Cell biology of vasopressin-induced water channels
加压素诱导的水通道的细胞生物学
基本信息
- 批准号:10005038
- 负责人:
- 金额:$ 52.33万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2012
- 资助国家:美国
- 起止时间:2012-09-20 至 2022-08-31
- 项目状态:已结题
- 来源:
- 关键词:ActinsAnimalsAntineoplastic AgentsApicalAreaBackBiological AssayBiologyBrattleboro RatsBypassCell Culture TechniquesCell LineCell membraneCell modelCell physiologyCellsCellular AssayCellular biologyChemicalsClinicalComplexCyclic AMP-Dependent Protein KinasesDataDehydrationDiseaseDrug ScreeningDuct (organ) structureEGF geneEndocytosisEndosomesEpidermal Growth Factor ReceptorEpidermal Growth Factor Receptor Tyrosine Kinase InhibitorEpithelial CellsEquilibriumErlotinibEventExocytosisFDA approvedFluorescenceFundingGoalsHumanHypertensionHyponatremiaIn SituIn VitroKidneyKidney Concentrating AbilityKnockout MiceKnowledgeLeadLiquid substanceLithiumMapsMembraneModelingMusMutateMutationOutputPathway interactionsPatternPharmaceutical PreparationsPharmacologyPhosphorylationPhosphotransferasesProcessProductionProtein phosphataseProteinsReceptor InhibitionReceptor SignalingRecyclingRoleSignal PathwaySignal TransductionSiteSmall Interfering RNASodiumTemperatureTestingTissuesUrineVasopressin ReceptorVasopressinsWaterWater consumptionWorkapical membraneaquaporin-2basedepolymerizationdesigndrug discoverydrug testingequilibration disorderhigh throughput screeningin vivoinhibitor/antagonistkidney celllive cell imagingmutantnovelnovel therapeuticspolymerizationpreventprotein expressionrenal epitheliumresponsesalt intakescreeningsildenafilsmall molecule librariestraffickingtranscytosistranslational medicinetyrphostin AG-490vasopressin resistant diabetes insipiduswater channel
项目摘要
PROJECT SUMMARY/ABSTRACT
We believe that a combination of informed, targeted and unbiased drug screening is necessary to devise strategies to
normalize water balance disorders, including nephrogenic diabetes insipidus and hyponatremia. The overall strategy
proposed in this renewal application is designed to facilitate this goal. Over the past three years of funding, we have
uncovered several important and previously unrecognized aspects of aquaporin 2 biology: 1) AQP2 catalyzes actin
depolymerization in response to AVP: 2) AQP2 trafficking to the apical membrane involves transient basolateral insertion
and redirection via transcytosis: 3) AQP2 recycles constitutively in the complete absence of any known phosphorylation
events. Aim 1 takes advantage of this new knowledge and interrogates the relationship between AQP2 phosphorylation,
actin organization, and the polarity of AQP2 membrane delivery and accumulation in renal epithelial cells. This approach
allows us to envisage more informed approaches to water balance disorders. Next, our use of high throughput chemical
screening in the previous funding cycle led us to discover that the FDA approved cancer drug Erlotinib, an EGFR
inhibitor, reduces urine output by 50% in lithium treated NDI mice. Aim 2 will explore the mechanism by which EGFR
inhibition alone, in the complete absence of VP, causes AQP2 phosphorylation and membrane accumulation in the
absence of PKA stimulation. We need to identify which signaling pathway is responsible in order to fully understand how
Erlotinib works in this setting, and to suggest alternative targeted approaches. Finally, our quest for additional new
compounds that modulate AQP2 membrane accumulation will continue in Aim 3, in which a fluorescence assay will be
used for unbiased screening of chemical libraries for inhibitors or stimulators of endocytosis. While endocytosis
inhibitors are candidates for use in NDI, specific stimulators of AQP2 endocytosis could be useful in conditions of water
overload that could lead to hyponatremia and even hypertension. We will also test exocytosis-inhibitor compounds that
were identified in our previous screen for their ability to prevent AQP2 membrane accumulation, also a feature of drugs
that would prevent water overloading. Our prior studies and the work proposed in this renewal application range from the
in vitro characterization of protein interactions, through cell culture assays, to whole animal studies. We have developed
new cell lines for high throughput chemical screens, a new AQP2-EGFP construct for live cell imaging, and we have a
newly-established colony of conditional vasopressin receptor knockout mice in our facility for in vivo drug testing. Our
work combines the need for a better understanding of basic mechanisms in order to drive translational medicine and
clinical advances, with a more direct drug discovery approach using novel cell assays.
项目总结/摘要
我们认为,有必要结合知情、有针对性和无偏见的药物筛选来制定战略,
使水平衡紊乱正常化,包括肾源性尿崩症和低钠血症。总体战略
本更新申请中提出的建议旨在促进这一目标。在过去三年的资助中,我们
揭示了水通道蛋白2生物学的几个重要和以前未被认识的方面:1)AQP 2催化肌动蛋白
AVP引起的解聚:2)AQP 2向顶膜的运输涉及短暂的基底外侧插入
3)AQP 2在完全不存在任何已知磷酸化的情况下组成性地聚集
事件目的1利用这一新的知识,并询问AQP 2磷酸化,
肌动蛋白组织,以及AQP 2膜传递和肾上皮细胞中积累的极性。这种方法
使我们能够设想更明智的方法来解决水平衡失调问题。接下来,我们使用高通量化学
在上一个资助周期的筛选中,我们发现FDA批准的抗癌药物厄洛替尼(EGFR)
在锂处理的NDI小鼠中,抑制剂使尿量减少50%。目的2:探讨EGFR在肿瘤细胞中的作用机制。
在完全不存在VP的情况下,单独的抑制导致AQP 2磷酸化和细胞膜积聚。
没有PKA刺激。我们需要确定哪个信号通路负责,以充分了解如何
厄洛替尼在这种情况下工作,并提出替代的靶向方法。最后,我们寻求更多新的
调节AQP 2膜积累的化合物将在目标3中继续,其中荧光测定将在
用于无偏筛选内吞作用的抑制剂或刺激剂的化学文库。虽然内吞作用
抑制剂是用于NDI的候选物,AQP 2内吞作用的特异性刺激剂可以在水的条件下使用
超负荷可能导致低钠血症甚至高血压。我们还将测试胞吐抑制剂化合物,
在我们之前的筛选中鉴定了它们阻止AQP 2膜积聚的能力,这也是药物的一个特征,
可以防止水超载。我们先前的研究和在这次更新申请中提出的工作范围从
从体外蛋白质相互作用的表征,通过细胞培养试验,到整个动物研究。我们已经开发
用于高通量化学筛选的新细胞系,用于活细胞成像的新AQP 2-EGFP构建体,我们有一个用于高通量化学筛选的新细胞系。
新建立的条件性血管加压素受体敲除小鼠群体在我们的设施,在体内药物测试。我们
工作结合了更好地理解基本机制的需要,以推动转化医学,
临床进展,使用新的细胞测定更直接的药物发现方法。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Dennis Brown其他文献
Dennis Brown的其他文献
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{{ truncateString('Dennis Brown', 18)}}的其他基金
Cell Biology of Vasopressin-induced Water Channels-Research Supplement
加压素诱导的水通道的细胞生物学-研究补充
- 批准号:
10835229 - 财政年份:2023
- 资助金额:
$ 52.33万 - 项目类别:
An Open-Labeled, Single Arm Phase 2 Efficacy and Safety Study of REM-001 Photodynamic Therapy (PDT) for Treatment of Cutaneous Metastatic Breast Cancer (CMBC)
REM-001 光动力疗法 (PDT) 治疗皮肤转移性乳腺癌 (CMBC) 的开放标记单臂 2 期疗效和安全性研究
- 批准号:
10699535 - 财政年份:2023
- 资助金额:
$ 52.33万 - 项目类别:
HD Upgrade to a Nikon A1R Confocal Imaging Platform
高清升级至尼康 A1R 共焦成像平台
- 批准号:
10415591 - 财政年份:2022
- 资助金额:
$ 52.33万 - 项目类别:
Defining protein:protein interactions for the regulation of renal V-ATPase function: role in expression, assembly and trafficking.
定义蛋白质:调节肾 V-ATP 酶功能的蛋白质相互作用:在表达、组装和运输中的作用。
- 批准号:
10670311 - 财政年份:2019
- 资助金额:
$ 52.33万 - 项目类别:
Defining protein:protein interactions for the regulation of renal V-ATPase function: role in expression, assembly and trafficking.
定义蛋白质:调节肾 V-ATP 酶功能的蛋白质相互作用:在表达、组装和运输中的作用。
- 批准号:
10454931 - 财政年份:2019
- 资助金额:
$ 52.33万 - 项目类别:
Defining protein:protein interactions for the regulation of renal V-ATPase function: role in expression, assembly and trafficking.
定义蛋白质:调节肾 V-ATP 酶功能的蛋白质相互作用:在表达、组装和运输中的作用。
- 批准号:
10207619 - 财政年份:2019
- 资助金额:
$ 52.33万 - 项目类别:
A Zeiss LSM800 confocal microscope with Airyscan
配备 Airyscan 的 Zeiss LSM800 共焦显微镜
- 批准号:
9075249 - 财政年份:2016
- 资助金额:
$ 52.33万 - 项目类别:
Cell Biology of Vasopressin-induced Water Channels
加压素诱导的水通道的细胞生物学
- 批准号:
10652774 - 财政年份:2012
- 资助金额:
$ 52.33万 - 项目类别:
Cell biology of vasopressin-induced water channels
加压素诱导的水通道的细胞生物学
- 批准号:
9176186 - 财政年份:2012
- 资助金额:
$ 52.33万 - 项目类别:
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