Development of an orexin-2 receptor selective agonist, [Ala11, d-Leu15]orexin-B

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Abstract

Investigation of l-alanine and d-amino acid replacement of orexin-B revealed that three l-leucine residues at the positions of 11, 14, and 15 in orexin-B were important to show selectivity for the orexin-2 receptor (OX2) over the orexin-1 receptor (OX1). l-Alanine substitution at position 11 and d-leucine substitution at positions 14 and 15 maintained the potency of orexin-B to mobilize [Ca2+]i in CHO cells expressing the OX2, while their potency for the OX1 was significantly reduced. In combined substitutions, we identified that [Ala11, d-Leu15]orexin-B showed a 400-fold selectivity for the OX2 (EC50=0.13 nM) over OX1 (EC50=52 nM). [Ala11, d-Leu15]orexin-B is a beneficial tool for addressing the functional roles of the OX2.

Abstract

High potent and selective orexin-2 receptor selective agonist peptides, [Ala11]orexin-B and [Ala11, d-Leu15]orexin-B, were found from systematic l-alanine and d-amino acid replacement of orexin-B.

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Peptide Preparation

Orexin-A and -B were purchased (Peptide Institute, Inc, Osaka, Japan). All orexin-B analogue peptides were synthesized on the solid support using a Fmoc/tBu strategy on Pioneer™ peptide synthesizer (Applied Biosystems, Foster City, CA, USA) or ACT357MPS (Advanced ChemTech, Louisville, KY, USA). Fmoc-NovaSyn® TGR resin (Novabiochem, Laufelfingen, Switzerland) was used as a starting material. All crude peptides were cleaved from resins with TFA/thioanisole/ethandithiol/m-cresol within 2 h,

Measurement of Intracellular Calcium Ion Concentrations

CHO-K1 cells expressing each subtype of orexin receptors (OX1 and OX2) were maintained by a general method. The intracellular calcium ion concentrations were measured by the method previously reported.3 In brief, CHO–K1 cells stably expressing human OX1 or OX2 receptors (OX1–CHO or OX2–CHO) were seeded into 96-well plates and incubated with a cytoplasmic calcium indicator, Fluo-3 AM. After free dye was washed away, the intracellular Ca2+ mobilization evoked by orexins and related peptides was

l-Alanine Scan of Orexin-B

To address the importance of each residue with minimal impact to the three-dimensional structure, alanine-scanned analogues of orexin-B were synthesized. Three alanine residues at positions 17, 22, and 23 in the orexin-B sequence were replaced with glycine. Their potency to mobilize intracellular Ca2+ concentrations in OX1–CHO and OX2–CHO cells were measured (Table 1). The substitutions at positions 1–9, 12–14, 16, and 17 did not significantly affect the intrinsic potency of orexin-B for both

d-Amino Acid Replacement of Orexin-B

To change the orientation of the amino acid side chain with minimal impact on the overall hydrophobicity and dipole moment, we conducted d-amino acid replacement (Table 2). d-Alanine was used as a substitute for glycine in the sequence of orexin-B. d-Amino acid substitutions at positions 23–28 showed significant reduction of the potency for both orexin receptors. In addition, d-leucine substitutions at positions 14 and 15 significantly improved the selectivity of orexin-B by 5- and 8-fold,

Combination Study on the Alanine Scan and d-Amino Acid Replacement

Each positional scan showed that three leucine residues, Leu11, Leu14, and Leu15, in the orexin-B sequence were important for the activation of the OX1, and that modification of these sites was relatively tolerable for the activation of the OX2. Based on these results, combination replacements of orexin-B were performed to determine whether or not synergistic effects would be observed (Table 3). Among the synthesized peptides, the combination of an l-alanine substitution at Leu11 and a d

Acknowledgments

We thank Shinnosuke Abe and Hirokazu Ohsawa for the analysis of mass spectra. We are also grateful to Ms. K. Marcopul and Ms. J. Lowry, Merck & Co., for her critical reading of this manuscript.

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