脂肪酸修饰24位赖氨酸可显著提高降钙素基因相关肽的血浆稳定性

doi:10.5246/jcps.2018.09.060

摘要/Abstract

摘要：

降钙素基因相关肽(CGRP)是一种广泛分布于神经系统的神经肽,由37个氨基酸组成。CGRP较短的半衰期阻碍了其研究和应用。本文利用固相多肽合成技术和无铜点击化学合成了多个脂肪酸修饰的CGRP衍生物,并对其进行了体外稳定性和活性的评价,筛选得到两种稳定性得到显著提高且具有高亲和力的衍生物。

关键词: 降钙素基因相关肽, 血浆半衰期, 脂肪酸

Abstract:

As a 37-amino acid vasoactive neuropeptide, calcitonin gene-related peptide (CGRP) is widely distributed in nervous systems. The studies and clinical applications of CGRP are limited by its peptide nature and short half-life. A series of peptide analogues of the α-form of CGRP were synthesized. Afterwards, by using in vitro metabolic and activity studies, we prepared two high affinity analogues with significantly improved plasma stability.

Key words: Calcitonin gene-related peptide, Half-life, Fatty acid

中图分类号:

R915

Supporting:

Figure S1. HPLC trace and ESI-MS data of peptide CGRP_Lys2-N₃. Linear gradient: 29%–54% solvent B over 30 min, GS-120-10-C18-Bio column, t_R = 9.12 min; Calcd for C₁₆₆H₂₇₃N₅₃O₅₁S: 3843.34 Da (average isotopes), [M+3H]³⁺ m/z = 1282.11, [M+4H]⁴⁺ m/z = 961.84; observed: 1282.20, 961.90.

Figure S2. HPLC trace and ESI-MS data of peptide CGRP_Lys4-N₃. Linear gradient: 12%–37% solvent B over 30 min, GS-120-10-C18-Bio column, t_R = 6.97 min; Calcd for C₁₆₆H₂₇₃N₅₃O₅₁S: 3843.34 Da (average isotopes), [M+3H]³⁺ m/z = 1282.11, [M+4H]⁴⁺ m/z = 961.84; observed: 1282.20, 961.90.

Figure S3. HPLC trace and ESI-MS data of peptide CGRP_Lys24-N₃. Linear gradient: 35%–60% solvent B over 30 min, GS-120-10-C18-Bio column, t_R = 12.07 min; Calcd for C₁₆₂H₂₆₂N₅₂O₅₁S₂: 3816.3 Da (average isotopes), [M+3H]³⁺ m/z = 1273.1, [M+4H]⁴⁺ m/z = 955.08; observed: 1273.35, 955.10.

Figure S4. HPLC trace and ESI-MS data of peptide CGRP_Lys35-N₃. Linear gradient: 28%–53% solvent B over 30min, GS-120-10-C18-Bio column, t_R = 10.5 min; Calcd for C₁₆₂H₂₆₂N₅₂O₅₁S₂: 3816.3 Da (average isotopes), [M+3H]³⁺ m/z = 1273.1, [M+4H]⁴⁺ m/z = 955.08; observed: 1273.20, 955.00.

Figure S5. HPLC trace and ESI-MS data of peptide analogue P-1. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 16.12 min; Calcd for C₂₀₃H₃₂₅N₅₅O₅₃S: 4416.23 Da (average isotopes), [M+3H]³⁺ m/z = 1473.08, [M+4H]⁴⁺ m/z = 1105.06; observed: 1473.18, 1105.22.

Figure S6. HPLC trace and ESI-MS data of peptide analogue P-2. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 16.62 min; Calcd for C₂₀₂H₃₂₁N₅₅O₅₂S₂: 4416.24 Da (average isotopes), [M+3H]³⁺ m/z = 1473.08, [M+4H]⁴⁺ m/z = 1105.06; observed: 1473.25, 1105.28.

Figure S7. HPLC trace and ESI-MS data of peptide analogue P-3. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 18.62 min; Calcd for C₂₀₀H₃₁₆N₅₄O₅₃S₂: 4389.17 Da (average isotopes), [M+3H]³⁺ m/z = 1464.05, [M+4H]⁴⁺ m/z = 1098.29; observed: 1464.24, 1098.35.

Figure S8. HPLC trace and ESI-MS data of peptide analogue P-4. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 19.95 min; Calcd for C₂₀₀H₃₁₆N₅₄O₅₃S₂: 4389.17 Da (average isotopes), [M+3H]³⁺ m/z = 1464.05, [M+4H]⁴⁺ m/z = 1098.29; observed: 1464.24, 1098.35.

Figure S9. HPLC trace and ESI-MS data of peptide analogue P-5. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 10.83 min; Calcd for C₂₀₃H₃₂₃N₅₅O₅₅S: 4446.21 Da (average isotopes), [M+3H]³⁺ m/z = 1483.07, [M+4H]⁴⁺ m/z = 1112.55; observed: 1483.27, 1112.78.

Figure S10. HPLC trace and ESI-MS data of peptide analogue P-6. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 11.50 min; Calcd for C₂₀₂H₃₁₉N₅₅O₅₄S₂: 4446.23 Da (average isotopes), [M+3H]³⁺ m/z = 1483.08, [M+4H]⁴⁺ m/z = 1112.55; observed: 1483.20, 1112.78.

Figure S11. HPLC trace and ESI-MS data of peptide analogue P-7. Linear gradient: 35%–70% solvent B over 30min, Proto 300 C4 column, t_R = 13.10 min; Calcd for C₂₀₀H₃₁₄N₅₄O₅₅S₂: 4419.16 Da (average isotopes), [M+3H]³⁺ m/z = 1474.05, [M+4H]⁴⁺ m/z = 1105.79; observed: 1474.19, 1106.04.

Figure S12. HPLC trace and ESI-MS data of peptide analogue P-8. Linear gradient: 35%–70% solvent B over 30min, Proto 300 C4 column, t_R = 13.65 min; Calcd for C₂₀₀H₃₁₄N₅₄O₅₅S₂: 4419.16 Da (average isotopes), [M+3H]³⁺ m/z = 1474.05, [M+4H]⁴⁺ m/z = 1105.79; observed: 1474.19, 1106.04.

Figure S13. HPLC trace and ESI-MS data of peptide analogue P-9. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 18.67 min; Calcd for C₂₀₅H₃₂₉N₅₅O₅₃S: 4444.28 Da (average isotopes), [M+3H]³⁺ m/z = 1482.43, [M+4H]⁴⁺ m/z = 1112.07; observed: 1482.64, 1112.34.

Figure S14. HPLC trace and ESI-MS data of peptide analogue P-10. Linear gradient: 35%–70% solvent B over 30min, Proto 300 C4 column, t_R = 19.35 min; Calcd for C₂₀₄H₃₂₅N₅₅O₅₂S₂: 4444.30 Da (average isotopes), [M+3H]³⁺ m/z = 1482.43, [M+4H]⁴⁺ m/z = 1112.08; observed: 1482.57, 1112.28.

Figure S15. HPLC trace and ESI-MS data of peptide analogue P-11. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 19.95 min; Calcd for C₂₀₂H₃₂₀N₅₄O₅₃S₂: 4417.23 Da (average isotopes), [M+3H]³⁺ m/z = 1473.41, [M+4H]⁴⁺ m/z = 1105.30; observed: 1473.56, 1105.53.

Figure S16. HPLC trace and ESI-MS data of peptide analogue P-12. Linear gradient: 35%–70% solvent B over 30 min, Proto 300 C4 column, t_R = 22.67 min; Calcd for C₂₀₂H₃₂₀N₅₄O₅₃S₂: 4417.23 Da (average isotopes), [M+3H]³⁺ m/z = 1473.41, [M+4H]⁴⁺ m/z = 1105.30; observed: 1473.56, 1105.53.

范子博, 周德敏. 脂肪酸修饰24位赖氨酸可显著提高降钙素基因相关肽的血浆稳定性[J]. 中国药学(英文版), 2018, 27(9): 589-599.

Zibo Fan, Demin Zhou. Modification at Lys24 with fatty acid generates active calcitonin gene-related peptide with much improved plasma stability[J]. Journal of Chinese Pharmaceutical Sciences, 2018, 27(9): 589-599.

参考文献

[1] Brain, S.D.; Cambridge, H. Calcitonin gene-related peptide: vasoactive effects and potential therapeutic role. Gen. Pharmacol. Vasc. Syst. 1996, 27, 607-611.

[2] Ma, H. Calcitonin gene-related peptide (CGRP). Nat. Sci. 2004, 2, 41-47.

[3] Brain, S.D.; Grant, A.D. Vascular Actions of Calcitonin Gene-Related Peptide and Adrenomedullin. Physiol. Rev. 2004, 84, 903-934.

[4] Supowit, S.C.; Rao, A.; Bowers, M.C.; Zhao, H.; Fink, G.; Steficek, B.; Patel, P.; Katki, K.A.; DiPette, D.J. Calcitonin gene-related peptide protects against hypertension-induced heart and kidney damage. Hypertension. 2005, 45, 109-114.

[5] Russell, F.A.; King, R.; Smillie, S.J.; Kodji, X.; Brain, S.D. Calcitonin Gene-Related Peptide: Physiology and Pathophysiology. Physiol. Rev. 2014, 94, 1099-1142.

[6] Nilsson, C.; Hansen, T.K.; Rosenquist, C.; Hartmann, B.; Kodra, J.T.; Lau, J.F.; Clausen, T.R.; Raun, K.; Sams, A. Long acting analogue of the calcitonin gene-related peptide induces positive metabolic effects and secretion of the glucagon-like peptide-1. Eur. J. Pharmacol. 2016, 773, 24-31.

[7] Miranda, L.P.; Shi, L.; Holder, J.R.; Wright, M.; Gegg, C.V.; Johnson, E.; Wild, K.; Stenkilsson, M.; Doellgast, G.; Manning, B.H.; Salyers, K. Peptide antagonists of the calcitonin gene-related peptide (CGRP) receptor with improved pharmacokinetics and pharmacodynamics: Peptide Antagonists to Calcitonin Gene-Related Peptide Receptor. Biopolymers. 2013, 100, 422-430.

[8] Watkins, H.A.; Rathbone, D.L.; Barwell, J.; Hay, D.L.; Poyner, D.R. Structure-activity relationships for α-calcitonin gene-related peptide. Br. J. Pharmacol. 2013, 170, 1308-1322.

[9] Aubdool, A.A.; Thakore, P.; Argunhan, F.; Smillie, S.J.; Schnelle, M.; Srivastava, S.; Alawi, K.M.; Wilde, E.; Mitchell, J.; Farrell-Dillon, K. A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy and Heart Failure. Circulation. 2017, 136, 367-383.

[10] Conner, A.C.; Hay, D.L.; Howitt, S.G.; Kilk, K.; Langel, Ü.; Wheatley, M.; Smith, D.M.; Poyner, D.R. Interaction of calcitonin-gene-related peptide with its receptors. Biochem. Soc. Trans. 2002, 30, 451-455.

[11] Negro, A.; Lionetto, L.; Simmaco, M.; Martelletti, P. CGRP receptor antagonists: an expanding drug class for acute migraine. Expert. Opin. Investig. Drugs. 2012, 21, 807-818.

[12] Chiba, T.; Yamaguchi, A.; Yamatani, T.; Nakamura, A.; Morishita, T.; Inui, T.; Fukase, M.; Noda, T.; Fujita, T. Calcitonin gene-related peptide receptor antagonist human CGRP-(8-37). Am. J. Physiol.-Endocrinol. Metab. 1989, 256, E331-E335.

[13] Miranda, L.P.; Holder, J.R.; Shi, L.; Bennett, B.; Aral, J.; Gegg, C.V.; Wright, M.; Walker, K.; Doellgast, G.; Rogers, R.; Li, H.; Valladares, V.; Salyers, K.; Johnson, E.; Wild, K. Identification of Potent, Selective, and Metabolically Stable Peptide Antagonists to the Calcitonin Gene-Related Peptide (CGRP) Receptor. J. Med. Chem. 2008, 51, 7889-7897.

[14] Agard, N.J.; Baskin, J.M.; Prescher, J.A.; Lo, A.; Bertozzi, C.R. A Comparative Study of Bioorthogonal Reactions with Azides. ACS Chem. Biol. 2006, 1, 644-648.

[15] Anseth, K.S.; Klok, H.A. Click Chemistry in Biomaterials, Nanomedicine, and Drug Delivery. Biomacromolecules. 2016, 17, 1-3.

[16] Mbua, N.E.; Guo, J.; Wolfert, M.A.; Steet, R.; Boons, G.J. Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC) Reveal New Features of Glycoconjugate Biosynthesis. ChemBioChem. 2011, 12, 1912-1921.