Name: identifying inhibitors of the hbx-ddb1 interaction using a split luciferase assay system
Uploaded: 2019-12-21T14:00:00
Description: Watch this Scientific Journal Video about Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System at JoVE.com

这项工作得到了教育部的助学金支持， 文化、体育、科学和技术，日本（#19H03430和#17K09405，#19J11829到K.S.），由创新领域科学研究资助（#18H05024到M.O.），由日本医学研究和发展厅肝炎研究计划，AMED（M.O.，#JP19fk021005），由创新开发和应用新药物乙型肝炎（#JP19fk0310102日本应用酶学基金会和小林癌症研究基金会（M.O.），GSK日本研究赠款2018年（K.S.），以及宫川纪念研究基金会（K.S.）的赠款。

注：图1A显示了裂解荧光素酶测定的示意图表示，如图1B所示。图1B概述了测定过程。 相互作用动力学可以实时测量，无需细胞分变。
1. 细胞制备
<ol><li>在Dulbeco的改性Eagle培养基（DMEM）中保持培养的HEK293T细胞，辅以10%v/v胎儿牛血清（FBS），1x青霉素/链霉素在37°C，在20%O2和5%CO2</sub...

<ol>
	<li>Tang, L. S. Y., Covert, E., Wilson, E., Kottilil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hepatitis+B+infection:+a+review.">Chronic hepatitis B infection: a review.</a> The Journal of the American Medical Association. 319, 1802-1813 (2018).</li><li>Sekiba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+pathogenesis:+Fresh+insights+into+hepatitis+B+virus+RNA.">Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA.</a> World Journal of Gastroenterology. 24, 2261-2268 (2018).</li><li>Slagle, B. L., Bouchard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+X+and+regulation+of+viral+gene+expression.">Hepatitis B virus X and regulation of viral gene expression.</a> Cold Spring Harbor Perspectives in Medicine. 6, a021402 (2016).</li><li>Murphy, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+Virus+X+Protein+Promotes+Degradation+of+SMC5/6+to+Enhance+HBV+Replication.">Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication.</a> Cell Reports. 16, 2846-2854 (2016).</li><li>Decorsi&#232;re, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+X+protein+identifies+the+Smc5/6+complex+as+a+host+restriction+factor.">Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.</a> Nature. 531, 386-389 (2016).</li><li>Niu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Smc5/6+complex+restricts+HBV+when+localized+to+ND10+without+inducing+an+innate+immune+response+and+is+counteracted+by+the+HBV+X+protein+shortly+after+infection.">The Smc5/6 complex restricts HBV when localized to ND10 without inducing an innate immune response and is counteracted by the HBV X protein shortly after infection.</a> PLoS One. 12, e0169648 (2017).</li><li>Sekiba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+HBV+Transcription+From+cccDNA+With+Nitazoxanide+by+Targeting+the+HBx-DDB1+Interaction.">Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx-DDB1 Interaction.</a> Cellular and Molecular Gastroenterology and Hepatology. 7, 297-312 (2019).</li><li>Eggers, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells.">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11, 400-408 (2015).</li><li>Zhang, J. H., Chung, T. D. Y., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> Journal of Biomolecular Screening. 4, 67-73 (1999).</li><li>Rao, V. S., Srinivas, K., Sujini, G. N., Kumar, G. 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我们开发了一种方便的筛选方法，使用裂光酶测定法来发现HBx-DDB1结合抑制剂。相互作用动力学可以在活细胞中实时检测，而无需细胞变热。抑制HBx-DDB1相互作用导致Smc5/6的恢复，导致抑制病毒转录，蛋白质表达和cccDNA生产7。这种新的抗病毒作用机制可以克服目前HBV疗法的不足。
虽然有许多方法可用于研究活细胞中的蛋白质-蛋白质相互作用，但检查这些相?...

乙型肝炎病毒（HBV）感染是全世界一个重大的公共卫生问题，每年估计有2.4亿人长期感染乙肝病毒，9万人死于感染并发症，包括肝硬化和肝细胞癌（HCC）1。虽然目前抗HBV治疗剂，核细胞（t）类比，足以抑制病毒逆转录，但它们很少实现病毒蛋白的消除，这是长期的临床目标。它们对病毒蛋白消除的不良影响是因为他们对肝细胞核2中表皮病毒共价闭合环DNA（cccDNA）小染色体的病毒转录缺乏直接影响。
HBV转录由HBV调节X（HBx）蛋白3激活。最近的研究表明，HBx通过劫持DDB1-CUL4-ROC1 E3泛素结合酶复合物4，5，6，降解染色体5/6（Smc5/6）的结构维持，这是阻止HBV转录从cccDNA的宿主限制因子。因此，促进来自cccDNA的病毒转录的关键步骤被认为是HBx-DDB1相互作用。能够抑制HBx和DDB1之间结合的化合物可以阻断病毒转录，事实上，硝基氧烷通过实验室7开发的筛选系统被确定为HBx-DDB1相互作用的抑制剂。
在这里，我们介绍我们方便的筛选系统，用于识别HBx-DDB1相互作用的抑制剂，它利用裂性荧光素酶补充测定7，8。裂化荧光素酶亚单位与HBx和DDB1融合，HBx-DDB1相互作用使亚单位接近，形成产生明亮发光信号的功能酶。由于亚单位之间的相互作用是可逆的，该系统可以检测迅速分离的HBx-DDB1蛋白（图1）。利用该系统，可以很容易地筛选大型化合物库，从而发现能够有效抑制HBx-DDB1相互作用的新型化合物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture microplate, 96 well, PS, F-BOTTOM</td>
			<td>Greiner-Bio-One GmbH</td>
			<td>655098</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6046</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Tocris Bioscience</td>
			<td>3176</td>
			<td></td>
		</tr>
		<tr>
			<td>Effectene transfection reagent</td>
			<td>Qiagen</td>
			<td>301425</td>
			<td>Includes DNA-condensation buffer, enhancer solution and transfection reagent</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Nichirei</td>
			<td>175012</td>
			<td></td>
		</tr>
		<tr>
			<td>GloMax 96 microplate luminometer</td>
			<td>Promega</td>
			<td>E6521</td>
			<td></td>
		</tr>
		<tr>
			<td>HBx&#8211;LgBit expressing DNA plasmid</td>
			<td>Our laboratory</td>
			<td></td>
			<td>Available upon request</td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBiT PPI starter systems</td>
			<td>Promega</td>
			<td>N2015</td>
			<td>Includes Nano-Glo Live Cell Reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11058021</td>
			<td>Described as &#34;buffered cell culture medium&#34; in the manuscript</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Takara</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Screen-Well FDA-approved drug library V2 version 1.0</td>
			<td>Enzo Life Sciences</td>
			<td>BML-2841</td>
			<td>Compounds used here were as follows: mequinol, mercaptopurine hydrate, mesna, mestranol, metaproterenol hemisulfate, metaraminol bitartrate, metaxalone, methacholine chloride, methazolamide, methenamine hippurate, methocarbamol, methotrexate, methoxsalen, methscopolamine bromide, methsuximide, methyclothiazide, methyl aminolevulinate&#183;HCl, methylergonovine maleate, metolazone, metyrapone, mexiletine&#183;HCl, micafungin, miconazole, midodrine&#183;HCl, miglitol, milnacipran&#183;HCl, mirtazapine, mitotane, moexipril&#183;HCl, mometasone furoate, mupirocin, nadolol, nafcillin&#183;Na, naftifine&#183;HCl, naratriptan&#183;HCl, natamycin, nebivolol&#183;HCl, nelarabine, nepafenac, nevirapine, niacin, nicotine, nilotinib, nilutamide, nitazoxanide, nitisinone, nitrofurantoin, nizatidine, nortriptyline&#183;HCl, olsalazine&#183;Na, orlistat, oxaprozin, oxtriphylline, oxybutynin Chloride, oxytetracycline&#183;HCl, paliperidone, palonosetron&#183;HCl, paromomycin sulfate, pazopanib&#183;HCl, pemetrexed disodium, pemirolast potassium, penicillamine, penicillin G potassium, pentamidine isethionate, pentostatin, perindopril erbumine, permethrin, perphenazine, phenelzine sulfate, phenylephrine, phytonadione, pimecrolimus, pitavastatin calcium, and podofilox</td>
		</tr>
		<tr>
			<td>SmBit&#8211;DDB1 expressing DNA plasmid</td>
			<td>Our laboratory</td>
			<td></td>
			<td>Available upon request</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying inhibitors of the hbx-ddb1 interaction using a split luciferase assay system

迫切需要新的治疗乙型肝炎病毒（HBV）感染的治疗方法。虽然目前可用的核细胞（t）ide类比能有效抑制病毒复制，但它们对从病毒共价闭环DNA（cccDNA）转录的病毒蛋白的表达没有直接影响。由于高病毒抗原负荷可能在这个慢性和HBV相关的致癌作用，HBV治疗的目标是根除病毒蛋白。HBV调节蛋白X（HBx）与宿主DNA损伤结合蛋白1（DDB1）蛋白结合，降解染色体5/6（Smc5/6）的结构维持，导致从cccDNA激活病毒转录。在这里，使用裂化荧光素酶补充测定系统，我们提出了一个全面的化合物筛选系统，以识别HBx-DDB1相互作用的抑制剂。我们的协议能够轻松检测活细胞内的相互作用动力学。这项技术可能成为发现治疗HBV感染的新治疗剂的关键测定方法。

使用该协议后的代表性结果如图2A，B所示。信背景比大于80，Z'因子9（高通量筛选的黄金标准质量指数）大于0.5，表明该检测系统对于高通量筛选是可以接受的。与对照组（仅限DMSO）相比，阈值设置为>40%抑制，我们确定硝基氧烷为候选药物7。使用这个系统，通过筛选其他更大的化合物库可以找到更好...

Watch this Scientific Journal Video about Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System at JoVE.com

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

在这里，我们提出了一种使用裂解荧光素酶检测系统来抑制HBx-DDB1相互作用的抗乙型肝炎病毒药物的方法。该系统易于检测蛋白质-蛋白质相互作用，适用于识别此类相互作用的抑制剂。

使用分体路西酶测定系统识别 HBx-DDB1 相互作用的抑制剂

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

HBX-DDB1相互作用是促进从ccDNA进行HPV转录的关键步骤，因此该协议可能成为发现HPV功能治疗新治疗剂的关键资产。我们的程序很简单，只需很短的时间进行筛选。我混合的相互作用可以实时检测，而无需细胞解解。此外，筛选质量令人满意，Z等分高。HBX-DDB1 相互作用的抑制导致 Smc5/6 的恢复，从而抑制 HPV 转录、蛋白质表达和 ccDNA 生产。这种新型的抗病毒作用机制可以克服目前HPV疗法的不足。蛋白质-蛋白质相互作用是药物靶点的重要类别。此处描述的基于分裂-荧光酶的酸，针对病毒和有S蛋白之间的相互作用，可能为开发其他传染病的治疗方法提供一种新的策略。尽管我们的协议简单易懂，但如果没有可视化演示，某些步骤可能很难重现。因此，可视化演示对理解我们的协议有很大的帮助。首先将5倍10至第5个 HEK293T 细胞播种到 100 毫米的培养皿中，并孵育在 37 摄氏度下过夜。第二天，稀释每微克的HBx-LgBit和SmBit-DDB1表达DNA质粒和DNA凝结缓冲液，总体积为300微升。加入16微升增强剂溶液，通过涡流混合管一秒钟。在室温下孵育样品三分钟，然后加入60微升转染试剂。旋转管10秒钟，再孵育样品8分钟。同时，用细胞从培养皿中吸出介质，用五毫升PVS洗涤。吸升PVS并添加7毫升DMEM。将三毫升DMEM加入导管，用转染复合物，上下移液器混合，并将混合物加入细胞中。然后，在37摄氏度和5%的二氧化碳下孵育细胞10小时。孵育后，取出已花的培养培养，用五毫升PVS清洗细胞。在0.25%的尝试素EDTA的一毫升下取出PVS，并在37摄氏度下孵育细胞以分离它们。接下来，加入四毫升DMEM，通过多次在细胞层表面移液来分散介质，并将悬浮液转移到管中。将细胞在500次G下离心5分钟，然后丢弃上最后一代，并在PVS的一毫升中重新悬浮细胞颗粒。重复四分法并丢弃上经剂。然后，用缓冲细胞培养培养剂重新悬浮细胞颗粒，辅以10%FBS，使每毫升100万个细胞的种子密度。将50微升的细胞悬浮液放入96井板的每个井中，将细胞返回37摄氏度的培养箱10小时。当细胞孵育时，将筛选化合物和溶剂稀释至13.5 X浓度。在每个井中加入12.5微升的发光基板，并在室温下孵育板5分钟。使用发光计测量基线亮度，并在后立即将化合物的五微升和受控的 DMSO 添加到每个井中。每30分钟测量一次发光两小时，然后计算与DMSO处理相比化合物的抑制作用。测量了基线发光信号，计算信号与背景比大于80。Z 等数值大于 0.5，表明此检测系统可接受高通量筛选。通过将阈值设置为大于 40% 的抑制比与仅 DMSO 控制相比，硝基多糖胺被确定为候选药物。我们之前通过用这种方法筛选反应性小规模化合物库，将硝氮氧化物确定为HBX-DDB1相互作用的抑制剂。使用硝基多糖酰胺，我们确认抑制HBX-DDB1相互作用，减少病毒转录，以及随后的病毒产品水平。必须事先确定分裂荧光素酶与目标蛋白质融合的最佳部分。在这种情况下，HPX 在 HPX 的 C 终点与大位融合，DDB1 的末元位将 DDB1 的 DDB1 融合到小位，提供了最佳结果。

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

科研

医学

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

利用经颅磁刺激，以研究人类的神经肌肉系统

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

术中的微妙的子宫内膜异位症的检测：A新型范式为相关联的与的的腹膜的完整性之亏损盆腔疼痛的的的的检测和治疗的

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

药品再利用产生假设使用“RE：精药品”系统

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

小鼠颞叶癫痫的 Radiotelemetry 模型及脑电图监测

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...