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

项目总结 人类非洲锥虫病(HAT)是一种由非洲人不同亚种引起的贫困疾病。 布鲁氏锥虫原生动物寄生虫。通过Tse-Tse Fly的叮咬传播，1级疾病是 以锥体为特征的，通过血液和淋巴系统传播，然后跨越 血脑屏障进入中枢神经系统，导致2期疾病。中枢神经系统的渗透与一种可怕的 一系列逐渐恶化并最终致命的心理和生理疾病。药效 治疗依赖于过时的、往往是有毒的药物，这些药物必须在医疗监督下服用，并 抵抗力是建立起来的，还是持续不断的威胁。需要新的药物。蛋白酶体在进化上是一种 细胞中保守的多亚基蛋白质复合体，调节正常的蛋白质周转和降解。 错误折叠的蛋白质。小分子抑制剂对布鲁氏毛滴虫蛋白酶体(Tb20S)的抑制作用 锥虫。然而，很少有关于蛋白水解性切割偏好或 Tb20S靶标的抑制谱。发现这些信息将支持 具有改进的特异性和有效性的蛋白酶体抑制剂，然后可以形成新治疗的基础 帽子的颜色。因此，在目标1中，我们将使用一个 这项技术被称为多重底物质谱仪(MSP-MS)。产生的数据将告知(I) 用来测定Tb20S活性的优化多肽底物的合成和(Ii)a 药物化学努力(目标3)合成具有更高特异性的新的Tb20S抑制剂。在目标2中，我们 将使用包含不同支架和反应基团的各种蛋白酶体抑制物库来筛选Tb20S 由内部和合作者开发的。产生的数据将告知(I)更广泛的化学物质 与抑制Tb20S有关的空间和(Ii)，再次，为目标3计划的药物化学方案。 对于目标3，我们将使用最高性能的Tb20S来筛选布氏锥虫和哺乳动物细胞系 了解寄生虫杀灭和细胞毒性之间的区别。这一目标还涉及一个 评估所有蛋白酶体抑制和细胞筛选数据的重点药物化学活动 以海洋天然产物Tb20S为骨架合成特异性更强的Tb20S抑制剂 在完成拟议的R21研究后，我们将确定一系列Tb20S 作为HAT的治疗药物，抑制剂准备进一步开发。

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.