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
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?…
Divulgaciones
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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Citar este artículo
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).