Activation and Regulation of the Understudied Kinome Using MIB/MS Technology
Activation and Regulation of the Understudied Kinome Using MIB/MS Technology
批准号:
9120936
负责人:
GARY L. JOHNSON
金额:
$39.51万
依托单位国家:
美国
项目类别:
财政年份:
2014
资助国家:
美国
项目状态:
已结题
起止时间:
2014-08-01 至 2018-04-30
关键词:
AddressAffinityAntibodiesBindingBiologicalBiological AssayBiologyBone MarrowBrainBreast Cancer geneCell LineCell physiologyCellsChemicalsChromatinClinicalCoupledCytoskeletonDDR1 geneDDR2 geneDNA DamageDevelopmentDiseaseERBB2 geneEnzymesEpigenetic ProcessFutureGene FusionGenetic ScreeningGlioblastomaGoalsHealthHeartHistocompatibility TestingHumanHuman Cell LineInformaticsKidneyKnowledgeLipidsLiverLungMAP2K1 geneMAPK1 geneMAPK3 geneMalignant neoplasm of lungMalignant neoplasm of pancreasMass Spectrum AnalysisMeasurementMeasuresMetabolicMethodsMusMutationNodalNormal tissue morphologyPancreasPatientsPharmaceutical PreparationsPhosphorylationPhosphotransferasesPrincipal Component AnalysisProtein IsoformsProtein KinaseProtein Kinase InhibitorsProteinsPublished CommentRNA InterferenceRNA interference screenReagentReceptor Protein-Tyrosine KinasesRegulationRenal Cell CarcinomaResearchRunningSepharoseSeriesSignal TransductionSystemTechnologyTissuesTumor Cell LineTumor TissueXenograft procedurebasebcr-abl Fusion Proteinscancer typedark matterdrug discoveryhigh throughput technologyhuman diseasein vivoinhibitor/antagonistkinase inhibitorknock-downleukemiamelanomanovel strategiesoverexpressionpreferenceprotein kinase inhibitorresponsetranscriptome sequencingtumor
中文摘要
描述:已经实施了一种新的方法来研究蛋白激酶的整体激活状态。我们的方法允许从细胞系、组织和肿瘤中分离和分析蛋白激酶,这些蛋白激酶在使用多重抑制物珠(MIB)的单次质谱分析中得到分析。MIBs由琼脂糖珠和共价固定化、连接子适应的激酶抑制剂的混合物组成。激酶捕获是可重复性的,是以下各项的函数
酶对不同固定化抑制剂的亲和力以及酶的激活状态
激活剂。我们合成的用于连接琼脂糖珠的抑制剂主要是1型抑制剂,它优先结合蛋白激酶的激活状态。广泛性
对MIB的表征表明,它们有很强的结合活性状态的激酶的偏好,而非活性的激酶结合得很差。使用MIB柱的分层珠子成分,我们能够在一次质谱分析中捕获和识别超过275个激酶。根据RNA-seq分析,MIB至少捕获了细胞系、肿瘤或正常组织中表达的动态组的75%-80%。重要的是,我们已经从细胞和组织裂解物中捕获并表征了多达275个蛋白激酶,其蛋白质含量低至500毫克。当组织受到限制时,这种程度的灵敏度对于动态组分析是必不可少的。因此,MIB/MS提供了一种无与伦比的、可扩展的、基于生物学的高通量技术来分析包括“非靶向和未被研究的”激酶在内的动态组的激活状态。对于许多“未被研究的”激酶,很少或根本没有试剂,从而限制了它们的研究,而MIB/MS克服了这些限制。MIB/MS还检测试剂无法区分密切相关的激酶的活性变化(例如,MEK1与MEK2和ERK1与ERK2的不同活性状态,它们是通过无法区分异构体的抗磷抗体一起评估的)。同样,我们可以很容易地区分相关受体酪氨酸激酶(RTK)的调节,如DDR1和DDR2,而对它们的研究试剂很少。AIMS将定义全面的动态组激活签名,其中包括糟糕的或未刻画的激酶。目的包括:1.利用MIB/MS对跨越不同癌症类型的一系列人类细胞系、人和小鼠肿瘤以及小鼠正常组织进行Key组激活状态的测定。2.使用针对蛋白质和脂蛋白激酶、细胞骨架、代谢调节剂、DNA损伤和表观遗传染色质修饰酶的特定化学探针来确定动态组对细胞生理扰动的反应。3.确定未被研究的激酶的RNAi敲除对激动组激活状态的影响。这些目的强调确定未被研究的激酶的激活状态,以及特定的未被研究的激酶是否作为响应特定细胞扰动的激活/抑制信号的一部分发挥作用。这一分析将确定未被研究的激酶活性受到调控的信号网络和细胞功能,并有助于内环境平衡控制机制。我们的目标是确定和验证哪些目前未被研究的激酶作为Kinome内的调节节点,并保证未来的化学探针开发。
英文摘要
DESCRIPTION: A novel approach has been implemented to study the activation state of protein kinases "en masse". Our methods allow isolation and analysis of protein kinases from cell lines, tissues and tumors assayed in a single mass spectrometry run using Multiplexed Inhibitor Beads (MIBs). MIBs consist of mixtures of Sepharose beads with covalently immobilized, linker adapted, kinase inhibitors. Kinase capture is reproducible and is a function of
affinity of kinases for the different immobilized inhibitors as well as the activation state of the
kinase. The inhibitors we have synthesized for linker conjugation to Sepharose beads are primarily type 1 inhibitors that preferentially bind activated states of protein kinases. Extensive
characterization of the MIBs has demonstrated they have a strong preference for binding the active state of kinases and inactive kinases bind poorly. Using a layered bead composition for MIB columns, we are able to capture and identify more than 275 kinases in a single mass spectrometry run. Based on RNA-seq analysis, MIBs are capturing at least 75-80% of the expressed kinome from cell lines, tumors or normal tissues. Importantly, we have captured and characterized up to 275 kinases with as little as 500 mg protein from cell and tissue lysates. This level of sensitivity is essential for kinome analysis when tissue is limiting. Thus, MIB/MS provides an unparalleled scalable biology- based high throughput technology to assay the activation state of the kinome including "untargeted and understudied" kinases. There simply are few or no reagents for many of the "understudied" kinases, thereby limiting their study and MIB/MS overcomes these limitations. MIB/MS also detects activity changes in kinases for which reagents cannot distinguish closely related kinases (e.g., different activity states of MEK1 vs. MEK2 and ERK1 vs. ERK2, which are assessed together by anti-phospho-antibodies that cannot distinguish isoforms). Similarly, we can easily distinguish the regulation of related receptor tyrosine kinases (RTKs), such as DDR1 and DDR2, for which there are few reagents for study. The aims will define comprehensive kinome activation signatures that include poorly or uncharacterized kinases. Aims include: 1. Determine the activation state of the kinome using MIB/MS for a series of human cell lines spanning different cancer types, human and mouse tumors, and normal tissues of the mouse. 2. Determine the response of the kinome to perturbation of cellular physiology using specific chemical probes targeting protein and lipid kinases, cytoskeleton, metabolic regulators, DNA damage and epigenetic chromatin modifying enzymes. 3. Determine the consequence of RNAi knockdown of understudied kinases on the activation state of the kinome. The aims emphasize determining the activation state of understudied kinases and if specific understudied kinases function as part of a signature of kinase activation/inhibition in response to specific cellular perturbations. This analysis will defne the signaling networks and cellular functions for which the activity of understudied kinases are regulated and contribute to homeostatic control mechanisms. The goal is to identify and validate which current understudied kinases function as regulatory nodes within the kinome and warrant future chemical probe development.
期刊论文(3)
专著(0)
科研奖励(0)
会议论文
DOI:
10.1002/cmdc.201700663
发表时间:
2018-01-08
期刊:
ChemMedChem
影响因子:
3.4
作者:
[Asquith CRM, Laitinen T, Bennett JM, Godoi PH, East MP, Tizzard GJ, Graves LM, Johnson GL, Dornsife RE, Wells CI, Elkins JM, Willson TM, Zuercher WJ]
通讯作者:
Zuercher WJ
Illuminating Function of the Understudied Druggable Kinome
-
批准号:10251291
-
项目类别:
-
资助金额:$225.18万
-
财政年份:2017
-
负责人:GARY L. JOHNSON
-
依托单位:
Illuminating Function of the Understudied Druggable Kinome
-
批准号:9762097
-
项目类别:
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资助金额:$226.6万
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财政年份:2017
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负责人:GARY L. JOHNSON
-
依托单位:
Illuminating Function of the Understudied Druggable Kinome
-
批准号:9453342
-
项目类别:
-
资助金额:$230.82万
-
财政年份:2017
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负责人:GARY L. JOHNSON
-
依托单位:
Illuminating Function of the Understudied Druggable Kinome
-
批准号:10015261
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项目类别:
-
资助金额:$225.92万
-
财政年份:2017
-
负责人:GARY L. JOHNSON
-
依托单位:
The adaptive kinome in pancreatic cancer
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批准号:8859584
-
项目类别:
-
资助金额:$57.37万
-
财政年份:2015
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负责人:GARY L. JOHNSON
-
依托单位:
Kinome Reprogramming in Response to Targeted Kinase Inhibitors
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批准号:8457042
-
项目类别:
-
资助金额:$26.78万
-
财政年份:2012
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负责人:GARY L. JOHNSON
-
依托单位:
Kinome Reprogramming in Response to Targeted Kinase Inhibitors
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批准号:8273335
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项目类别:
-
资助金额:$27.89万
-
财政年份:2012
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负责人:GARY L. JOHNSON
-
依托单位:
Kinome Reprogramming in Response to Targeted Kinase Inhibitors
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批准号:8607578
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项目类别:
-
资助金额:$27.72万
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财政年份:2012
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负责人:GARY L. JOHNSON
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依托单位:
Molecular Therapeutics
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批准号:8340206
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项目类别:
-
资助金额:$18.8万
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财政年份:2011
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负责人:GARY L. JOHNSON
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依托单位:
Regulation of Sequential Protein Kinase Pathways
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批准号:8038148
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项目类别:
-
资助金额:$10.14万
-
财政年份:2010
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负责人:GARY L. JOHNSON
-
依托单位:
Receptor Interaction with GTP-Regulatory Proteins
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批准号:7867340
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项目类别:
-
资助金额:$36.32万
-
财政年份:2009
-
负责人:GARY L. JOHNSON
-
依托单位:
Function of MEKK3 and its osmosensing scaffold protein
-
批准号:7889368
-
项目类别:
-
资助金额:$10.38万
-
财政年份:2009
-
负责人:GARY L. JOHNSON
-
依托单位:
Defining Signal Network Plasticity
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批准号:7498599
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项目类别:
-
资助金额:$7.32万
-
财政年份:2008
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负责人:GARY L. JOHNSON
-
依托单位:
EFFICACY OF COORDINATED SUPPORTIVE CARE IN GYNECOLOGIC ONCOLOGY PATIENTS
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批准号:7608096
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项目类别:
-
资助金额:$0.08万
-
财政年份:2007
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负责人:GARY L. JOHNSON
-
依托单位:
EFFICACY OF COORDINATED SUPPORTIVE CARE IN GYNECOLOGIC ONCOLOGY PATIENTS
-
批准号:7378108
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项目类别:
-
资助金额:$2.08万
-
财政年份:2006
-
负责人:GARY L. JOHNSON
-
依托单位:
Inhibitor Screens for Five MAPK Kinase Kinases Important in Human Disease
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批准号:7169344
-
项目类别:
-
资助金额:$18.25万
-
财政年份:2006
-
负责人:GARY L. JOHNSON
-
依托单位:
EFFICACY OF COORDINATED SUPPORTIVE CARE IN GYNECOLOGIC ONCOLOGY PATIENTS
-
批准号:7203341
-
项目类别:
-
资助金额:$4.33万
-
财政年份:2005
-
负责人:GARY L. JOHNSON
-
依托单位:
UNC Center for Nanosensing the Stressome (RMI)
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批准号:6930731
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项目类别:
-
资助金额:$7.3万
-
财政年份:2004
-
负责人:GARY L. JOHNSON
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依托单位:
Efficacy of Coordinated Supportive Care in Gynecologic Oncology Patients
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批准号:6981289
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项目类别:
-
资助金额:$2.78万
-
财政年份:2004
-
负责人:GARY L. JOHNSON
-
依托单位:
Function of MEKK3 and its osmosensing scaffold protein
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批准号:8123425
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项目类别:
-
资助金额:$31.14万
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财政年份:2003
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负责人:GARY L. JOHNSON
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依托单位:
海外基金