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A Surface Plasmon Resonance Method to Study HCV NS5B Inhibitors
采用表面等离子体共振研究HCV NS5B抑制因子   

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Abstract

Surface Plasmon Resonance (SPR) technology is a well-established platform used to evaluate the kinetic parameters of protein-small molecule interactions. Below, we describe the use of the ProteOn XPR36 biosensor from Bio-Rad (Hercules, CA) to evaluate the binding of small molecule inhibitors to recombinant NS5B protein. The high pI (> 9) of this construct allows for chemical immobilization using HEPES-buffered saline at pH 7.5. This is in contrast to traditional biosensor protocols that use both low pH and ionic strength. The use of a more physiological buffer to immobilize this enzyme leads to improved surface activity.

Keywords: Surface Plasmon Resonance (SPR)(表面等离子体共振(SPR)), ProteOn XPR36(质子xpr36), BIosensor(生物传感器), NS5B(NS5B), Small molecule inhibitor(小分子抑制剂)

Materials and Reagents

  1. HCV NS5B inhibitors
  2. Purified recombinant HCV NS5B ΔC21 soluble protein [cloned and purified according to Boyce et al. (2014) and Hung et al. (2011)]
  3. ProteOn GLH Sensor Chip (Bio-Rad Laboratories, catalog number: 176-5013 )
  4. HEPES solution (1 M) (Sigma-Aldrich, catalog number: H3537-1L )
  5. MgCl2 (1 M) (Sigma-Aldrich, catalog number: M1028-100ML )
  6. EDTA (0.5 M) (Sigma-Aldrich, catalog number: E7889-100ML )
  7. NaCl (5 M) (Sigma-Aldrich, catalog number: S6546-1L )
  8. KCl (2 M) (Life Technologies, Ambion®, catalog number: AM9640G )
  9. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (Sigma-Aldrich, catalog number: C4706-10G )
  10. Surfactant P20 (10% v/v) (General Electric Company, catalog number: BR-1000-54 )
  11. DMSO (Sigma-Aldrich, catalog number: 472301-100ML )
  12. ProteOn Amine Coupling Kit (Bio-Rad Laboratories, catalog number: 176-2410 ) containing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC or EDC), sulfo-N-hydroxysuccinimide (Sulfo-NHS) and ethanolamine-HCl
  13. Preconditioning Reagents (see Recipes)
  14. Immobilization Buffer (see Recipes)
  15. Running Buffer (see Recipes)
  16. Running Buffer with 5% DMSO (see Recipes)

Equipment

  1. ProteOn Standard & Deep-Well Microplates (Bio-Rad Laboratories, catalog numbers: 176-6020 and 176-6023 )
  2. ProteOn Microplate Sealing Film (Bio-Rad Laboratories, catalog number: 176-6040 )
  3. Nalgene Rapid-Flow Filter Units and Bottle Top Filters, PES Membrane, Sterile (VWR International, catalog number: 16211-056 ) (for the 1 L size)
  4. ProteOn XPR36 instrument (Bio-Rad Laboratories)
  5. BenchTop Centrifuge for microfuge tubes
  6. BenchTop Centrifuge to spin ProteOn 96-well assay plate

Software

  1. ProteOn Manager Software Version 3.1.0.6
  2. Scrubber software designed for ProteOn data analysis (BioLogic Software)

Procedure

  1. Prime the system with distilled water. Precondition a GLH chip using 1-min pulses each of 100 mM HCl, 50 mM NaOH, 0.5% SDS and 10% DMSO using a flow rate of 30 µl/min in both the horizontal and vertical directions. Seal all microplates used with ProteOn sealing film.
  2. Prime the system with Running Buffer. Activate all 6 ligand channels (L1-L6) in the vertical direction using EDC and sulfo-NHS as prescribed by the manufacturer using a flow rate of 30 µl/min and a contact time of 5 min.
  3. Dilute NS5B protein in Immobilization Buffer to a concentration of 30 µg/ml and inject using a flow rate of 30 µl/min for 5 min in the vertical direction into the ligand channel(s) to obtain 5,000-10,000 RU of immobilized protein (see Notes). Leave the L1 channel open as a reference channel.
  4. When calculating the theoretical maximum response, Rmax, the activity of the protein should be taken into account. Rmax is calculated as RL * (MWA/MWL) * n, where RL is equal to the amount of ligand immobilized, MWA is the molecular weight of the analyte, MWL is the molecular weight of the ligand, and n is the number of binding sites per ligand molecule. Assuming single-site binding (i.e. n = 1), a small molecule analyte having an average molecular weight of 500 Da interacting with a surface comprised of 10,000 RU of NS5B ΔC21 (M.W.~ 64,300 Da) should yield a theoretical Rmax of ~ 78 RU (i.e. 10,000 RU x 500 Da/64,300 Da) or 39 RU if the surface is only 50% active.
  5. Deactivate all vertical channels (L1-L6) by injecting ethanolamine-HCl for 5 min at flow rate of 30 µl/min.
  6. Rotate the multi-channel module (MCM) as all subsequent injections will occur in the horizontal direction. Prime the system with Running Buffer containing 5% DMSO and also use this buffer to inject buffer three times to help stabilize the system after rotation.
  7. To account for excluded volume effects, a set of five solutions between 4.5% and 5.5% DMSO (DMSO concentration series) are injected for the software to generate a DMSO excluded volume calibration (EVC) curve. Prepare a 5.5% DMSO solution by adding 15 µl of 100% DMSO to 3,000 µl of Running Buffer with 5% DMSO, and a 4.5% DMSO solution by adding 333 µl of Running Buffer to 3,000 µl of Running Buffer with 5% DMSO. Mix the 5.5% and 4.5% DMSO solutions according to the volumes prescribed below to obtain the five different solutions. Five separate injections are performed to generate a DMSO concentration series each corresponding to the injection of one of the DMSO solutions. The software will use this series of injections to generate a DMSO calibration curve to correct for excluded volume effects.
    5.5% DMSO buffer   4.5% DMSO buffer
    1200 µl                      0 µl
    900 µl                        300 µl
    600 µl                        600 µl
    300 µl                        900 µl
    0 µl                           1200 µl
  8. When designing the analyte injection sequence, include buffer injections between every injection of compound (or as space permits in the 96-well plate).
  9. Thaw compounds, typically stored in 100% DMSO in microfuge tubes at -20 °C, and quickly spin them in a microfuge centrifuge.
  10. Serially dilute the compounds in DMSO to achieve a set of five concentrations (four-fold dilutions in this example) at 20 times the desired final assay concentration. Transfer the diluents to wells in the microplate containing an appropriate volume of Running Buffer to yield a final DMSO concentration of 5% (i.e. matching the % of DMSO in Running Buffer with DMSO). These will be injected as Analytes A2-A6. Add 100% DMSO to the same volume of Running Buffer to the well corresponding to injection of Analyte A1 to yield a final DMSO concentration of 5%. Injection from well A1 will serve as a blank and be used to double-reference the data.
  11. Centrifuge the plate using a quick 15-30 second pulse to ensure collection of any compound solution stuck to the walls of the microplate well.
  12. Inject compounds at 100 µl/min for a contact time of 90 sec and for variable dissociation times ranging between 60 sec and one hour (as required by individual compounds). Absent any knowledge about a compound’s affinity, preliminary experiments involving injection at a single concentration may have to be performed in order to gauge optimal dissociation times for each compound.
  13. Data can be analyzed using ProteOn Software but can also be analyzed using a version of Scrubber software designed for ProteOn data. Double reference all signals (using L1 & A1) and correct for excluded volume effects by applying the DMSO calibration curve.
  14. Sensorgrams for all the concentrations tested should be fit simultaneously, i.e. globally. In general, data should be always first fit to a simple 1:1 kinetic model which yields the equilibrium dissociation constant (KD), the maximum binding capacitance of the surface Rmax, (which may differ from the theoretical maximum binding capacitance) and the rate constants of association (ka) and dissociation (kd). In a simple 1:1 kinetic model, single values of ka, kd and Rmax are obtained. Both Scrubber and ProteOn fitting routines solve for the parameters ka and kd. KD is then calculated as the ratio of kd/ka. Use of other more complex models that require more than one value for any of these parameters should be explicitly stated. Filibuvir binding to NS5B ΔC21 (see Figure 1) is an example of a small molecule binding showing simple 1:1 kinetics.


    Figure 1. Filibuvir Binding to NS5B ΔC21. Sensorgrams of Filibuvir binding are shown. Filibuvir was injected for a contact time of 90 sec at concentrations of (from top to bottom) 4 µM (orange), 1 µM (pink), 0.25 µM (green), 0.0625 µM (blue) and 0.0156 µM (cyan). Dissociation was monitored for 180 sec. The fit to the data are shown as solid lines.

Notes

The physiological immobilization buffer used can readily pre-concentrate sufficient NS5B enzyme onto the carboxyl surface. The ligand activity increased from 45% to 99% for NS5B ΔC21 when opting to dilute protein in HEPES-buffered saline rather than standard low pH (5.5) 10 mM sodium acetate immobilization buffer.

Recipes

  1. Preconditioning Reagents
    100 mM HCl
    50 mM NaOH
    0.5% SDS
    10% DMSO
  2. Immobilization Buffer
    10 mM HEPES (pH 7.5)
    150 mM NaCl
    Filter sterilize (0.45 μm)
  3. Running Buffer
    50 mM HEPES
    5 mM MgCl2
    10 mM KCl
    1 mM EDTA
    1 mM TCEP
    0.01% P20
    Adjust pH to 7.5 with NaOH
    Add dH2O to the desired volume
    Filter sterilize (0.45 μm)
  4. Running Buffer with 5% DMSO
    Add 100% DMSO to Running Buffer to a final concentration of 5% DMSO
    For example, add 50 ml of DMSO to Running Buffer to a total volume of 1 L.

Acknowledgments

A brief description of this protocol has previously appeared in Boyce et al. (2014).

References

  1. Boyce, S. E., Tirunagari, N., Niedziela-Majka, A., Perry, J., Wong, M., Kan, E., Lagpacan, L., Barauskas, O., Hung, M., Fenaux, M., Appleby, T., Watkins, W. J., Schmitz, U. and Sakowicz, R. (2014). Structural and regulatory elements of HCV NS5B polymerase - beta-loop and C-terminal tail - are required for activity of allosteric thumb site II inhibitors. PLoS One 9(1): e84808.
  2. Hung, M., Wang, R. and Liu, X. (2011). Preparation of HCV NS3 and NS5B proteins to support small-molecule drug discovery. Curr Protoc Pharmacol Chapter 13: Unit13B 16.




简介

表面等离子体共振(SPR)技术是一个成熟的平台,用于评估蛋白质 - 小分子相互作用的动力学参数。 下面,我们描述使用来自Bio-Rad(Hercules,CA)的ProteOn XPR36生物传感器来评价小分子抑制剂与重组NS5B蛋白的结合。 该构建体的高pI(> 9)允许使用pH7.5的HEPES缓冲盐水进行化学固定。 这与使用低pH和离子强度的传统生物传感器方案相反。 使用更多生理缓冲液来固定该酶导致改善的表面活性。

关键字:表面等离子体共振(SPR), 质子xpr36, 生物传感器, NS5B, 小分子抑制剂

材料和试剂

  1. HCV NS5B抑制剂
  2. 纯化的重组HCV NS5BΔC21可溶性蛋白[根据Boyce等人(2014)和Hung等人(2011)克隆和纯化]
  3. ProteOn GLH传感器芯片(Bio-Rad Laboratories,目录号:176-5013)
  4. HEPES溶液(1M)(Sigma-Aldrich,目录号:H3537-1L)
  5. MgCl 2(1M)(Sigma-Aldrich,目录号:M1028-100ML)
  6. EDTA(0.5M)(Sigma-Aldrich,目录号:E7889-100ML)
  7. NaCl(5M)(Sigma-Aldrich,目录号:S6546-1L)
  8. KCl(2M)(Life Technologies,Ambion ,目录号:AM9640G)
  9. 三(2-羧乙基)膦盐酸盐(TCEP)(Sigma-Aldrich,目录号:C4706-10G)
  10. 表面活性剂P20(10%v/v)(General Electric Company,目录号:BR-1000-54)
  11. DMSO(Sigma-Aldrich,目录号:472301-100ML)
  12. 含有1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDAC或EDC),磺基-N-羟基琥珀酰亚胺(Sulfo-NHS)和乙醇胺-HCl的蛋白质偶联试剂盒(Bio-Rad Laboratories,目录号:176-2410)
  13. 预处理试剂(参见配方)
  14. 固定缓冲液(参见配方)
  15. 运行缓冲区(参见配方)
  16. 用5%DMSO运行缓冲液(见配方)

设备

  1. ProteOn标准& 深孔微孔板(Bio-Rad Laboratories,目录号:176-6020和176-6023)
  2. Proteon微孔板密封膜(Bio-Rad Laboratories,目录号:176-6040)
  3. Nalgene快速过滤器和瓶顶过滤器,PES膜,无菌(VWR国际,目录号:16211-056)(1升尺寸)
  4. ProteOn XPR36仪器(Bio-Rad Laboratories)
  5. 微量离心管的BenchTop离心机
  6. BenchTop离心机旋转ProteOn 96孔测定板

软件

  1. Proteon管理软件版本3.1.0.6
  2. 用于ProteOn数据分析(BioLogic软件)的Scrubber软件

程序

  1. 用蒸馏水灌注系统。 使用100mM HCl,50mM NaOH,0.5%SDS和10%DMSO各1分钟脉冲,使用30μl/min的流速在水平和垂直方向上预处理GLH芯片。 密封所有与ProteOn密封膜一起使用的微孔板
  2. 使用运行缓冲区填充系统。使用制造商规定的EDC和sulfo-NHS在垂直方向上激活所有6个配体通道(L1-L6),流速为30μl/min,接触时间为5分钟。
  3. 在固定化缓冲液中稀释NS5B蛋白至浓度为30μg/ml,并以30μl/min的流速在垂直方向上注入配体通道5分钟,以获得5,000-10,000RU的固定化蛋白质(参见笔记)。将L1通道打开为参考通道。
  4. 当计算理论最大响应时,应考虑蛋白质的活性。 R sub max被计算为R sub(MW sub/MW sub)* n,其中R sub L sub等于固定的配体的量,MW sub是分析物的分子量,MW sub是配体的分子量,并且n是每个配体分子的结合位点的数目。假设单位点结合(即n = 1),具有500Da的平均分子量的小分子分析物与由10,000RU的NS5BΔC21(MW〜64,300Da)组成的表面相互作用应当产生约78RU(即10,000RU/500Da/64,300Da)的理论R submax或如果表面仅为50%活性的39RU。
  5. 通过以30μl/min的流速注射乙醇胺-HCl 5分钟,停用所有垂直通道(L1-L6)。
  6. 旋转多通道模块(MCM),因为所有后续进样都将在水平方向上进行。用含有5%DMSO的运行缓冲液填充系统,并使用该缓冲液注入缓冲液三次 有助于在旋转后稳定系统
  7. 为了说明排除的体积效应,注射4.5%和5.5%之间的一组五种溶液(DMSO浓度系列)用于软件以产生DMSO排除体积校准(EVC)曲线。通过向3,000μl含有5%DMSO的运行缓冲液中加入333μl运行缓冲液,向15μl含有5%DMSO的运行缓冲液和4.5%DMSO溶液中加入15μl100%DMSO制备5.5%DMSO溶液。根据下面所述的体积混合5.5%和4.5%DMSO溶液以获得五种不同的溶液。进行五次单独的注射以产生各自对应于一种DMSO溶液的注射的DMSO浓度系列。软件将使用这一系列进样来生成DMSO校准曲线,以校正排除的体积效应 5.5%DMSO缓冲液4.5%DMSO缓冲液 1200μl                     0微升
    900μl                      300微升
    600μl                      600μl
    300μl                      900μl
    0μl                          1200μl
  8. 当设计分析物注射序列时,在每次注射化合物之间包括缓冲液注射(或在96孔板中的空间允许)。
  9. 解冻化合物,通常储存在-20℃的微量离心管中的100%DMSO中,并在微量离心机中快速旋转它们。
  10. 在DMSO中连续稀释化合物,以达到所需最终测定浓度的20倍的一组五个浓度(在本实施例中为四倍稀释)。将稀释液转移到含有适当体积的运行缓冲液的微量培养板中的孔中,以产生5%的最终DMSO浓度(即,匹配运行缓冲液中的DMSO与DMSO的%)。这些将作为分析物A2-A6注入。向相同体积的运行缓冲液中加入100%DMSO至对应于注射分析物A1的孔,得到5%的最终DMSO浓度。从井A1的注入将作为空白并用于双参考数据。
  11. 使用快速15-30秒脉冲离心板,以确保收集粘在微孔板孔壁上的任何化合物溶液。
  12. 以100μl/min注射化合物,接触时间为90秒,可变解离时间为60秒至1小时(根据单个化合物的需要)。在没有关于化合物的亲和力的任何知识的情况下,可能必须进行涉及以单一浓度注射的初步实验,以便测量每种化合物的最佳解离时间。
  13. 数据可以使用ProteOn软件分析,但也可以使用为ProteOn数据设计的Scrubber软件版本进行分析。双重参考所有信号(使用L1和A1),并通过应用DMSO校准曲线校正排除的体积效应
  14. 所有测试浓度的传感图应当同时拟合,即全局。一般来说,数据应始终首先适合于产生平衡解离的简单的1:1动力学模型 常数(K D),表面的最大结合电容R submax(其可以不同于理论最大结合电容)和缔合的速率常数( k a )和解离( k d )。在简单的1:1动力学模型中,单个值 , k d < max 。 Scrubber和ProteOn拟合程序都解决了参数 k a 和 k d 。然后将K 计算为 k / k a 的比率。应明确说明使用其他需要多个值的更复杂模型。菲律宾与NS5BΔC21结合(见图1)是显示简单的1:1动力学的小分子结合的实例。


    图1. Filibuvir与NS5BΔC21的结合。 显示Filibuvir结合的传感图。在(从顶部到底部)4μM(橙色),1μM(粉红色),0.25μM(绿色),0.0625μM(蓝色)和0.0156μM(青色)的浓度下,将Filibuvir注射90秒的接触时间。监测解离180秒。数据的拟合以实线显示。

笔记

使用的生理固定缓冲液可以容易地将足够的NS5B酶预浓缩在羧基表面上。当选择在HEPES缓冲盐水中稀释蛋白质而不是标准低pH(5.5)10mM乙酸钠固定缓冲液时,NS5BΔC21的配体活性从45%增加至99%。

食谱

  1. 预处理试剂
    100 mM HCl
    50 mM NaOH
    0.5%SDS
    10%DMSO
  2. 固定缓冲液
    10mM HEPES(pH7.5) 150mM NaCl 过滤灭菌(0.45μm)
  3. 运行缓冲区
    50 mM HEPES
    5mM MgCl 2/
    10 mM KCl
    1mM EDTA
    1 mM TCEP
    0.01%P 2 O 用NaOH调节pH至7.5 将dH 2 O添加到所需的量
    过滤灭菌(0.45μm)
  4. 用5%DMSO运行缓冲液
    向运行缓冲液中加入100%DMSO至最终浓度为5%DMSO 例如,将50ml DMSO加入运行缓冲液至总体积为1L。

致谢

此协议的简要描述以前出现在Boyce等人(2014)。

参考文献

  1. Boyce,SE,Tirunagari,N.,Niedziela-Majka,A.,Perry,J.,Wong,M.,Kan,E.,Lagpacan,L.,Barauskas,O.,Hung,M.,Fenaux, ,Appleby,T.,Watkins,WJ,Schmitz,U.and Sakowicz,R。(2014)。 HCV NS5B聚合酶(β环和C末端尾)的结构和调节元件是 活性的变构拇指位点II抑制剂。 9(1):e84808。
  2. Hung,M.,Wang,R。和Liu,X。(2011)。 制备用于支持小分子药物发现的HCV NS3和NS5B蛋白。 Curr Protoc Pharmacol 第13章:Unit13B 16.




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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wong, M. and Papalia, G. A. (2014). A Surface Plasmon Resonance Method to Study HCV NS5B Inhibitors . Bio-protocol 4(4): e1044. DOI: 10.21769/BioProtoc.1044.
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