|
|
药学学报, 2021, 56(8): 2252-2259 |
|
|
引用本文： |
|
|
谭瑶, 杨奕帅, 褚召莉, 长孙东亭, 朱晓鹏, 罗素兰. α-芋螺毒素[A10L]PnIA荧光探针的设计合成及活性研究[J]. 药学学报, 2021, 56(8): 2252-2259. |
|
|
TAN Yao, YANG Yi-shuai, CHU Zhao-li, ZHANGSUN Dong-ting, ZHU Xiao-peng, LUO Su-lan. Design, synthesis, and activity study of α-conotoxin[A10L]PnIA fluorescent probe[J]. Acta Pharmaceutica Sinica, 2021, 56(8): 2252-2259. |
|
|
|
|
|
|
|
| α-芋螺毒素[A10L]PnIA荧光探针的设计合成及活性研究 |
|
| 谭瑶1, 杨奕帅1, 褚召莉1, 长孙东亭1, 朱晓鹏1,2*, 罗素兰1,2* |
|
1. 海南大学热带生物资源教育部重点实验室, 海南大学生命科学与药学院, 海南 海口 570228;
2. 广西大学医学院, 广西 南宁 530004 |
| 摘要: |
| α7烟碱型乙酰胆碱受体（nAChR）广泛分布于中枢和外周神经系统，与多种神经系统疾病、炎症反应密切相关。α-芋螺毒素[A10L]PnIA作为靶向α7 nAChR的拮抗剂，对研究α7 nAChR相关生理、病理过程具有重要作用。利用荧光素5-羧基四甲基罗丹明标记[A10L]PnIA，体外两步法氧化折叠获得活性肽（[A10L]PnIA-F）。利用非洲爪蟾卵母细胞表达体系和双电极电压钳技术检测[A10L]PnIA-F荧光肽的活性。同时，利用小鼠巨噬细胞和CCK8检测其细胞毒性。合成的[A10L]PnIA-F荧光肽，质谱鉴定其分子质量为2 077.28 Da，与理论值一致；电生理测定其对鼠源α7 nAChR的半阻断剂量（IC50）为17.32 nmol·L-1，较[A10L]PnIA本体（IC50，13.84 nmol·L-1）基本保持一致；细胞毒检测结果显示，在5 nmol·L-1～10 μmol·L-1的浓度范围内，对小鼠巨噬细胞的生长无显著抑制。结果表明α-芋螺毒素荧光探针[A10L]PnIA可以为α7 nAChR相关的神经生理、病理机制的研究提供工具。 |
| 关键词:
α7烟碱型乙酰胆碱受体
α-芋螺毒素[A10L]PnIA
荧光探针
电生理活性
|
|
| Design, synthesis, and activity study of α-conotoxin[A10L]PnIA fluorescent probe |
|
| TAN Yao1, YANG Yi-shuai1, CHU Zhao-li1, ZHANGSUN Dong-ting1, ZHU Xiao-peng1,2*, LUO Su-lan1,2* |
|
1. Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China;
2. Medical School, Guangxi University, Nanning 530004, China |
| Abstract: |
| α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems, and is closely related to a variety of neurological diseases and inflammation response. α-Conotoxin[A10L]PnIA, as an antagonist targeting α7 nAChR, plays an important role in studying the physiological and pathological processes involved in α7 nAChR.[A10L]PnIA was labeled with fluorescein 5-carboxytetramethylrho-damine, and the active peptide ([A10L]PnIA-F) was obtained by a two-step oxidative folding procedure in vitro. The Xenopus oocyte expression system and the two-electrode voltage clamp technique were used to identify the potency of[A10L]PnIA-F fluorescent peptide, and its cytotoxicity was detected by mouse macrophages and CCK8 method. The molecular weight of[A10L]PnIA-F fluorescent peptide was identified by mass spectrometry as 2 077.28 Da, which was consistent with the theoretical value. Electrophysiological determination of its halfmaximal inhibitory concentration (IC50) for α7 nAChR is 17.32 nmol·L-1, which is consistent with[A10L]PnIA (IC50, 13.84 nmol·L-1). The cytotoxicity test results showed that within the concentration range of 5 nmol·L-1 to 10 μmol·L-1, there was no significant inhibition on the growth of mouse macrophages. The results showed that the α-conotoxin fluorescent probe[A10L]PnIA could provide pharmacological tools for the research of α7 nAChRrelated neurophysiological and pathological mechanisms. |
| Key words:
α7 nicotinic acetylcholine receptor
α-conotoxin[A10L]PnIA
fluorescent probe
electrophysio-logical activity
|
|
| 收稿日期: 2021-04-12
|
| DOI: 10.16438/j.0513-4870.2021-0528 |
| 基金项目: 海南省基础与应用基础研究计划（自然科学领域）高层次人才项目（2019RC072）；国家自然科学基金资助项目（31760249，81872794）. |
通讯作者: 朱晓鹏,Tel:86-898-66289538,E-mail:biozxp@163.com;罗素兰,Tel:86-898-66289538,E-mail:luosulan2003@163.com
Email: biozxp@163.com;luosulan2003@163.com |
|
|
|
|
|
|
|
参考文献: |
|
|
[1] Millar NS, Gotti C. Diversity of vertebrate nicotinic acetylcho-line receptors[J]. Neuropharmacology, 2009, 56:237-246.
[2] Kabbani N, Nichols RA. Beyond the channel:metabotropic signaling by nicotinic receptors[J]. Trends Pharmacol Sci, 2018, 39:354-366.
[3] Ma KG, Qian YH. Alpha 7 nicotinic acetylcholine receptor and its effects on Alzheimer's disease[J]. Neuropeptides, 2019, 73:96-106.
[4] Wallace TL, Porter RH. Targeting the nicotinic alpha7 acetylcho-line receptor to enhance cognition in disease[J]. Biochem Pharmacol, 2011, 82:891-903.
[5] Hajiasgharzadeh K, Somi MH, Sadigh-eteghad S, et al. The dual role of alpha7 nicotinic acetylcholine receptor in inflammationassociated gastrointestinal cancers[J]. Heliyon, 2020, 6:e03611.
[6] Kanaoka Y, Koga M, Sugiyama K, et al. Varenicline enhances oxidized LDL uptake by increasing expression of LOX-1 and CD36 scavenger receptors through alpha7 nAChR in macrophages[J]. Toxicology, 2017, 380:62-71.
[7] Ulleryd MA, Mjornstedt F, Panagaki D, et al. Stimulation of alpha 7 nicotinic acetylcholine receptor (alpha7nAChR) inhibits atherosclerosis via immunomodulatory effects on myeloid cells[J]. Atherosclerosis, 2019, 287:122-133.
[8] Dehkordi O, Rose JE, Balan KV, et al. Co-expression of nAChRs and molecules of the bitter taste transduction pathway by epithelial cells of intrapulmonary airways[J]. Life Sci, 2010, 86:281-288.
[9] Fujii T, Mashimo M, Moriwaki Y, et al. Expression and function of the cholinergic system in immune cells[J]. Front Immunol, 2017, 8:1085.
[10] de jonge WJ, van der zanden EP, The FO, et al. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway[J]. Nat Immunol, 2005, 6:844-954.
[11] Fujii Y, Fujigaya H, Moriwaki Y, et al. Enhanced serum antigenspecific IgG1 and proinflammatory cytokine production in nicotinic acetylcholine receptor α7 subunit gene knockout mice[J]. J Neuroimmunol, 2007, 189:69-74.
[12] Wang H, Yu M, Ochani M, et al. Nicotinic acetylcholine receptor ahira unit is an essential regulator of inflammation[J]. Nature, 2003, 421:384-388.
[13] Zhang Q, Zhang YJ, Chen XW, et al. Role of alpha7 nicotinic acetylcholine receptor in lung inflammation and carcinogenesis[J]. Chin J Clin Oncol (中国肿瘤临床), 2020, 47:851-855.
[14] Chen J, Cheuk IWY, Shin VY, et al. Acetylcholine receptors:key players in cancer development[J]. Surg Oncol, 2019, 31:46-53.
[15] Egleton RD, Brown KC, Dasgupta P. Nicotinic acetylcholine receptors in cancer:multiple roles in proliferation and inhibition of apoptosis[J]. Trends Pharmacol Sci, 2008, 29:151-158.
[16] Zhu XP, Yu JP, Hu YY, et al. Analgesic activity of α-conotoxin LtIA[J]. Chin Pharm J (中国药学杂志), 2016, 51:1941-1946.
[17] Zhangsun DT, Wu Y, Zhu XP, et al. Sensitivity of α -conotoxin TxID on stoichiometry of α3β4 nicotinic acetylcholine receptors[J]. Chin Pharm J (中国药学杂志), 2016, 51:802-808.
[18] Zhangsun DT, Wu Y, Zhu XP, et al. Antagonistic activity of α-conotoxin TxIB isomers on rat and human α6/α3β2β3 nicotinic acetylcholine receptors[J]. Chin Pharm J (中国药学杂志), 2017, 52:574-580.
[19] Ellison M, Haberlandt C, Gomez-Casati ME, et al. R-RgIA:a novel conotoxin that specifically and potently blocks the α9α10 nAChR[J]. Biochemistry, 2006, 45:1511-1517.
[20] Armishaw C, Jensen AA, Balle T, et al. Rational design of alphaconotoxin analogues targeting alpha7 nicotinic acetylcholine receptors:improved antagonistic activity by incorporation of proline derivatives[J]. J Biol Chem, 2009, 284:9498-9512.
[21] Boaro A, Ageitos L, Torres M, et al. Light-emitting probes for labeling peptides[J]. Cell Reports Phys Sci, 2020, 1:100257.
[22] Kong J, Wang Y, Qi W, et al. Green fluorescent protein inspired fluorophores[J]. Adv Colloid Interface Sci, 2020, 285:102286.
[23] Chileveru HR, Lim SA, Chairatana P, et al. Visualizing attack of Escherichia coli by the antimicrobial peptide human defensin 5[J]. Biochemistry, 2015, 54:1767-1777.
[24] Zhao C, Mendive-tapia L, Vendrell M. Fluorescent peptides for imaging of fungal cells[J]. Arch Biochem Biophys, 2019, 661:187-195.
[25] El-Mounadi K, Islam KT, Hernandez-Ortiz P, et al. Antifungal mechanisms of a plant defensin MtDef4 are not conserved between the ascomycete fungi Neurospora crassa and Fusarium graminearum[J]. Mol Microbiol, 2016, 100:542-559.
[26] Islam KT, Shah DM, El-mounadi K. Live-cell imaging of fungal cells to investigate modes of entry and subcellular localization of antifungal plant defensins[J]. J Vis Exp, 2017:e55995.
[27] Zerfas BL, Trader DJ. Monitoring the immunoproteasome in live cells using an activity-based peptide-peptoid hybrid probe[J]. J Am Chem Soc, 2019, 141:5252-5260.
[28] Liu Y, Liu Z, Wang Y, et al. A tetrathiafulvalene-L-glutamine conjugated derivative as a supramolecular gelator for embedded C60 and absorbed rhodamine B[J]. New J Chem, 2020, 44:14151-14160.
[29] Kubin RF, Fletcher AN. Fluorescence quantum yields of some rhodamine dyes[J]. J Lumin, 1983, 27:455-462.
[30] Shin VY, Wu WK, Ye YN, et al. Nicotine promotes gastric tumor growth and neovascularization by activating extracellular signalregulated kinase and cyclooxygenase-2[J]. Carcinogenesis, 2004, 25:2487-2495.
[31] Zheng Y, Ritzenthaler JD, Roman J, et al. Nicotine stimulates human lung cancer cell growth by inducing fibronectin expression[J]. Am J Respir Cell Mol Biol, 2007, 37:681-690.
[32] Yang T, Xiao T, Sun Q, et al. The current agonists and positive allosteric modulators of alpha7 nAChR for CNS indications in clinical trials[J]. Acta Pharm Sin B, 2017, 7:611-622.
[33] Li Q, Yang TY, Xue Y, et al. The structure-activity relationships of novel α7 nicotinic acetylcholine receptor agonists based on indolizine scaffold[J]. Acta Pharm Sin (药学学报), 2016, 51:1584-1594.
[34] Kong FJ, Ma LL, Zhang HH, et al. Alpha 7 nicotinic acetylcho-line receptor agonist GTS-21 mitigates isoflurane-induced cognitive impairment in aged rats[J]. J Surg Res, 2015, 194:255-261.
[35] Mashimo M, Takeshima S, Okuyama H, et al. alpha7 nAChRs expressed on antigen presenting cells are insensitive to the conventional antagonists alpha-bungarotoxin and methyllycaconitine[J]. Int Immunopharmacol, 2020, 81:106276.
[36] Witayateeraporn W, Arunrungvichian K, Pothongsrisit S, et al. alpha7-Nicotinic acetylcholine receptor antagonist QND7 suppresses non-small cell lung cancer cell proliferation and migration via inhibition of Akt/mTOR signaling[J]. Biochem Biophys Res Commun, 2020, 521:977-983.
[37] Hone AJ, Rueda-ruzafa L, Gordon TJ, et al. Expression of alpha3beta2beta4 nicotinic acetylcholine receptors by rat adrenal chromaffin cells determined using novel conopeptide antagonists[J]. J Neurochem, 2020, 154:158-176.
[38] Luo SL, Zhu XP, Yu JP, et al. Pharmacological activities of α3β2 and α3β4 nicotinic acetycholine receptors with different α and β subunit stoichiometries[J]. Chin J Pathophysiol (中国病理生理杂志), 2017, 33:961-967. |
|
|
|
|
|
|
|
|
|
|
|
|