Name: a semi-high-throughput adaptation of the nadh-coupled atpase assay for screening small molecule inhibitors
Uploaded: 2019-08-17T14:00:00
Description: Watch this Scientific Journal Video about A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors at JoVE.com

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。

1. 制备库存溶液和试剂
<ol><li>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。</li>
	<li>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。</li>
	<li>制备含有70mM 3-（N-变形）丙烷酸（MOPS）、10mM MgCl 2、0.9mM乙...

<ol>
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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?...

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。

<table border="0" cellpadding="0" cellspacing="0" style="width:956px;" width="955">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">384-well Low Flange Black Flat Bottom Polystyrene NBS Microplate</td>
			<td style="width:147px;">Corning</td>
			<td style="width:123px;">3575</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">ATP (Adenosine 5&#8242;-triphosphate disodium salt hydrate)</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Aurora FRD-IB Dispenser</td>
			<td>Aurora Discovery, Inc.</td>
			<td>00017425</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Biomek NXP Multichannel Laboratory Automation Workstation</td>
			<td>Beckman Coulter</td>
			<td>A31841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blebbistatin</td>
			<td style="width:147px;">AMRI</td>
			<td style="width:123px;">N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA (Bovine Serum Albumin, Protease-Free)</td>
			<td>Akron Biotech</td>
			<td>AK1391&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Centrifuge 5430 R, refrigerated, with Rotor FA-35-6-30</td>
			<td>Eppendorf</td>
			<td>022620663</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Centrifuge 5430, non-refrigerated, with Rotor A-2-MTP</td>
			<td>Eppendorf</td>
			<td>022620568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO (Dimethyl sulfoxide)</td>
			<td>&#160;Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">DTT (DL-Dithiothreitol)</td>
			<td>&#160;Sigma</td>
			<td>D5545</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">E1 ClipTip Multichannel Pipette; 384-format; 8-channel</td>
			<td>Thermo Scientific</td>
			<td>4672010</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">E1 ClipTip Multichannel Pipette; 96-format; 8-channel</td>
			<td>Thermo Scientific</td>
			<td>4672080</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid)</td>
			<td>&#160;Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">EnVision 2104 Multilabel Plate Reader</td>
			<td>PerkinElmer</td>
			<td>2104-0010</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Glycerol</td>
			<td>&#160;Sigma</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">LDH (L-Lactic Dehydrogenase from rabbit muscle)</td>
			<td>Sigma</td>
			<td>L1254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2.6H2O (Magnesium chloride hexahydrate)</td>
			<td>&#160;Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Microplate Shaker</td>
			<td>VWR</td>
			<td>&#160; 12620-926&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate, 384 well, PP, Small Volume, Deep Well, Natural</td>
			<td>Greiner Bio-One</td>
			<td>784201</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">MOPS (3-(N-Morpholino)propanesulfonic acid)</td>
			<td>&#160;Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Myosin Motor Protein (full length) (Bovine cardiac muscle)</td>
			<td>Cytoskeleton&#160;</td>
			<td>MY03</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Myosin Motor Protein (full length) (Rabbit skeletal muscle)</td>
			<td>Cytoskeleton&#160;</td>
			<td>MY02</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">NADH (&#946;-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate)</td>
			<td>Sigma</td>
			<td>N8129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaN3 (Sodium azide)</td>
			<td>&#160;Sigma</td>
			<td>71289</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">NaOH (Sodium hydroxide)</td>
			<td>&#160;Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Optical Filter CFP 470/24nm (Emission)</td>
			<td>PerkinElmer</td>
			<td>2100-5850</td>
			<td>Barcode 240</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Optical Filter Fura2 380/10nm (Excitation)</td>
			<td>PerkinElmer</td>
			<td>2100-5390</td>
			<td>Barcode 112</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Optical Module: Beta Lactamase</td>
			<td>PerkinElmer</td>
			<td>2100-4270</td>
			<td>Barcode 418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OriginPro 2017 software</td>
			<td>OriginLab</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">para-Aminoblebbistatin</td>
			<td style="width:147px;">AMRI</td>
			<td style="width:123px;">N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">para-Nitroblebbistatin</td>
			<td style="width:147px;">AMRI</td>
			<td style="width:123px;">N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">PEP (Phospho(enol)pyruvic acid monopotassium salt)</td>
			<td>Sigma</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">PK (Pyruvate Kinase from rabbit muscle)</td>
			<td>Sigma</td>
			<td>P9136</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Rabbit Muscle Acetone Powder</td>
			<td>Pel Freez Biologicals</td>
			<td>41995-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

a semi-high-throughput adaptation of the nadh-coupled atpase assay for screening small molecule inhibitors

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）。

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...

Watch this Scientific Journal Video about A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors at JoVE.com

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

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

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。

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

该协议适用于旨在开发 ATPase 抑制剂的广泛筛选项目，无论是作为基础研究的探针，还是作为潜在临床化合物的探针。此方法已针对半高通量筛选应用进行了优化。它还被设计为避免一些常见的伪影，它不需要处理危险材料。在我们的实验室中，我们一直在使用这种检测来开发新的和特定的非肌肉肌素II抑制剂，用于治疗甲基安非他明使用障碍。从理论上讲，这种方法可以很容易地应用于任何产生ATP的酶。可视化演示有助于澄清所有重要的技术细节。首先，通过稀释行动素缓冲液中的浓缩蛋白库存溶液，为每384孔黑色聚苯乙烯微孔板准备4，500微升20微摩尔稀释的蛋白溶液。通过上下移液 30 次彻底混合溶液，以打破行为素丝，降低粘度和异质性。然后离心溶液，去除任何沉淀的蛋白质。小心地将上清液转移到干净的离心管中。将3，252.9微升肌素缓冲液组合到每个检测板的15毫升锥形离心管中，为涉及骨骼肌素II、171.4微升的LDH溶液和171.4微升的PK溶液进行测定，以准备主酶混合物。将 162.1 微升 ATP、162.1 微升 PEP 和 324.1 微升 NADH 溶液组合在 15 毫升锥形离心管中，为每个板准备主基板混合。此时不要添加 actin 以避免聚合和沉淀。要创建七步串行一到两个 NADH 稀释校准，请准备 8 个 1.5 毫升微离心管。将 12.3 微升的 NADH 库存溶液与 257.7 微升的明辛缓冲液混合在第一管中，以制作 250 微摩尔溶液。然后将135微升的米奥辛缓冲液放入剩余的7个管子中。将溶液的135微升从第一管转移到第二管中，然后通过移液混合。重复，直到到达第七管。使用最后一根管子作为无 NADH 控制，仅带缓冲液。始终在复合板上包括正负控制，以及用于检测板上校准的 NADH 稀释。这些控件对于在数据分析期间识别工件非常重要。使用八通道移液器，将 20 微升的 NADH 校准解决方案转移到检测板中三分的第一排。现在添加4.2微升的骨骼肌肌素II到酶混合物。漩涡简单。在配药前将明西辛加入酶混合物中。稀释的明蛋白溶液的长期孵育可能导致因降水而丧失活性，并且与塑料表面有非特异性结合。将制备的明素酶混合物装入自动分配器的样品容器之一。在板中，除第一排外，将8.4微升的明辛酶混合到检测板的每个井中。然后将检测板和复合板放在配备 100 纳米引脚工具头的自动液体处理系统的实验室设备定位器上。然后在软件中运行适当的方法，将100纳米的溶液从准备好的复合板转移到测定板。接下来，将检测板放在微孔板摇床上，在 1，200 rpm 的室温下摇动一分钟。将离心剂溶液的4，052微升加入基板混合和涡流中。为了开始酶反应，使用自动分配器将11.6微升的物基板混合到检测板的每个井中，第一排除外。在微孔板摇床上，在室温下以 1，200 rpm 摇动检测板一分钟。以101倍g离心检测板，30秒。使用 380 纳米、10 纳米带宽度激励滤波器和 470 纳米、24 纳米带宽度发射滤波器以及 425 纳米切断二色镜进行测定。在运行检测之前，优化闪烁数量、检测器增益、板尺寸和测量高度。在高浓度模式下运行测量。确保板读卡器的内部温度稳定在 25 摄氏度。将板装入板读卡器并摇动 30 秒，使每个井中的液体表面形状类似，并允许板达到测量温度。以 45 秒的间隔扫描板，并记录 NADH 荧光 30 分钟。使用导出的数据，绘制每个井的观测荧光强度与时间的关系。执行简单的线性回归，以确定每一井的荧光响应的斜率和截距。斜率与 NADH 消耗率成正比，这可用作 ATP 消耗率的非常好的近似值。截距与测量开始时的 NADH 浓度成正比。然后，根据 NADH 的浓度绘制板第一行获得的截距，为 NADH 构建校准曲线。截距估计真正的荧光强度在开始，比原始荧光强度读取在开始的平均值更有信心。执行简单的线性回归以获取 NADH 校准线的斜率和截距。在这里，截距描述了没有 NADH 存在的荧光背景信号，而斜率对应于一个摩尔 NADH 溶液的外推理论荧光强度。要将荧光变化转换为 ATP 消耗率，请将 NADH 校准线的斜率除以其余孔的荧光响应的斜率。接下来，根据抑制剂的浓度绘制 ATP 消耗率。要确定抑制常数，请使用任何能够非线性曲线拟合的适当统计软件，如 Origin 2017。选择非线性曲线拟合，使剂量-响应数据与对应于简单一对一结合均衡模型的二次方程保持一对一。Y 是 ATP 消耗率。Y-最小值是无抑制剂的ATP消费率。Y-最大值是理论ATP消费率在100%抑制。t 和 t 分别是明蛋白酶和抑制剂的总浓度。KI 是抑制常数。在这项研究中，骨骼和心肌肌素II ATPase反应显示线性荧光强度痕迹，无论使用的肌素或抑制剂的存在。基础和活性 ATPase 率显示线性依赖骨骼或心肌肌素 II 的浓度。筛查窗口系数或 Z 因子 0.78 表示可靠的检测，具有非常分离的阳性和负对。布莱比斯塔汀、准氨基苯甲酸素和准硝基比斯塔汀在报告溶解度值或低于其报告的溶解度值时显示定期剂量反应曲线。心脏和骨骼肌的抑制常数通过拟合一个表示简单平衡结合模型的数据的二次方程来确定。使用骨骼肌素II在 ATPase 测定中获得的 blebbistatin 的荧光强度轨迹显示正常线性递减信号，具体取决于存在抑制剂的数量。然而，在 blebistatin 的溶解性之上，观察到信号的意外增加很可能是由于形成明亮的荧光 blebistatin 晶体。在具有正常原荧光强度痕迹的准硝基硝基丁的情况下，由于降水，获得上述溶解度的反应速率与确定的剂量-反应曲线不同。通过使用这种方法，我们开发了新的，有效的，选择性的明蛋白抑制剂，可以用作科学研究的科学工具或作为未来的直接候选者。始终建议使用特定于感兴趣的酶的其他 ATPase 或其他功能检测来区分真阳性和假阳性命中。所有蛋白质都结合塑料表面。此问题可以严重影响结果，特别是当蛋白质存在于低浓度。始终避免不必要的液体处理，并使用非结合管。替代 ATPase 检测通常需要处理硫酸、有毒物质或放射性物质。相比之下，NADH相关 ATPase 检测仅需要无毒性或非常低毒性的试剂。

Research

JoVE Journal

Biochemistry

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

科研

生物化学

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

热通菌膜-双磷酶抑制剂的筛选

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

细胞性谷胱甘肽S-转移酶潜在抑制剂的分光光度筛选

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

开发用于快速定量检测小分子化合物的横向流动免疫色谱带

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...