The catalytic core of the proteasome as a drug target to treat Human African Trypanosomiasis

项目摘要 人类非洲锥虫病（HAT）是一种由非洲锥虫的各种亚种引起的贫困疾病。 布氏锥虫原生寄生虫。通过叮咬传播的tse-tse苍蝇，第一阶段的疾病是 其特征在于通过血液和淋巴系统传播的锥虫，然后穿过血液和淋巴系统。 血脑屏障进入中枢神经系统，导致2期疾病。中枢神经系统的浸润与一种可怕的 一系列逐渐恶化并最终致命的心理和生理疾病。药物 治疗依赖于过时的并且通常有毒的药物，这些药物必须在医疗监督下施用， 是建立了抵抗还是持续的威胁需要新的药物。蛋白酶体是一种进化- 一种细胞中保守的多亚基蛋白复合物，调节正常的蛋白质周转和降解， 错误折叠的蛋白质抑制T.布氏蛋白酶体（Tb20S）与小分子抑制剂杀死 锥虫然而，很少有关于蛋白水解切割偏好或蛋白水解酶的信息。 Tb20S靶标的抑制曲线。发现这些信息将有助于开发 具有改进的特异性和效力的蛋白酶体抑制剂，然后可以形成新治疗的基础 的帽子。因此，在目的1中，我们将使用一种新的方法来全面地分析Tb20S的切割特异性。 这是一种称为多重底物质谱分析（MSP-MS）的技术。所产生的数据将告知（i） 合成优化的肽基底物，用其可以测量Tb20S的活性，和（ii） 药物化学努力（目标3）合成具有改进的特异性的新的Tb20S抑制剂。在目标2中， 将用含有不同支架和反应基团的各种蛋白酶体抑制剂文库筛选Tb20S 由内部和合作者开发的。产生的数据将为（i）更广泛的化学品 空间与抑制Tb 20S和（ii），再次，药物化学计划的目标3。 对于目标3，我们将用表现最好的Tb20S筛选布氏锥虫和哺乳动物细胞系 抑制剂，以了解寄生虫杀灭和细胞毒性之间的差异。这一目标还涉及一个 集中的药物化学活动，评估所有的蛋白酶体抑制和细胞筛选数据， 合成具有改进的特异性的Tb20S抑制剂，其使用被称为 卡马霉素B。待建议的R21研究完成后，我们会发现一系列Tb20S 抑制剂准备作为HAT的治疗剂进一步开发。

PROJECT SUMMARY Human African Trypanosomiases (HAT) is a disease of poverty caused by various sub-species of the Trypanosoma brucei protozoan parasite. Transmitted by the bite of the tse-tse fly, Stage 1 disease is characterized by trypanosomes that disseminate through the blood and lymphatic systems, and then cross the blood-brain barrier into the CNS to cause Stage 2 disease. Infiltration of the CNS is associated with a frightening array of progressively deteriorating and eventually lethal psychological and physiological disorders. Drug therapy relies on antiquated and often toxic drugs that must be administered under medical supervision and for which resistance is established or a constant threat. New drugs are needed. The proteasome is an evolutionarily- conserved, multi-subunit protein complex in the cell that regulates normal protein turnover and degradation of misfolded proteins. Inhibition of the T. brucei proteasome (Tb20S) with small molecule inhibitors kills trypanosomes. However, there is little information regarding the proteolytic cleavage preferences or the inhibition profile of the Tb20S target. Uncovering this information would support the development of proteasome inhibitors with improved specificity and potency that could then form the basis for a new treatment of HAT. Accordingly, in Aim 1 we will comprehensively profile the cleavage specificity of Tb20S using a technology called multiplex substrate profiling by mass spectrometry (MSP-MS). The data arising will inform (i) the synthesis of optimized peptidyl substrates with which the activity of Tb20S can be measured and (ii) a medicinal chemistry effort (Aim 3) to synthesize new Tb20S inhibitors with improved specificity. In Aim 2, we will screen Tb20S with various proteasome inhibitor libraries containing diverse scaffolds and reactive groups that have been developed in-house and by collaborators. The data arising will inform (i) the broader chemical space associated with inhibition of Tb20S and (ii), again, the medicinal chemistry program planned for Aim 3. For Aim 3, we will screen both Trypanosoma brucei and mammalian cell lines with the top performing Tb20S inhibitors to understand the differential between parasite-killing and cytotoxicity. This Aim also involves a focused medicinal chemistry campaign that evaluates all of the proteasome inhibition and cell screening data to synthesize Tb20S inhibitors with improved specificity using the scaffold of the marine natural product known as carmaphycin B. Upon completion of the proposed R21 studies, we will have identified a series of Tb20S inhibitors ready for further development as therapeutics for HAT.