HYPOTHEMYCIN TARGETS IN HUMAN CELLS

人类细胞中的次霉素靶点

• 批准号: 8363820
• 负责人: Jack Taunton
• 金额: $ 0.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363820
• 关键词: Active Sites; African Trypanosomiasis; Biological Factors; Cells; Chemistry; Collaborations; Cysteine; Funding; Grant; Human; In Vitro; Label; Mass Spectrum Analysis; Methods; National Center for Research Resources; Parasites; Parents; Pharmaceutical Preparations; Phosphotransferases; Principal Investigator; Research; Research Infrastructure; Resources; Role; Sampling; Source; System; Trypanosoma brucei brucei; United States National Institutes of Health; Work; analog; cost; hypothemycin; in vivo; kinase inhibitor; small molecule; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Hypothemycin is a natural product known to irreversibly inhibit a subset of kinases that contain a non-conserved cysteine in their active site. Although its in vitro selectivity profile is known, what it targets in a more "realistic", in vivo context remains mostly unknown. We have recently synthesized an analog of hypothemycin that mirrors the activity of its parent and allows us to use Click Chemistry to identify those targets that it covalently labels. In collaboration with the UCSF Mass Spectrometry Facility, we have used this molecule to identify several essential kinases in Trypanosoma brucei, the causative agent of African sleeping sickness, of which only one was known and studied before. We continue to use this molecule to better understand the roles of these targets in the parasite. Also by applying the methods developed through our work with T. brucei, we plan to develop a list of human kinases labeled by our hypothemycin probe in vivo and compare that to what has been generated in vitro. This allows us to explore the in vivo selectivity profiles of other small molecule kinase inhibitors, including approved drugs, and begin to resolve the disconnect between in vitro and in vivo profiles. Mass spectrometry provides the sensitivity necessary to detect low abundance kinases and the ability to identify multiple kinases within the same sample and to quantify our results. This makes mass spectrometry an important tool for us to effectively explore and understand how kinase inhibitor selectivity in vivo compares to in vitro systems.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 Hypothemycin是一种天然产物，已知可不可逆地抑制活性位点含有非保守半胱氨酸的激酶亚类。 尽管其体外选择性特征是已知的，但其在更“现实”的体内环境中靶向什么仍然是未知的。 我们最近合成了一种hypothemycin的类似物，它反映了它的母体的活性，并允许我们使用点击化学来识别它共价标记的那些靶点。 在与UCSF质谱设施的合作中，我们使用这种分子来鉴定布氏锥虫中的几种必需激酶，布氏锥虫是非洲昏睡病的病原体，其中只有一种是已知的，以前也进行了研究。我们继续使用这种分子来更好地了解这些靶标在寄生虫中的作用。同样，通过应用我们与T.布氏杆菌，我们计划开发一个由我们的hypothemycin探针在体内标记的人类激酶列表，并将其与体外产生的激酶进行比较。 这使我们能够探索其他小分子激酶抑制剂的体内选择性概况，包括已批准的药物，并开始解决体外和体内概况之间的脱节。 质谱法提供了检测低丰度激酶所需的灵敏度，以及在同一样品中鉴定多种激酶并量化结果的能力。这使得质谱法成为我们有效探索和理解体内激酶抑制剂选择性与体外系统相比的重要工具。