药学学报, 2021, 56(9): 2325-2334
引用本文:
方家豪, 曹雨虹, 何宇臻, 洪战英*, 柴逸峰. 膜蛋白稳定技术及其在药物筛选中的应用进展[J]. 药学学报, 2021, 56(9): 2325-2334.
FANG Jia-hao, CAO Yu-hong, HE Yu-zhen, HONG Zhan-ying*, CHAI Yi-feng. Advancements in stabilization technologies for membrane protein and its application in drug screening[J]. Acta Pharmaceutica Sinica, 2021, 56(9): 2325-2334.

膜蛋白稳定技术及其在药物筛选中的应用进展
方家豪, 曹雨虹, 何宇臻, 洪战英*, 柴逸峰
海军军医大学药学院, 上海 200433
摘要:
膜蛋白承担了生物膜的主要功能,是新药研发最重要的靶点群,大约60%的药物以膜蛋白为靶点。由于膜蛋白在水溶液中有明显的聚集和变性倾向,在体外很难模拟维持膜蛋白正确构象的类膜环境,导致膜蛋白的结构和功能以及相关配体药物的研究远远滞后于水溶性蛋白。膜蛋白稳定技术对于建立高专属性、高灵敏度和高通量的膜蛋白配体药物筛选方法具有重要的意义。本文综述了目前用于稳定分离纯化膜蛋白的一些技术,包括洗涤剂、人造膜、聚合物、慢病毒颗粒等,以及这些技术在药物筛选中的具体应用。
关键词:    膜蛋白      稳定技术      洗涤剂      人造膜      聚合物      慢病毒颗粒      药物筛选     
Advancements in stabilization technologies for membrane protein and its application in drug screening
FANG Jia-hao, CAO Yu-hong, HE Yu-zhen, HONG Zhan-ying*, CHAI Yi-feng
School of Pharmacy, Naval Medical University, Shanghai 200433, China
Abstract:
Membrane proteins are the main undertakers of biofilm function, and also the most important target group for innovative drug discovery and research. About 60% of drugs targets are membrane proteins. Due to the obvious aggregation and denaturation tendency of membrane proteins in aqueous solution, it is difficult to simulate the membrane like environment to maintain the correct conformation of membrane proteins in vitro, which results in the slower-growing research on the structure and function of membrane proteins and related ligand drugs than that of water-soluble proteins. Membrane protein stabilization technology is the premise of establishing high specificity, high sensitivity and high throughput drug screening methods for membrane protein ligands, which is of great significance. In this paper, some techniques for stable separation and purification of membrane proteins are reviewed, including detergents, artificial membranes, polymers, lentiviral particles and so on, as well as their specific applications in drug screening.
Key words:    membrane protein    stabilization technology    detergent    artificial membrane    polymer    lentivirus particles    drug screening   
收稿日期: 2021-04-20
DOI: 10.16438/j.0513-4870.2021-0573
基金项目: 国家自然科学基金资助项目(81872829,81673386).
通讯作者: 洪战英,Tel:86-21-81871269,E-mail:hongzhy001@163.com
Email: hongzhy001@163.com
相关功能
PDF(626KB) Free
打印本文
0
作者相关文章
方家豪  在本刊中的所有文章
曹雨虹  在本刊中的所有文章
何宇臻  在本刊中的所有文章
洪战英*  在本刊中的所有文章
柴逸峰  在本刊中的所有文章

参考文献:
[1] Lappano R, Maggiolini M. G protein-coupled receptors:novel targets for drug discovery in cancer[J]. Nat Rev Drug Discov, 2011, 10:47-60.
[2] Santos R, Ursu O, Gaulton A, et al. A comprehensive map of molecular drug targets[J]. Nat Rev Drug Discov, 2017, 16:19-34.
[3] Wu CP, Ambudkar VS. The pharmacological impact of ATP-binding cassette drug transporters on vemurafenib-based therapy[J]. Acta Pharm Sin B, 2014, 4:105-111.
[4] Minuesa G, Huber-Ruano I, Pastor-Anglada M, et al. Drug uptake transporters in antiretroviral therapy[J]. Pharmacol Ther, 2011, 132:268-279.
[5] Hauser AS, Attwood MM, Rask-Andersen M, et al. Trends in GPCR drug discovery:new agents, targets and indications[J]. Nat Rev Drug Discov, 2017, 16:829-842.
[6] Yang C, He B, Dai W, et al. The role of caveolin-1 in the biofate and efficacy of anti-tumor drugs and their nano-drug delivery systems[J]. Acta Pharm Sin B, 2021, 11:961-977.
[7] He Y, Wang K, Yan N. The recombinant expression systems for structure determination of eukaryotic membrane proteins[J]. Protein Cell, 2014, 5:658-672.
[8] Das M, Du Y, Mortensen JS, et al. Correction:trehalose-cored amphiphiles for membrane protein stabilization:importance of the detergent micelle size in GPCR stability[J]. Org Biomol Chem, 2019, 17:4919-4920.
[9] Fiori MC, Jiang Y, Zheng W, et al. Polymer nanodiscs:discoidal amphiphilic block copolymer membranes as a new platform for membrane proteins[J]. Sci Rep, 2017, 7:15227-15236.
[10] Bada Juarez JF, Harper AJ, Judge PJ, et al. From polymer chemistry to structural biology:the development of SMA and related amphipathic polymers for membrane protein extraction and solubilisation[J]. Chem Phys Lipids, 2019, 221:167-175.
[11] Heym RG, Hornberger WB, Lakics V, et al. Label-free detection of small-molecule binding to a GPCR in the membrane environment[J]. Biochim Biophys Acta, 2015, 1854:979-986.
[12] Anandan A, Vrielink A. Detergents in membrane protein purification and crystallisation[J]. Adv Exp Med Biol, 2016, 922:13-28.
[13] Lichtenberg D, Ahyayauch H, Alonso A, et al. Detergent solubilization of lipid bilayers:a balance of driving forces[J]. Trends Biochem Sci, 2013, 38:85-93.
[14] Kotov V, Bartels K, Veith K, et al. High-throughput stability screening for detergent-solubilized membrane proteins[J]. Sci Rep, 2019, 9:10379-10398.
[15] Iwata S. Methods and Results in Crystallization of Membrane Proteins[M]. La Jolla:International Unversity Line Press, 2003:106-114.
[16] Newstead S, Kim H, Von Heijne G, et al. High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae[J]. Proc Natl Acad Sci U S A, 2007, 104:13936-13941.
[17] Newstead S, Ferrandon S, Iwata S. Rationalizing alpha-helical membrane protein crystallization[J]. Protein Sci, 2008, 17:466-472.
[18] Hiller S, Garces RG, Malia TJ, et al. Solution structure of the integral human membrane protein VDAC-1 in detergent micelles[J]. Science, 2008, 321:1206-1210.
[19] Metola A, Bouchet AM, Alonso-Mario M, et al. Purification and characterization of the colicin A immunity protein in detergent micelles[J]. Biochim Biophys Acta Biomembr, 2017, 1859:2181-2192.
[20] Mcgregor CL, Chen L, Pomroy NC, et al. Lipopeptide detergents designed for the structural study of membrane proteins[J]. Nat Biotechnol, 2003, 21:171-176.
[21] Das M, Mahler F, Hariharan P, et al. Diastereomeric cyclopentane-based maltosides (CPMs) as tools for membrane protein study[J]. J Am Chem Soc, 2020, 142:21382-21392.
[22] Hardy D, Bill RM, Rothnie AJ, et al. Stabilization of human multidrug resistance protein 4(MRP4/ABCC4) using novel solubilization agents[J]. SLAS Discov, 2019, 24:1009-1017.
[23] Hyoung EB, Du Y, Hariharan P, et al. Asymmetric maltose neopentyl glycol amphiphiles for a membrane protein study:effect of detergent asymmetricity on protein stability[J]. Chem Sci, 2019, 10:1107-1116.
[24] Hussain H, Du Y, Tikhonova E, et al. Resorcinarene-based facial glycosides:implication of detergent flexibility on membrane-protein stability[J]. Chemistry, 2017, 23:6724-6729.
[25] Ehsan M, Du Y, Mortensen JS, et al. Self-assembly behavior and application of terphenyl-cored trimaltosides for membrane-protein studies:impact of detergent hydrophobic group geometry on protein stability[J]. Chem Eur J, 2019, 25:11545-11554.
[26] Sadaf A, Ramos M, Mortensen JS, et al. Conformationally restricted monosaccharide-cored glycoside amphiphiles:the effect of detergent headgroup variation on membrane protein stability[J]. ACS Chem Biol, 2019, 14:1717-1726.
[27] Hussain H, Helton T, Du Y, et al. A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability[J]. Analyst, 2018, 143:5702-5710.
[28] Rothnie AJ. Detergent-free membrane protein purification[J]. Methods Mol Biol, 2016, 1432:261-267.
[29] Reeves JP. Formation and properties of thin-walled phospholipid vesicles[J]. J Cell Physiol, 1969, 73:49-60.
[30] Traïkia M, Warschawski DE, Recouvreur M, et al. Formation of unilamellar vesicles by repetitive freeze-thaw cycles:characterization by electron microscopy and 31P-nuclear magnetic resonance[J]. Eur Biophys J, 2000, 29:184-195.
[31] Pott T, Bouvrais H, Méléard P. Giant unilamellar vesicle formation under physiologically relevant conditions[J]. Chem Phys Lipids, 2008, 154:115-119.
[32] Johnson ZL, Lee SY. Liposome reconstitution and transport assay for recombinant transporters[J]. Methods Enzymol, 2015, 556:373-383.
[33] Hunte C, Richers S. Lipids and membrane protein structures[J]. Curr Opin Struct Biol, 2008, 18:406-411.
[34] Kurisu G, Zhang H, Smith JL, et al. Structure of the cytochrome b6f complex of oxygenic photosynthesis:tuning the cavity[J]. Science, 2003, 302:1009-1014.
[35] Jidenko M, Nielsen R, Sorensen T, et al. Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae[J]. Proc Natl Acad Sci U S A, 2005, 102:11687-11691.
[36] Komiya M, Kato M, Tadaki D, et al. Advances in artificial cell membrane systems as a platform for reconstituting ion channels[J]. Chem Rec, 2020 Jul, 20:730-742.
[37] Sanders CR, Prosser RS. Bicelles:a model membrane system for all seasons?[J]. Structure, 1998, 6:1227-1234.
[38] Leitz AJ, Bayburt TH, Barnakov AN, et al. Functional reconstitution of Beta2-adrenergic receptors utilizing self-assembling nanodisc technology[J]. BioTechniques, 2006, 40:601-602.
[39] Nath A, Atkins WM, Sligar SG. Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins[J]. Biochemistry, 2007, 46:2059-2069.
[40] Yeh V, Lee TY, Chen CW, et al. Highly efficient transfer of 7TM membrane protein from native membrane to covalently circularized nanodisc[J]. Sci Rep, 2018, 8:13501-13510.
[41] Yao Y, Nisan D, Fujimoto LM, et al. Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL[J]. Protein Expr Purif, 2016, 122:56-63.
[42] Nasr ML, Baptista D, Strauss M, et al. Covalently circularized nanodiscs for studying membrane proteins and viral entry[J]. Nat Methods, 2017, 14:49-52.
[43] Parmar MJ, Lousa Cde M, Muench SP, et al. Artificial membranes for membrane protein purification, functionality and structure studies[J]. Biochem Soc Trans, 2016, 44:877-882.
[44] Dorr JM, Koorengevel MC, Schafer M, et al. Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel:the power of native nanodiscs[J]. Proc Natl Acad Sci U S A, 2014, 111:18607-18612.
[45] Das A, Zhao J, Schatz GC, et al. Screening of type I and II drug binding to human cytochrome P450-3A4 in nanodiscs by localized surface plasmon resonance spectroscopy[J]. Anal Chem, 2009, 81:3754-3759.
[46] Swainsbury DJK, Scheidelaar S, Foster N, et al. The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes[J]. Biochim Biophys Acta Biomembr, 2017, 1859:2133-2143.
[47] Jamshad M, Lin YP, Knowles TJ, et al. Surfactant-free purification of membrane proteins with intact native membrane environment[J]. Biochem Soc Trans, 2011, 39:813-818.
[48] Knowles TJ, Finka R, Smith C, et al. Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer[J]. J Am Chem Soc, 2009, 131:7484-7485.
[49] Xue M, Cheng L, Faustino I, et al. Molecular mechanism of lipid nanodisk formation by styrene-maleic acid copolymers[J]. Biophys J, 2018, 115:494-502.
[50] Dörr JM, Scheidelaar S, Koorengevel MC, et al. The styrene-maleic acid copolymer:a versatile tool in membrane research[J]. Eur Biophys J, 2016, 45:3-21.
[51] Gulati S, Jamshad M, Knowles TJ, et al. Detergent free purification of ABC transporters[J]. Biochem J, 2014, 461:269-278.
[52] Long AR, Brien C, Malhotra K, et al. A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs[J]. BMC Biotechnol, 2013, 13:41-51.
[53] Smirnova IA, Sjöstrand D, Li F, et al. Isolation of yeast complex IV in native lipid nanodiscs[J]. Biochim Biophys Acta, 2016:2984-2992.
[54] Jamshad M, Charlton J, Lin YP, et al. G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent[J]. Biosci Rep, 2015, 35:1-10.
[55] Logez C, Damian M, Legros C, et al. Detergent-free isolation of functional g protein-coupled receptors into nanometric lipid particles[J]. Biochemistry, 2016:38-48.
[56] Morrison KA, Akram A, Mathews A, et al. Membrane protein extraction and purification using styrene-maleic acid (SMA) co-polymer:effect of variations in polymer structure[J]. Biochem J, 2016, 473:4349-4360.
[57] Lee SC, Knowles TJ, Postis VL, et al. A method for detergent-free isolation of membrane proteins in their local lipid environment[J]. Nat Protoc, 2016, 11:1149-1162.
[58] Ravula T, Hardin NZ, Ramadugu SK, et al. pH Tunable and divalent metal ion tolerant polymer lipid nanodiscs[J]. Langmuir, 2017, 33:10655-10662.
[59] Hall SCL, Tognoloni C, Charlton J, et al. An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles[J]. Nanoscale, 2018, 10:10609-10619.
[60] Oluwole AO, Danielczak B, Meister A, et al. Solubilization of membrane proteins into functional lipid-bilayer nanodiscs using a diisobutylene/maleic acid copolymer[J]. Angew Chem Int Ed Engl, 2017, 56:1919-1924.
[61] Danielczak B, Meister A, Keller S. Influence of Mg2+ and Ca2+ on nanodisc formation by diisobutylene/maleic acid (DIBMA) copolymer[J]. Chem Phys Lipids, 2019, 221:30-38.
[62] Gulamhussein AA, Uddin R, Tighe BJ, et al. A comparison of SMA (styrene maleic acid) and DIBMA (di-isobutylene maleic acid) for membrane protein purification[J]. Biochim Biophys Acta Biomembr, 2020, 1862:183281-183311.
[63] Lavington S, Watts A. Detergent-free solubilisation & purification of a G protein coupled receptor using a polymethacrylate polymer[J]. Biochim Biophys Acta Biomembr, 2021, 1863:183441-183450.
[64] Yasuhara K, Jin A, Ravula T, et al. Spontaneous lipid nanodiscs fomation by amphiphilic polymethacrylate copolymers[J]. J Am Chem Soc, 2017, 139:18657-18663.
[65] Martínez-Muoz L, Barroso R, Guedán Paredes A, et al. Methods to immobilize GPCR on the surface of SPR sensors[J]. Methods Mol Biol, 2015, 1272:173-188.
[66] Hoffman TL, Canziani G, Jia L, et al. A biosensor assay for studying ligand-membrane receptor interactions:binding of antibodies and HIV-1 Env to chemokine receptors[J]. Proc Natl Acad Sci U S A, 2000, 97:11215-11220.
[67] Gong J, Chen Y, Pu F, et al. Understanding membrane protein drug targets in computational perspective[J]. Curr Drug Targets, 2019, 20:551-564.
[68] Hou X, Sun M, Bao T, et al. Recent advances in screening active components from natural products based on bioaffinity techniques[J]. Acta Pharm Sin B, 2020, 10:1800-1813.
[69] Junko J, Toshihiro O, Masatoshi N. One hour in vivo-like phenotypic screening system for anti-cancer drugs using a high precision surface plasmon resonance device[J]. Anal Sci, 2018, 34:1189-1194.
[70] Wang Y, Guo H, Feng Z, et al. PD-1-targeted discovery of peptide inhibitors by virtual screening, molecular dynamics simulation, and surface plasmon resonance[J]. Molecules, 2019, 24:3784-3791.
[71] Zhang F, Wang S, Yin L, et al. Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging[J]. Anal Chem, 2015, 87:9960-9965.
[72] Wang YZ, Liu X, Way G, et al. An in vitro Förster resonance energy transfer-based high-throughput screening assay identifies inhibitors of SUMOylation E2 Ubc9[J]. Acta Pharmacol Sin, 2020, 41:1497-1506.
[73] Song D, Yu W, Ren Y, et al. Discovery of bazedoxifene analogues targeting glycoprotein 130[J]. Eur J Med Chem, 2020, 199:112375-112433.
[74] Komolov KE, Senin II, Philippov PP, et al. Surface plasmon resonance study of G protein/receptor coupling in a lipid bilayer-free system[J]. Anal Chem, 2006, 78:1228-1234.
[75] Oshima A, Hirano-Iwata A, Mozumi H, et al. Reconstitution of human ether-a-go-go-related gene (hERG) Channels in microfabricated silicon chips[J]. Anal Chem, 2013, 85:4363-4369.
[76] Tadaki D, Yamaura D, Araki S, et al. Mechanically stable solvent-free lipid bilayers in nano- and micro-tapered apertures for reconstitution of cell-free synthesized hERG channels[J]. Sci Rep, 2017, 7:17736-17745.
[77] Maynard JA, Lindquist NC, Sutherland JN, et al. Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins[J]. Biotechnol J, 2010, 4:1542-1558.
[78] Rich RL, Miles AR, Gale BK, et al. Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies[J]. Anal Biochem, 2009, 386:98-104.
[79] Xu H, Hill JJ, Michelsen K, et al. Characterization of the direct interaction between KcsA-Kv1.3 and its inhibitors[J]. Biochim Biophys Acta, 2015, 1848:1974-1980.
[80] Cao Y, Cao Y, Shi Y, et al. Surface plasmon resonance biosensor combined with lentiviral particle stabilization strategy for rapid and specific screening of P-glycoprotein ligands[J]. Anal Bioanal Chem, 2021, 413:2021-2031.
[81] Chen L, Lv D, Wang S, et al. Surface plasmon resonance-based membrane protein-targeted active ingredients recognition strategy:construction and implementation in ligand screening from herbal medicines[J]. Anal Chem, 2020, 92:3972-3980.