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Bioengenharia

一种快速定量荧光方法蛋白靶向小分子药物筛选

Published: October 16, 2015
doi:
10.3791/53261
, , , ,
1Institute of Materials Research and Engineering,Agency for Science, Technology and Research (A*STAR), 2School of Chemical & Biomedical Engineering,Nanyang Technological University

Summary

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Abstract

我们表明，用于确定小药物分子的结合亲和力与靶蛋白通过形成荧光金纳米簇金（Au NCS）加载药物的蛋白质内，基于由在Au纳米晶发射的差分荧光信号一个新的药物筛选方法。白蛋白蛋白质如人血清白蛋白（HSA）和牛血清白蛋白（BSA）被选择作为模型蛋白质。四个小分子药物 （如布洛芬，华法令，苯妥英，和磺胺）不同的结合亲和力到白蛋白的蛋白质进行测试。已经发现，荧光金NC的药物加载白蛋白蛋白质内的变性条件（即60℃或在尿素的存在下）下的形成速度比形成在原始蛋白（不含药物）慢。而且，这样形成的NC的荧光强度被发现负相关对这些药物的结合亲和力到白蛋白的蛋白质。特别是，越高药物 – 蛋​​白结合亲和性，较慢的凹NC的形成速率，并因此所得的Au NC的低级荧光强度观察。所得的Au NC的荧光强度因此提供测试了不同药物的相对结合强度的简单量度。这种方法也可延伸通过简单地改变在固定的蛋白质浓度预装在蛋白质的药物含量测定的具体药物-蛋白结合常数（K D）。测量结果与使用其它声望，而是更复杂的方法获得的值相匹配良好。

Introduction

血清白蛋白例如人血清白蛋白（HSA）和牛血清白蛋白（BSA）是在血浆中最丰富的蛋白，并在维持血液舱的渗透压发挥至关重要的作用。他们也被确认为载体蛋白为低水溶性的小分子，如类固醇，脂肪酸，甲状腺激素，以及各种各样的药物。的结合性（例如，结合位点，结合亲和力或强度）这些分子与血清白蛋白的形成在药动学的一个重要课题。1-4几种分析方法已经开发，研究不同的药物的结合性质的血清白蛋白，如X射线晶体学，5,6-核磁共振（NMR），7-11和表面等离子体共振（SPR），12,13等然而，这些方法是通过任一个繁琐和耗时的分析处理（ 例如受限的，单晶的X-射线crystallo生长图形研究），专业和昂贵的设备要求（SPR），或需要昂贵的同位素标记（NMR）进行检测。因此，非常需要开发为小分子药物筛选的替代方法以快速，直接的，并且成本效益的方式。

金纳米簇金（Au NCS）是一种特殊类型的纳米材料，其包含几个到几十个金属原子的有尺寸小于2纳米。14-17他们已经吸引了广泛的研究兴趣较小的由于它们的离散和尺寸相关的电子结构，18个， 19和分子状吸收和排放。20-23这种独特的材料性质，特别是强的荧光，已经发现不同的应用，如感测和成像的生物系统。24-32超小型荧光灯的Au NC的可使用的功能的蛋白质合成，如血清白蛋白，如模板33在一个典型的蛋白质为模板合成的Au NC的，一定量的Au盐的被封装的蛋白质内，随后通过蛋白质本身降低。该蛋白质的还原能力是由于构成功能的氨基酸残基（例如，酪氨酸），可以通过增加溶液的pH值至碱性激活。解折叠蛋白质结构被认为是对金NC的形成中的关键步骤。这是因为在未折叠蛋白，多种还原剂官能团可以暴露于封装的金盐。蛋白质解折叠，可以通过热处理或暴露于变性剂来实现的。小分子药物的引入也可影响展开的过程，也就是修改中点变性温度和展开的焓。34,35所有这些因素的影响，又可以通过荧光的Au纳米晶的形成动力学得到反映并表现在所得的Au NC的荧光强度。36

e_content“>此视频通过在更高的温度（60℃）在载有药物的白蛋白的蛋白质合成的Au纳米晶或在变性剂（如尿素）所得的Au NC的的荧光强度的存在表明药物筛选的方法是信号读出。首先，金的NC被合成的温度为60℃或在尿素的存在下处理的HSA和BSA的模板，以展示如何蛋白解折叠（通过热处理或变性剂诱导的）影响的Au纳米晶二的形成动力学，的Au NC的合成蛋白质的模板预装不同的药物，及对所得的Au NC的相对荧光强度的载药效果进行了研究，它们提供了相对结合强度的量度。最后，在Au NC-药物筛选协议被修改为药物-蛋白结合常数（K D）的通过改变预装在固定浓度的蛋白质的药物含量定量测定。

Protocol

注意：使用前请咨询了所有相关化学品的安全数据表（SDS）。的药物筛选试验涉及纳米材料的合成和处理相比，他们散装对方也可以有额外的危害。整个实验过程中要实行请确保所有必要的管制措施，包括利用工程控制（通风柜）和个人防护装备（PPE， 例如 ，安全长裤，闭趾鞋，化学防护手套和安全眼罩）的。 1.前处理化学试剂用于药物筛选前体金NC的合成<ol…

Representative Results

蛋白折叠为蛋白质为模板的金NC的形成的重要过程，因为蛋白质的多反应性官能团（例如，酪氨酸残基）可以暴露于降低封装的金离子，从而加速的Au纳米晶的形成速率。加热和外部变性剂是促进蛋白质折叠过程中的两个共同的装置。 图1展示了加热的影响，并增加外部变性剂对Au NC的形成动力学，使用HSA作为模型蛋白质。加热对Au NC的形成动力学的影响，首先，通过测量光致发?…

Discussion

有需要在该方法中，以被突出显示的几个关键步骤。在筛选不同的小分子药物的相对结合亲和力，对协议的步骤3.1.2，3.1.3，3.1.4和关键是要取得良好的结果，显示了相对结合强度相一致的趋势。在这些步骤中，测量添加化学品和绘图反应液的行动应尽快以减少时滞效应和测量添加化学品和绘图反应液应实行以确保一致性的相同的序列。在测量布洛芬与HSA的结合常数的协议，步骤3.2.4朝向杀敌</s…

Declarações

The authors have nothing to disclose.

Acknowledgements

Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

Materials

Gold (III) chloride solution, 30% Sigma-Aldrich 484385 Corrosive, irritant
Human serum albumin, 96% Sigma-Aldrich A1887
Bovine Serum albumin, 96% Sigma-Aldrich A2153
Ibuprofen, 98% Sigma-Aldrich I4883 
warfarin, 98% Sigma-Aldrich A2250
phenytoin Sigma-Aldrich PHR1139
sulphanilamide, 99% Sigma-Aldrich S9251
dimethyl sulfoxide Sigma-Aldrich D8418
urea Sigma-Aldrich U5128
Sodium hydroxide Sigma-Aldrich 221465
Magnetic stirrer IKA RT5
Microplate reader Tecan Infinite M200
384-well plate Corning
5 mL air displacement pipette Eppendorf
1000 mL air displacement pipette Eppendorf
100 mL air displacement pipette Eppendorf
5000 mL Eppendorf tips
1000 mL Eppendorf tips
100 mL Eppendorf tips
1.5 mL micro tube Eppendorf
20 mL glass vial with screw cap
4 mL glass vial with screw cap
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Tags

Keywords: Fluorimetric MethodProtein-targetingSmall Molecule Drug ScreeningFluorescent Gold NanoclustersAlbumin ProteinsDrug-protein Binding AffinityDrug-protein Binding Constant
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Yu, Y., New, S. Y., Lin, J., Su, X., Tan, Y. N. A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening. J. Vis. Exp. (104), e53261, doi:10.3791/53261 (2015).
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