HYPOTHEMYCIN TARGETS IN HUMAN CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 次霉素是一种天然产物，已知能不可逆转地抑制活性部位含有非保守半胱氨酸的一组激酶。虽然它的体外选择性是已知的，但在更“现实”的体内背景下它的靶点仍然大多是未知的。我们最近合成了一种低霉素的类似物，它反映了其母体的活性，并允许我们使用点击化学来识别那些它共价标记的靶点。在与加州大学旧金山分校的质谱学设施的合作下，我们已经使用这个分子来鉴定布鲁氏锥虫的几个必要的激酶，布鲁氏锥虫是非洲昏睡病的病原体，以前只有一个已知和研究过。通过应用我们与布氏锥虫合作开发的方法，我们希望在体内开发出一份由我们的降霉素探针标记的人类激酶清单，并将其与体外产生的结果进行比较。作为后续，我们还希望探索其他小分子激酶抑制剂的体内选择性图谱，包括已批准的药物，并开始解决体外和体内图谱之间的脱节。 质谱学提供了检测低丰度激酶所需的灵敏度，以及识别同一样本中的多个激酶并量化我们的结果的能力。这使得质谱学成为我们有效探索和理解体内和体外系统中的激酶抑制剂选择性的重要工具。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. By applying the methods developed through our work with T. brucei, we hope to develop a list of human kinases labeled by our hypothemycin probe in vivo and compare that to what has been generated in vitro. As a follow up, we also hope 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.