Name: assessing cellular target engagement by shp2 (ptpn11) phosphatase inhibitors
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Description: Watch this Scientific Journal Video about Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors at JoVE.com

这项工作得到了国家卫生研究院赠款1R21CA195422（L.T.）、爱泼斯坦家庭基金会奖（至N.D.P.C.）和NCI癌症中心支持赠款P30CA030199的支持。此外，该项目由国家癌症研究所、国家卫生研究院的联邦资金提供，根据《化学生物学联合会合同第12号》提供。HHSN261200800001E。本出版物的内容不一定反映卫生与服务部的观点或政策，提及商品名称、商业产品或组织也不意味着得到美国政府的认可。内容完全由作者负责，不一定代表国家卫生研究院的官方观点。

1. 细胞培养和试剂的准备
<ol><li>配配 10% 胎儿牛血清、1x 抗生素/抗菌剂、20 mM HEPES 和 1 mM 丙酮酸钠的 500 mL 生长介质。储存在4°C。</li>
	<li>从冷冻原库存瓶中解冻蜂窝热移试剂（EA试剂、解液缓冲液和基材）。</li>
	<li>将试剂和缓冲液分配为2 mL等分，储存在-20°C。 注：避免冻结/解冻，以便可重复性，仅使用测定程序所需的试剂量。</li>
</ol>2. HEK293T电池的生长和维护
<ol><li>从...

<ol>
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我们提出了一个目标参与性测定，可以确认小分子与细胞中的SHP2磷酸酶的直接结合。该测定可以区分低亲和力抑制剂和高亲和力抑制剂，更重要的是，证实SHP099型的类体抑制剂对高致癌SHP2-E76K突变体缺乏效力。这种小型化测定的优势在于它能够集成到SHP2抑制剂筛选运动中。检测方法通过未知化学物质确认细胞内与SHP2结合的能力，是保证有效部署筛选资源的重要能力。总体而言，小型化协议是可?...

酪氨酸磷酸化在细胞1、2,的信号转导中起着重要的作用。这种转化后修饰由蛋白质酪氨酸激酶（PTKs）催化，由蛋白质酪氨酸磷酸盐（PTPs）逆转。因此，异常PTK或PTP功能导致许多遗传或后天人类疾病3，4，5，6。,5,63,Src-同源性2（SH2）包含磷酸酶2（SHP2）是一种广泛表达的非受体类型PTP编码的原生基因PTPN117，是许多生理过程的关键调节器，涉及信号转导通过激活Ras/Raf/ERK，PI3K/Akt，或JAK/STAT信号通路8。通常，SHP2 活动受到严格调节，以防止异常信令。在基础条件下，SHP2由其N端子SH2域自动抑制，该域阻止访问催化磷酸酶域内的活性位点（图1A）9，10。9,10Figure 1A细胞活化后，酪氨酸磷酸化结合蛋白会招募SHP2，使其采用活性构象，其中活性位点现在可进入其基质。在许多癌症中，SHP2活性升高。PTPN11中的体动增益（GOF）突变主要在白血病中得到识别，并防止N-SH2域与磷酸酶域结合，导致形成活性SHP2（图1B）11。11PTPN11 中的细菌性 GOF突变导致 50% 的努南综合征病例，这是一种发育障碍，恶性肿瘤风险增加 12。在PPN11突变很少的实体肿瘤中，磷酸化结合蛋白的含量更高，导致SHP2活性增强（图1C）。SHP2对于免疫检查点信号也很重要，因为BTLA或PD-1等检查点受体招募SHP2去磷化关键信号分子，防止免疫细胞激活13、14、15。13,14,15
以小分子为目标PPT一直是一个挑战，因为PTP的活性位点具有高度的保存和高充电性;针对活动位点的抑制剂通常有效，但表现出较差的选择性和口服生物利用度16，17，18，19，20，21，22。16,17,18,19,20,21,22事实上，许多报告的SHP2抑制剂在细胞23中缺乏选择性和缺乏疗效。最近，SHP2的异体抑制剂具有良好的效力和优异选择性的报告（例如，SHP09924和RMC-455025），并引发了人们对SHP2抑制剂的重新兴趣。基于SHP099和RMC-4550的化合物目前处于第一阶段临床试验中，用受体酪氨酸激酶（RTK）通路激活26，27治疗实体肿瘤。虽然具有开创性，这些化合物是无效的许多SHP2的致癌突变体，推动白血病发生在大量的血液癌患者28，29，30。28,29,30SHP099样化合物对SHP2致致致变异的缺乏效力源于其独特的等量性机制，因为它们通过结合和稳定SHP2突变体中被破坏的不活跃的闭合构象抑制SHP2活性。此外，根据最近的一份报告31，适应抵抗机制在治疗SHP099样抑制剂的患者是相当可想而知的。因此，开发针对其主动、开放状态的下一代SHP2抑制剂是一个激烈研究的主题。
新型SHP2抑制剂在细胞中的表征是铅优化过程的一个重要方面。关键的，经过验证的靶向作用，抑制剂在生理条件下提供一个额外的信心水平，药用化学资源被有效地部署在具有有希望的细胞疗效的化合物上。过去，已经开发出几种评估小分子抑制剂与靶点结合的方法，主要针对蛋白激酶32。为了开发SHP2细胞靶点接触性测定，我们利用细胞热移检测33。这种测定类似于蛋白质34的体外热移（PTS）测定，监测目标蛋白质的热稳定性，这种稳定性通常由小分子的结合改变。原始检测是一种低通量检测，利用抗体来量化目标蛋白水平。或者，我们选择了最近报道的热移位测定的变种，利用β-半乳糖酶片段补充（EFC）测定（图2）35 。35对于这些实验，感兴趣的蛋白质在细胞中表达为N-或C-终端融合蛋白，携带增强的ProLabel标签（ePL，β-半乳糖酶的42个氨基酸片段）。然后将细胞转移到384孔PCR兼容板，并孵育感兴趣的化合物。热循环器用于对细胞施加温度梯度，其蛋白质会随着温度的升高而变性和聚合。这些细胞的热稳定性取决于其温度的升高。候选化合物结合和稳定感兴趣的蛋白质的能力将导致该蛋白质的热稳定性增加。因此，在细胞分解后，那些由候选化合物稳定下来的标记蛋白将在高于通过车辆控制孵育的细胞标记蛋白的高温下留在溶液中。报告器酶接受器（EA）能够补充可溶性ePL标记蛋白，从而使用发光基β-半乳糖酶活性。
我们最近开发了一个强大的细胞热移测定野生型SHP2（SHP2-WT）和频繁的SHP2致癌变种（SHP2-E76K）在小型384井格式36。在这里，我们报告此检测的详细协议，该检测可可靠地检测SHP2抑制剂在细胞中的目标参与度，并证明抑制剂效力与细胞热移数据之间的高度相关性。一般检测工作流如图3 所示。我们的平台使用N-终端标记全长ePL-SHP2融合蛋白。有关生成相应的 pICP-ePL-N-SHP2-WT 和 pICP-ePL-N-SHP2-E76K 表达质粒，请参阅我们最近的出版物36。此测定可以使用热梯度进行，以建立 SHP2 热剖面，并确定存在或不存在抑制剂的 SHP2 熔化温度。一旦建立了热剖面，也可以在等温条件下进行，从而允许抑制剂剂量反应评估。下面描述了这两种类型的实验。

<table>
	<tbody>
		<tr>
			<td>384-well gradient equipped thermocycler</td>
			<td>Eppendorf AG</td>
			<td>X50h</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well low dead volume microplate Echo qualified</td>
			<td>Beckman Coulter, Inc.</td>
			<td>LP-0200</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>Greiner Bio-One</td>
			<td>657 160</td>
			<td>Sterile with lid</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (Anti Anti) 100 X</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>Countess II FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium 1X + GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>10566-016</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Echo acoustic liquid handler</td>
			<td>Beckman Coulter, Inc.</td>
			<td>Echo 550</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic multichannel pipette</td>
			<td>Thermo Fisher Scientific</td>
			<td>E1 ClipTip</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140-079</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-680</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>InCell Pulse starter kit</td>
			<td>Eurofins DiscoverX Corp.</td>
			<td>94-4007</td>
			<td>Components include EA buffer, lysis buffer, and substrate</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan Trading AG</td>
			<td>Spark</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel solution trough</td>
			<td>Thermo Fisher Scientific</td>
			<td>S253012005</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-010</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Thermal microplate sealer</td>
			<td>Agilent Technologies, Inc.</td>
			<td>PlateLoc</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagents</td>
			<td>Polyplus Transfection</td>
			<td>jetPRIME</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Twin.tec 384 real-time PCR plates</td>
			<td>Eppendorf AG</td>
			<td>30132734</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cellular target engagement by shp2 (ptpn11) phosphatase inhibitors

Src-同源性2（SH2）域包含磷酸酶2（SHP2）， 由PTPN11 原生基因编码，是受体酪氨酸激酶（RTK）驱动细胞信号的关键中介，促进细胞生存和增殖。此外，SHP2由免疫检查点受体招募，以抑制B和T细胞活化。异常SHP2功能已牵连到许多癌症的发展，进展和转移。事实上，小分子SHP2抑制剂最近已进入临床试验，用Ras/Raf/ERK通路活化治疗实体肿瘤，包括具有一些致癌Ras突变的肿瘤。然而，目前SHP2抑制剂的一类对白血病中频繁发生的SHP2致致致变异无效，而针对致癌SHP2的特定小分子的发育是当前研究的主题。大多数药物发现运动涉及细胞细胞酸蛋白（如 SHP2）的一个常见问题是，推动化学发现的主要检测方法通常是体外检测，不报告候选化合物的细胞靶度。为了提供一个测量细胞靶位参与度的平台，我们开发了野生型和突变型SHP2细胞热移测定。这些测定能够可靠地检测SHP2抑制剂在细胞中的目标接触。在这里，我们提供了该测定的综合方案，为SHP2抑制剂的评估和表征提供了宝贵的工具。

SHP2-WT的热梯度实验产生了一个西格莫德细胞热轮廓，其熔化过渡范围很窄，对于折叠蛋白来说，这是典型的和一致的（图4A）。SHP2 由三个独立的域组成：两个 SH2 域和催化域（图 1）。在自动抑制闭合构象这些域中，这些域是自关联的;在热剖面廓实验中观察到的熔化过渡大概反映了细胞中的这种状态。在10μM下与同性抑制剂SHP099的SHP2-WT的孵育，将SHP2-W...

Watch this Scientific Journal Video about Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors at JoVE.com

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

评估候选抑制剂在完整细胞中参与目标的能力对药物的发现至关重要。该协议描述了一个384井格式的细胞热移检测，可靠地检测针对野生型SHP2或其致癌变异的抑制剂的细胞靶接触。

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

酪氨酸磷酸酶SHP2是细胞信号的关键中介，促进细胞生存和增殖。我的团队专注于寻找用于治疗癌症的小分子SHP2抑制剂。我们的技术提供了一种快速而强大的方法来建立细胞的培养剂，以及小分子SHP2抑制剂在细胞中的目标接触。这就可以保证，发现资源是高效定向的。SHP2 是癌症治疗的诱人目标。和几个SHP2抑制剂，都在临床试验中。我们的技术，以促进发现下一代抑制剂与改进的疗效配置文件。研究助理塞莱斯特·罗梅罗和研究助理教授道格拉斯·谢夫勒将展示我们的实验室。使用三毫升细胞分离试剂，开始从板中分离 HEK293 T 细胞。用12毫升的生长介质稀释细胞，并在1，400倍G的离心中收集细胞4分钟。将细胞味觉重新在10毫升的生长介质中，用锥板蓝色和细胞计数器测量细胞的浓度和生存能力。板70万指数级生长细胞进入六井培养板的每个井，并在37摄氏度和5%的二氧化碳下孵育24小时。第二天，将两微克血浆DNA稀释成200微升的转染缓冲液。漩涡血浆DNA10秒，在1，400次G中，4分钟。在稀释的DNA中加入四微升转染试剂。旋转10秒钟，重复离心。在23摄氏度下孵育DNA10分钟，然后将转染混合物加入到6个井板中附着的 HEK293 T 细胞中，并将细胞再返回到培养箱24小时。为了准备检测板，在DMSO中稀释抑制剂溶液，其库存浓度为10毫摩尔，并分配到384孔低死量源板中，立即使用。在低于 0.5% DMSO 的目标最终体积下，使用液体处理器将所需体积的抑制剂或车辆放入 384 个实时 PCR 板中。最终体积小于 0.5%。使用带惰性气体净化的板密封器密封板。准备转染细胞孵育生长培养培养，并在37摄氏度的水浴中出售分离试剂。从培养箱中取出细胞，轻轻地从井中吸出培养物。将 0.3 毫升的细胞分离试剂添加到每个井中，然后来回轻轻摇动板，以彻底覆盖板底的表面。在23摄氏度下孵育板两分钟。在每个井中加入一毫升生长介质，轻轻移液井中的细胞。然后将它们转移到15毫升猎鹰离心管中离心机在1，400倍G的细胞4分钟收集细胞。轻轻吸气介质，然后将细胞颗粒重新在两毫升的生长介质中。使用 try 泛蓝色和单元格计数器确保细胞生存能力大于 90%。稀释细胞，每微升125个细胞的浓度，保持细胞悬浮不超过两个小时，以实现最佳生存能力。对于使用SHP2抑制剂进行孵育，将细胞分配到无菌单通道溶液槽中。将先前准备的 384 孔实时 PCR 板在 2，500 次 G 下离心 5 分钟，然后取出密封件并使用 125 微升多通道移液器，将稀释的 5 微升的电池添加到所需的孔中。在42倍G下离心30秒，不带盖子，然后盖上盖子，在37摄氏度和5%的二氧化碳下孵育板一小时。根据热轮廓梯度或等温实验的手稿方向对热循环器进行编程。启动热循环器热脉冲程序，将检测板放在热块上。程序完成后，拆下检测板。用五微升的许可检测主混合补充检测板的每个井。将板在42倍G下离心30秒，并在黑暗中储存在23摄氏度，30至60分钟。使用具有优化集成时间的微孔板读取器测量化学发光。野生型SHP2的热梯度实验，产生了一个西格莫德细胞热轮廓，其熔化过渡范围很窄，这是典型的折叠蛋白。野生SHP2型与同体抑制剂SHP099的孵育，稳定到热剖面到显著和可测量的程度。SHP2 的稳定性，也跟踪与 SHP099 的功效，如同量性化合物。在10微摩尔，更强大的RMC-4550，产生比SHP099野生类型SHP2更大的稳定程度。由于其独特的机制，SHP099像等体抑制剂对许多SHP2致癌突变体，包括SHP2-E76K效果较低。与这些已知特性一致，仅观察到SHP099的SHP2-E76K的边际热稳定性。EPL标签蛋白的表达水平可以显著影响信号强度。用于瞬态和稳定集成野生类型细胞的热剖面。在相同的资产条件下显示。不同曲线的规范化提供了可比较的热剖面，揭示了 EFC 检测系统的广泛范围。利用热循环器能力，在 384 井板的短轴上产生热梯度。可以在单个板上执行一系列等温滴定。在最佳温度下，使用全 10 点剂量响应的等温滴定产生一个有用的 EC50，用于 RMC-4550。这种资产的原理可用于监测由PROTAC化合物诱导的细胞中蛋白质的降解。我们使用细胞塞尔玛移位技术来发现突变SHP2和白血病的抑制剂，针对频繁SHP2致致突变体的药物将对这些癌症的治疗产生重大影响。

Research

JoVE Journal

Cancer Research

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

科研

癌症研究

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

用抗体阵列对信号转导通路的审问对酪氨酸激酶抑制剂的抗性评价

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

脱细胞和细胞肺模型研究肿瘤转移

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

A Spheroid Killing Assay by CAR T Cells

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

利用微阵列研究微环境对癌症细胞表型的影响

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

通过 SHP2 （PTPN11）磷酸酶抑制剂评估细胞目标参与度