Özet

细胞膜亲和色谱柱鉴定与固定化托肌球蛋白激酶受体B相互作用的特化植物代谢物

Published: January 19, 2022
doi:

Özet

该协议描述了用含有功能性跨膜滋养肌球蛋白激酶受体B蛋白的固定化细胞膜片段制备细胞膜亲和色谱(CMAC)柱。还解释了使用CMAC色谱柱鉴定与这些受体相互作用并存在于复杂天然混合物中的特化植物代谢物。

Abstract

由植物,真菌,细菌和海洋无脊椎动物合成的化学物质一直是新药命中和先导物的丰富来源。他汀类药物、青霉素、紫杉醇、雷帕霉素或青蒿素等药物,通常用于医疗实践,已首先从天然产物中鉴定和分离出来。然而,从天然来源鉴定和分离具有生物活性的特化代谢物是一个具有挑战性且耗时的过程。传统上,在提取生物质之后,从复杂的混合物中分离和纯化单个代谢物。随后,在功能测定中测试分离的分子以验证其生物活性。在这里,我们介绍了使用细胞膜亲和色谱(CMAC)色谱柱直接从复杂混合物中鉴定生物活性化合物。CMAC色谱柱允许鉴定与嵌入其天然磷脂双层环境中的固定化功能性跨膜蛋白(TMP)相互作用的化合物。这是一种靶向的方法,需要知道TMP,其活性打算用新发现的小分子候选药物进行调节。在该协议中,我们提出了一种用固定化肌球蛋白激酶受体B(TrkB)制备CMAC柱的方法,该方法已成为许多神经系统疾病药物发现的可行靶标。在本文中,我们提供了一个详细的方案,使用过表达TrkB受体的神经母细胞瘤细胞系将CMAC柱与固定的TrkB受体组装在一起。我们进一步介绍了研究色谱柱功能及其在鉴定与TrkB受体相互作用的专用植物代谢物中的应用的方法。

Introduction

植物混合物富含药理活性化合物1,可作为鉴定新药命中和先导联2345的良好来源。从天然产物中发现新药是一种富有成效的方法,许多目前批准的药物来自自然界中首先鉴定的化合物。天然化合物的化学多样性很难与人造化学合成分子库相匹配。许多天然化合物与人类蛋白质靶标相互作用并调节人类蛋白质靶标,并且可以被认为是进化优化的类药物分子6。这些天然化合物特别适合于药物先导物鉴定,以用于神经系统疾病6。目前FDA批准的两种用于治疗阿尔茨海默病(AD)的药物来自天然生物碱,即:加兰他敏和卡巴拉汀(毒扁豆碱的衍生物)6。L-DOPA 是目前帕金森病最常用的处方药,首先从蚕豆(Vicia faba L.)中鉴定出来。7.高臼和赖脲,多巴胺能受体激动剂是来自寄生真菌Claviceps purpurea8的天然麦角生物碱的衍生物。利血平,一种从印度蛇根(Rauvolfia serpentina (L.) Benth. ex Kurz)分离出来的生物碱,是最早的抗精神病药物之一9.最近,失调的免疫反应和全身炎症已经与许多神经系统疾病的发展有关,例如重度抑郁症或神经退行性疾病10。已发现植物性饮食与其他生活方式干预相结合可以改善老年人的认知和功能能力11,12131415161718192021.已经发现属于三萜烯和多酚的某些亲电分子在体外和体内模型12调节炎症反应。例如,含有α,β不饱和羰基(例如姜黄素,肉桂醛)或异硫氰酸酯基团(例如萝卜硫素)的天然化合物会干扰Toll样受体-4(TLR4)二聚化,从而抑制小鼠白细胞介素-3依赖性pro-B细胞系中促炎细胞因子的下游合成1222.流行病学证据强烈指出,存在于复杂食物基质中的膳食植物化学物质也可能构成新药先导物的可行来源6

鉴定植物提取物(包括植物性食物)中存在的生物活性分子的主要障碍之一是所研究样品的复杂性。传统上,单个化合物被分离,纯化,然后测试生物活性。这种方法通常导致鉴定最丰富和最有特征的化合物。没有明确分子靶点的表型药物发现方法依赖于复杂混合物的生物引导分馏23。在这种方法中,将提取物分馏成不太复杂的亚级分,随后在表型测定中进行测试。活性化合物的分离和纯化由测定中验证的生物活性指导。对特定药物靶标的特性的了解可以显着加快复杂混合物中存在的药理活性化合物的鉴定。这些方法通常基于分子靶标的固定化,例如酶,在固体表面上,如磁珠23。固定的靶标随后用于筛选实验,从而分离出与靶标相互作用的化合物。虽然这种方法已被广泛用于鉴定靶向胞质蛋白的化合物,但它在鉴定与跨膜蛋白(TMP)相互作用的化学物质方面不太常见23。TMP固定化的另一个挑战源于蛋白质的活性取决于其与细胞膜磷脂和双层中其他分子(如胆固醇2324)的相互作用。在试图固定跨膜靶标时,重要的是要保留蛋白质与其天然磷脂双层环境之间的这些微妙相互作用。

在细胞膜亲和色谱(CMAC)中,细胞膜片段,而不是纯化的蛋白质,被固定在人工膜(IAM)固定相颗粒23上。通过将磷脂酰胆碱类似物共价键合到二氧化硅上来制备IAM固定相。最近开发了新型的IAM固定相类,其中游离胺和硅醇基团是端封的(IAM.个人电脑。DD2 粒子)。在CMAC色谱柱制备过程中,细胞膜片段通过吸附固定在IAM颗粒的表面上。

迄今为止,CMAC色谱柱已被用于固定不同类别的TMP,包括离子通道(例如烟碱受体),GPCRs(例如,阿片受体),蛋白质转运蛋白(例如,p-糖蛋白)等固定化的蛋白质靶标已用于表征药效学(例如,解离常数,Kd)或确定与靶标相互作用的小分子配体的结合动力学(k 和k),以及鉴定复杂基质中存在的潜在新药先导物的过程24.在这里,我们介绍了具有固定化对肌球蛋白激酶受体B(TrkB)的CMAC柱的制备,该受体已成为许多神经系统疾病的药物发现的可行靶标。

先前的研究表明,脑源性神经营养因子(BDNF)/ TrkB通路的激活与某些神经系统疾病的改善有关,例如AD或重性抑郁障碍25262728。据报道,在AD中,BDNF水平及其受体TrkB表达降低,类似的减少损害了AD29动物模型中的海马功能。据报道,AD患者的血清和大脑中BDNF水平降低303132。发现Tau过表达或高磷酸化可下调原代神经元和AD动物模型中的BDNF表达333435。此外,据报道,BDNF对β-淀粉样蛋白诱导 的体外体内36的神经毒性具有保护作用。直接将BDNF施用于大鼠大脑被证明可以增加认知受损动物的学习和记忆力37。BDNF / TrkB成为改善神经和精神疾病的有效目标,包括AD2838。靶向BDNF / TrkB信号通路以开发AD中的疗法可能会增强我们对疾病的理解39。不幸的是,BDNF本身不能用作治疗,因为它的药代动力学性质差和不良副作用40。TrkB / BDNF途径的小分子激活剂已被探索为潜在的TrkB配体414243。在测试的小分子激动剂中,7,8-二羟基黄酮(7,8-DHF)已被证明可以激活BDNF / TrkB途径41444546。7,8-DHF(R13;4-氧代-2-苯基-4H-铬-7,8-二基双(甲基氨基甲酸酯))的衍生物目前正在考虑作为AD47的可能药物。最近,研究表明,几种抗抑郁药通过直接与TrkB结合并促进BDNF信号传导起作用,进一步强调了追求TrkB作为治疗各种神经系统疾病的有效靶点的重要性48

该协议描述了组装功能TrkB柱和TrkB-NULL阴性对照柱的过程。使用已知的天然产物小分子配体:7,8-DHF来表征色谱柱。此外,我们描述了筛选复杂基质的过程,以植物提取物为例,用于鉴定与TrkB相互作用的化合物。

Protocol

1. SH-SY5Y神经母细胞瘤细胞的细胞培养(TrkB和TrkB-NULL(亲本)细胞系) 注意:细胞系(SH-SY5Y细胞系(TrkB,BR6)和SH-SY5Y亲本细胞系(TrkB NULL))49,50 是从Kerafast购买的。培养的细胞被用作跨膜受体的来源,以固定用于制备CMAC柱。以下步骤描述了如何获得细胞膜片段和组装功能性CMAC柱。 通过在37°C的潮湿气氛中?…

Representative Results

按照该方案,组装了两个CMAC色谱柱:一个具有固定化的SH-SY5Y神经母细胞瘤细胞膜片段,具有过度表达的TrkB,另一个具有SH-SY5Y TrkB-NULL细胞膜片段。正确组装的CMAC柱如图 1 所示,细胞膜碎片固定所涉及的步骤如图 2所示。 自从在IAM上固定TrkB受体以来。个人电脑。以前没有尝试过DD2色谱固定相,使用标记配体的抗体染色和额亲和色?…

Discussion

鉴定存在于特化代谢物的复杂混合物中的活性化合物23是一项非常具有挑战性的任务。传统上,分离单个化合物,并在不同的测定中测试其活性。这种方法既耗时又昂贵,并且经常导致分离和鉴定最丰富和最有特征的化合物23。目前使用的高通量筛选测定法严重依赖于筛选具有已知靶点的组合化学文库,并且不是设计用于鉴定和分离复杂混合物中存在的生物活性…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

Z.C.A.得到了土耳其科学技术研究委员会(TUBITAK)2219-国际博士后研究奖学金计划的支持。本出版物中报告的研究得到了美国国立卫生研究院国家免费和中西医结合中心的支持,奖励号为1R41AT011716-01。这项工作也得到了美国药物学研究协会入门补助金的部分支持,Regis Technologies向L.C.拨款。内容完全由作者负责,并不一定代表美国国立卫生研究院的官方观点。

Materials

7-8 Dihydroxyflavone hydrate Sigma-Aldrich D5446-10 mg ≥98% (HPLC)
Adenosine 5'-triphosphate (ATP) disodium salt hydrate Sigma-Aldrich A2383-1 g
Ammonium acetate VWR Chemicals BDH BDH9204-500 g
BDNF antibody Invitrogen PA5-15198-400 μL Primary antibody; 2 mg/mL of concentration
Benzamidine hydrochloride hydrate Sigma-Aldrich B6506-25 g
Brain derived neurotrophic factor (BDNF) human Sigma-Aldrich B3795-10 μg Recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture
Calcium chloride VWR Analytical BDH9224-1 kg
Cholic acid sodium salt Alfa Aesar J62050-100 g
Dounce homogenizer VWR 71000-516 40 mL, 285 mm (overall lenght), 32 x 140 mm (O.D. x H)
Ethanol Sigma-Aldrich 493511
Ethylenediaminetetraacetic acid (EDTA) VWR Analytical BDH-9232-500 g
Fetal bovine serum Sigma-Aldrich F2442-500 mL sterile-filtered, suitable for cell culture
G418 disulfate salt solution Sigma-Aldrich G8168-100 mL 50 mg/mL in H2O, 0.1 μm filtered, suitable for cell culture
Glycerol VWR Life Science E520-100 mL
Immobilized artificial membrane (IAM.PC.DD2) Regis Technologies, Inc. 1-771050-500
Magnesium chloride hexahydrate VWR Analytical BDH9244-500 mL
Methanol Sigma-Aldrich 322425
Nikon Plan Fluor Nikon Confocal laser scanning microscope
Normal goat serum (10%) Life Technologies 50197Z
Penicillin-Streptomycin Sigma-Aldrich P4333-100 mL
Phenylmethanesulfonyl fluoride (PMSF) Thermo Scientific 36978-5 g
Phosphate buffered saline (PBS) VWR Life Science K812-500 mL 1x
Potassium chloride VWR Chemicals BDH 0395-1 kg
Protease inhibitor cocktail VWR Life Science Ambreso M221-1 mL Proteomics grade, containing 50 mM AEBSF, 30 µM aprotonin, 1 mM bestatin, 1 mM E-64 and 1 mM leupeptin
RPMI-1640 medium Sigma-Aldrich R8758-500 mL with L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Secondary antibody goat anti-rabbit IgG (H+L) Invitrogen Alexa Flour Plus 488 A32731
SH-SY5Y Neuroblastoma cell lines expressing Trk-B Kerafast ECP007
SH-SY5Y Trk-NULL cell line Kerafast ECP005
Snake skin dialysis tubing Thermo Scientific 88245 10K MWCO, 35 mm dry I.D.
Sodium azide Sigma-Aldrich S2002
Sodium chloride BDH VWR Analytical BDH9286-2.5 kg
Tricorn 5/20 column GE Healthcare 24-4064-08
Tris-HCl VWR Life Science 0497-1 kg
Trypsin-EDTA solution Sigma-Aldrich T4049-500 mL 0.25%, sterile-filtered, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA

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Arituluk, Z. C., Adhikari, B., Maitra, U., Goodman, C., Ciesla, L. M. Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B. J. Vis. Exp. (179), e63118, doi:10.3791/63118 (2022).

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