-natural amino acids Abu, 3Abz, 4Abz, Acp, Aep, ␤Ala, 3Amb, 4Amb, Isn, and Ahx were coupled manually directly at the Rink amide resin (30 mg, resin loading 0.6 mmol/g) after removal of the Fmoc group with 30% piperidine in N,N-dimethylformamide twice for 20 min.…”

“…The coupling reaction was performed twice with four equivalents of the Fmoc-protected un-natural amino acids, which were activated by 4 equivalents of 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium and 8 equivalents of N,N-diisopropyl-ethylamin for 1 h. Completeness of the reaction was analyzed by a ninhydrin assay (24). The core segment wFw was synthesized as described previously (23). The peptide mimetics were cleaved from the resin in one step using trifluoroacetic acid, precipitated from ice-cold diethyl ether, washed, and finally lyophilized.…”

Ghrelin Receptor Inverse Agonists: Identification of an Active Peptide Core and Its Interaction Epitopes on the Receptor

“…Peptide Synthesis. The inverse agonist peptides were synthesized by solid-phase technique on an automated multiple peptide synthesizer (Syro-MultiSynTech, Bochum, Germany) by using the Rink amide resin (30 mg; resin loading, 0.6 mmol/g) as described recently (Lang et al, 2004). All peptides were cleaved from the resin in one step with the use of TFA, precipitated from ice-cold diethyl ether, washed, and finally lyophilized.…”