IDENTIFICATION OF ELECTROPHILICLY MODIFIED PROTEINS IN EUKARYOTIC CELLS

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。 我们已经合成了一种不可逆的抑制剂，它能与多种蛋白激酶结合。 这种化合物FSPP共价修饰保守的催化赖氨酸活性位点，并携带炔丙基基团，以在蛋白质修饰后通过铜催化的“点击化学”连接不同的荧光和亲和标签。“FSPP抑制机制的共价性质有助于鉴定其激酶靶点。 我们正在利用这种化合物的化学性质来探测真核寄生虫布氏锥虫（Trypanosoma brucei）的激酶，这种寄生虫是非洲昏睡病的病原体，寻找新的潜在治疗靶点。 FSPP抑制布氏锥虫生长在一微摩尔，我们正试图确定必要的激酶介导的这种毒性使用亲和纯化和质谱。 此外，我们正在使用竞争性标记策略，以确定潜在的可逆活性位点定向抑制剂相同的激酶。 当用FSPP和可逆竞争剂共处理细胞裂解物时，与FSPP共享靶标的非共价抑制剂将减缓或停止那些靶标的化学修饰速率。 我们相信，这一战略将确定新的治疗目标，这种疾病，而在同一时间提供有价值的结构活性关系，为未来的药物开发。

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. We have synthesized an irreversible inhibitor that binds to a variety of protein kinases. This compound, FSPP, covalently modifies a conserved catalytic lysine active site and carries a propargyl group to attach diverse fluorescent and affinity tags after protein modification via copper catalyzed "click chemistry." The covalent nature of FSPP's inhibitory mechanism facilitates identification of its kinase targets. We are using this compound's chemical properties to probe the kinases of the eukaryotic parasite Trypanosoma brucei, the causative agent of African sleeping sickness, for new potential therapeutic targets. FSPP inhibits T brucei growth at one micro molar, and we are attempting to identify the essential kinase(s) mediating this toxicity using affinity purification and mass spectrometry. Additionally we are using a competitive labeling strategy to identify potential reversible active site directed inhibitors of the same kinases. Non-covalent inhibitors sharing targets with FSPP will slow or halt the rate of those targets' chemical modification when cell lysates are co-treated with FSPP and the reversible competitor. We believe this strategy will identify novel therapeutic targets for this disease while at the same time providing valuable structure activity relationships for future drug development.