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Biochimica

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

Published: August 17, 2019
doi:
10.3791/60017
, , , ,
1Department of Molecular Medicine,The Scripps Research Institute, 2Department of Neuroscience,The Scripps Research Institute, 3Laboratory of Molecular Physiology, NHLBI,National Institutes of Health

Summary

一种烟酰胺腺苷二核苷酸（NADH）与ATPase的检测已应用于小分子肌苷抑制剂的半高通量筛选。此动力学测定以 384 孔微孔板格式运行，总反应体积仅为每孔 20 μL。该平台应适用于几乎任何ADP生产酶。

Abstract

ATPase酶利用三磷酸腺苷中储存的游利用能量，催化体内各种不会自发发生的内发性生化过程。这些蛋白质对细胞生活的各个方面都至关重要，包括新陈代谢、细胞分裂、对环境变化的反应和运动。这里提出的协议描述了一种烟酰胺腺苷二核苷酸（NADH）耦合ATPase测定，该检测已适用于小分子ATPase抑制剂的半高通量筛选。该测定已应用于心脏和骨骼肌肌苷II，两个基于行为蛋白的分子运动ATPases，作为原理证明。ATP的水解通过测定中的酶反应与NADH的氧化结合。首先，由 ATPase 生成的 ADP 通过丙酮激酶 （PK） 再生为 ATP。PK催化磷酸二苯丙酸酯（PEP）与丙酮类酯的平行过渡。随后，乳酸通过乳酸脱氢酶（LDH）降低为乳酸盐，同时催化NADH的氧化。因此，ATP浓度的下降与NADH浓度的下降直接相关，随后是NADH内在荧光的变化。只要在反应系统中有PEP，ADP浓度仍然很低，避免由其自己的产品抑制ATPase酶。此外，ATP 浓度几乎保持不变，产生线性时间课程。荧光被持续监测，从而便于估计数据的质量，并有助于过滤出潜在的伪影（例如，由复合沉淀或热变化引起的）。

Introduction

肌苷是水解三磷酸腺苷（ATP）的机械化学能量传感器，用于在真核细胞1、2中沿活性素细胞骨架的丝状物产生定向运动。它们在结构和动力学上都适应了细胞内的各种功能，如细胞器的传输、肌肉收缩或细胞骨骼张力的产生1，2。肌苷超级家族由属于人类基因组3、4中12个不同肌苷类的+40肌苷基因表示。肌苷类的成员在一系列高度多样化的疾病中扮演着不同的角色，如几种癌症、神经系统疾病、骨骼肌病和肥大性心肌病5、6。鉴于这些分子马达的生理和病理功能很多，它们越来越被公认为各种条件的药物靶点，这并不奇怪。最近，在发现新的肌苷抑制剂8、9、10和活化剂11方面取得了重大进展，并改善了现有抑制剂12的特性。13,14,15.

烟酰胺腺苷二核苷酸（NADH）耦合ATPase测定早已用于测量各种酶的ATPase活性，如肉质性视网膜Ca2+泵ATPase 16，DNA修复ATPase Rad5417，AAA+ATPase p9718或微管马达运动体19。测定采用 ATP 再生循环。ATPase产生的二磷酸腺苷（ADP）通过丙酮酸激酶（PK）再生为ATP，将一个磷酸二醇酸（PEP）分子并联转化为丙酮酸盐。随后，通过乳酸脱氢酶（LDH）将丙酮酸酯降低为乳酸盐。这反过来又氧化了NADH的一个分子到NAD。因此，NADH浓度作为时间的函数的降低等于ATP水解率。只要 PEP 可用，ATP 再生循环就使 ATP 浓度几乎保持不变，ADP 浓度处于低水平。这导致线性时间课程，使得确定初始反应速率变得简单，并有助于避免ADP19对产品抑制。虽然NADH耦合ATPase测定已经适应了96井格式20，但高反应量（±150 μL）由于试剂需求较高，使得它不太适合快速筛选大量化合物。替代方法，如麦芽绿测定19，21，它依赖于检测由ATPase酶产生的磷酸盐，被证明更适合小型化和高通量筛选22,23,24.然而，端点测定更有可能受到几个工件的影响（下文讨论），如果没有全日制课程，这些伪像可能仍未被发现。

在这里，NADH耦合ATPase测定已针对小分子抑制剂的半高通量筛选进行了优化。骨骼和心肌肌苷II和肌苷抑制剂blebbisatin8，副阿米诺巴他丁13和准硝基布利他妥丁12用于证明测定的力量，它依赖于NADH荧光作为读出。该协议适用于筛选专注于任何ADP生产酶的项目。

Protocol

1. 制备库存溶液和试剂 通过在蒸馏水中溶解结晶DTT至最终浓度为1000 mM，制备二硫二硫醇（DTT）库存溶液。使用 1 M NaOH 溶液将 pHH 调整为 7.0。在-20°C下，以-20°C的分量和储存。 通过将蒸馏水中的结晶ATP溶解至100 mM的最终浓度，制备ATP库存溶液。使用 1 M NaOH 溶液将 pHH 调整为 7.0。在-20°C下，以-20°C的分量和储存。 制备含有70mM 3-（N-变形）丙烷酸（MOPS）、10mM MgCl 2、0.9mM乙…

Representative Results

用于筛选实验的典型板布局图如图1所示。第一行和最后一行分别保留用于NADH校准和正控制（20μM准阿米诺比沙丁，0.5%DMSO）。其余行（B到O）用于测试化合物的抑制活性。在这里，从DMSO中的10 mM化合物浓度开始的15步序1：2稀释被制备并从复合板转移到测定板，使测定板上的最高最终化合物浓度为50μM（以0.5%DMSO为单位）。两行用于获取一种化合…

Discussion

协议中的关键步骤

通过运行多个仅带负控制（无抑制剂的 ATPase 反应）的板，优化板材布局。仔细检查结果，查看反应速率的模式。例如，这些可能由”非结合”板的亲水表面涂层的边缘效应和/或缺陷引起。如果观察到图案，请更改板类型和/或板布局，以尽量减少伪影。例如，典型的剂量反应曲线（带三重的 16 浓度，总计 48 点）可以排列在 384 孔板上的三列或两?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家神经疾病和中风研究所和国家药物滥用研究所NS096833（CAM）的资助。

Materials

384-well Low Flange Black Flat Bottom Polystyrene NBS Microplate Corning 3575
ATP (Adenosine 5′-triphosphate disodium salt hydrate) Sigma A7699
Aurora FRD-IB Dispenser Aurora Discovery, Inc. 00017425
Biomek NXP Multichannel Laboratory Automation Workstation Beckman Coulter A31841
Blebbistatin AMRI N/A Custom synthesis
BSA (Bovine Serum Albumin, Protease-Free) Akron Biotech AK1391 
Centrifuge 5430 R, refrigerated, with Rotor FA-35-6-30 Eppendorf 022620663
Centrifuge 5430, non-refrigerated, with Rotor A-2-MTP Eppendorf 022620568
DMSO (Dimethyl sulfoxide)  Sigma D2650
DTT (DL-Dithiothreitol)  Sigma D5545
E1 ClipTip Multichannel Pipette; 384-format; 8-channel Thermo Scientific 4672010
E1 ClipTip Multichannel Pipette; 96-format; 8-channel Thermo Scientific 4672080
EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid)  Sigma E3889
EnVision 2104 Multilabel Plate Reader PerkinElmer 2104-0010
Glycerol  Sigma G2025
LDH (L-Lactic Dehydrogenase from rabbit muscle) Sigma L1254
MgCl2.6H2O (Magnesium chloride hexahydrate)  Sigma M2670
Microplate Shaker VWR   12620-926 
Microplate, 384 well, PP, Small Volume, Deep Well, Natural Greiner Bio-One 784201
MOPS (3-(N-Morpholino)propanesulfonic acid)  Sigma M1254
Myosin Motor Protein (full length) (Bovine cardiac muscle) Cytoskeleton  MY03
Myosin Motor Protein (full length) (Rabbit skeletal muscle) Cytoskeleton  MY02
NADH (β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate) Sigma N8129
NaN3 (Sodium azide)  Sigma 71289
NaOH (Sodium hydroxide)  Sigma S8045
Optical Filter CFP 470/24nm (Emission) PerkinElmer 2100-5850 Barcode 240
Optical Filter Fura2 380/10nm (Excitation) PerkinElmer 2100-5390 Barcode 112
Optical Module: Beta Lactamase PerkinElmer 2100-4270 Barcode 418
OriginPro 2017 software OriginLab N/A
para-Aminoblebbistatin AMRI N/A Custom synthesis
para-Nitroblebbistatin AMRI N/A Custom synthesis
PEP (Phospho(enol)pyruvic acid monopotassium salt) Sigma P7127
PK (Pyruvate Kinase from rabbit muscle) Sigma P9136
Rabbit Muscle Acetone Powder Pel Freez Biologicals 41995-2
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Riferimenti

  1. Heissler, S. M., Sellers, J. R. Kinetic Adaptations of Myosins for Their Diverse Cellular Functions. Traffic. 17 (8), 839-859 (2016).
  2. Hartman, M. A., Spudich, J. A. The myosin superfamily at a glance. Journal of Cell Science. 125 (Pt 7), 1627-1632 (2012).
  3. Berg, J. S., Powell, B. C., Cheney, R. E. A millennial myosin census. Molecular Biology of the Cell. 12 (4), 780-794 (2001).
  4. Sebe-Pedros, A., Grau-Bove, X., Richards, T. A., Ruiz-Trillo, I. Evolution and classification of myosins, a paneukaryotic whole-genome approach. Genome Biology and Evolution. 6 (2), 290-305 (2014).
  5. Newell-Litwa, K. A., Horwitz, R., Lamers, M. L. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities. Disease Models & Mechanisms. 8 (12), 1495-1515 (2015).
  6. He, Y. M., Gu, M. M. Research progress of myosin heavy chain genes in human genetic diseases. Yi Chuan. 39 (10), 877-887 (2017).
  7. Rauscher, A. A., Gyimesi, M., Kovacs, M., Malnasi-Csizmadia, A. Targeting Myosin by Blebbistatin Derivatives: Optimization and Pharmacological Potential. Trends in Biochemical Sciences. 43 (9), 700-713 (2018).
  8. Straight, A. F., et al. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science. 299 (5613), 1743-1747 (2003).
  9. Sirigu, S., et al. Highly selective inhibition of myosin motors provides the basis of potential therapeutic application. Proceedings of the National Academy of Sciences of the United States of America. 113 (47), E7448-E7455 (2016).
  10. Green, E. M., et al. A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice. Science. 351 (6273), 617-621 (2016).
  11. Morgan, B. P., et al. Discovery of omecamtiv mecarbil the first, selective, small molecule activator of cardiac Myosin. ACS Medicinal Chemistry Letters. 1 (9), 472-477 (2010).
  12. Kepiro, M., et al. para-Nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor. Angewandte Chemie International Edition. 53 (31), 8211-8215 (2014).
  13. Varkuti, B. H., et al. A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative. Scientific Reports. 6, 26141 (2016).
  14. Verhasselt, S., et al. Discovery of (S)-3′-hydroxyblebbistatin and (S)-3′-aminoblebbistatin: polar myosin II inhibitors with superior research tool properties. Organic and Biomolecular Chemistry. 15 (9), 2104-2118 (2017).
  15. Verhasselt, S., Roman, B. I., Bracke, M. E., Stevens, C. V. Improved synthesis and comparative analysis of the tool properties of new and existing D-ring modified (S)-blebbistatin analogs. European Journal of Medicinal Chemistry. 136, 85-103 (2017).
  16. Warren, G. B., Toon, P. A., Birdsall, N. J., Lee, A. G., Metcalfe, J. C. Reconstitution of a calcium pump using defined membrane components. Proceedings of the National Academy of Sciences of the United States of America. 71 (3), 622-626 (1974).
  17. Kiianitsa, K., Solinger, J. A., Heyer, W. D. Rad54 protein exerts diverse modes of ATPase activity on duplex DNA partially and fully covered with Rad51 protein. Journal of Biological Chemistry. 277 (48), 46205-46215 (2002).
  18. Hanzelmann, P., Schindelin, H. Structural Basis of ATP Hydrolysis and Intersubunit Signaling in the AAA+ ATPase p97. Structure. 24 (1), 127-139 (2016).
  19. Hackney, D. D., Jiang, W. Assays for kinesin microtubule-stimulated ATPase activity. Methods in Molecular Biology. 164, 65-71 (2001).
  20. Kiianitsa, K., Solinger, J. A., Heyer, W. D. NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Analytical Biochemistry. 321 (2), 266-271 (2003).
  21. Carter, S. G., Karl, D. W. Inorganic phosphate assay with malachite green: an improvement and evaluation. Journal of Biochemical and Biophysical Methods. 7 (1), 7-13 (1982).
  22. Henkel, R. D., VandeBerg, J. L., Walsh, R. A. A microassay for ATPase. Analytical Biochemistry. 169 (2), 312-318 (1988).
  23. Rowlands, M. G., et al. High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Analytical Biochemistry. 327 (2), 176-183 (2004).
  24. Rule, C. S., Patrick, M., Sandkvist, M. Measuring In Vitro ATPase Activity for Enzymatic Characterization. Journal of Visualized Experiments. (114), 54305 (2016).
  25. Pardee, J. D., Spudich, J. A. Purification of muscle actin. Methods in Cell Biology. 24, 271-289 (1982).
  26. Zhang, J. H., Chung, T. D., Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of Biomolecular Screening. 4 (2), 67-73 (1999).
  27. Kovacs, M., Toth, J., Hetenyi, C., Malnasi-Csizmadia, A., Sellers, J. R. Mechanism of blebbistatin inhibition of myosin II. Chem Journal of Biological Chemistry. 279 (34), 35557-35563 (2004).
  28. Allingham, J. S., Smith, R., Rayment, I. The structural basis of blebbistatin inhibition and specificity for myosin II. Nature Structural & Molecular Biology. 12 (4), 378-379 (2005).
  29. Kettlun, A. M., et al. Purification and Characterization of 2 Isoapyrases from Solanum-Tuberosum Var Ultimus. Phytochemistry. 31 (11), 3691-3696 (1992).
  30. Hulme, E. C., Trevethick, M. A. Ligand binding assays at equilibrium: validation and interpretation. British Journal of Pharmacology. 161 (6), 1219-1237 (2010).
  31. Motulsky, H. J., Neubig, R. R. Analyzing binding data. Current Protocols in Neuroscience. 52 (1), 7.5.1-7.5.65 (2010).
  32. Sehgal, P., Olesen, C., Moller, J. V. ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay. Methods in Molecular Biology. 1377, 105-109 (2016).
  33. Solinger, J. A., Lutz, G., Sugiyama, T., Kowalczykowski, S. C., Heyer, W. D. Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament. Journal of Molecular Biology. 307 (5), 1207-1221 (2001).
  34. Banik, U., Roy, S. A continuous fluorimetric assay for ATPase activity. Biochemistry Journal. 266 (2), 611-614 (1990).
  35. Xiao, Y. X., Yang, W. X. KIFC1: a promising chemotherapy target for cancer treatment?. Oncotarget. 7 (30), 48656-48670 (2016).
  36. See, S. K., et al. Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity. ACS Chemical Biology. 11 (1), 53-60 (2016).
  37. Datta, A., Brosh, R. M. New Insights Into DNA Helicases as Druggable Targets for Cancer Therapy. Frontiers in Molecular Biosciences. 5, 59 (2018).

Tags

Keywords: ATPase AssayNADH-coupledSemi-high-throughput ScreeningActinMyosin IIEnzyme InhibitorsCalibrationPositive And Negative Controls
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Radnai, L., Stremel, R. F., Sellers, J. R., Rumbaugh, G., Miller, C. A. A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors. J. Vis. Exp. (150), e60017, doi:10.3791/60017 (2019).
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