High throughput screening assays to identify chemical probes targeting Hectd3, an E3 ubiquitin ligase implicated in multiple sclerosis

摘要：多发性硬化症是一种慢性自身免疫性炎症性疾病，与脱髓鞘有关。 中枢神经系统(CNS)，仍然是一个公共卫生问题。目前还没有已知的治疗多发性 硬化症。尽管有几种疾病修正治疗(DMT)，但多发性硬化症的复发 DMT经常发生，往往会导致严重的不良反应，如肝功能衰竭和胎儿结局。小说 多发性硬化症患者需要通过治疗来减轻疾病负担。最近，我们发表了一篇 Hectd3是一种E3泛素连接酶，主要在免疫系统的T细胞中表达，在免疫系统中起着关键的作用。 实验性自身免疫性脑脊髓炎(EAE)在人类多发性硬化小鼠模型致病中的作用 硬化症。具体地说，我们发现Hectd3通过调节EAE中致病的Th17效应器反应来控制 MALT1和STAT3以非降解的方式泛素化，导致MALT1和STAT3的稳定。在……里面 此外，Hectd3介导的STAT3多泛素化促进了STAT3的激活。此外，Hectd3缺乏 小鼠表现出EAE疾病评分、Th17细胞致病性和效应Th17细胞因子的降低。 此外，Hectd3缺乏导致Th17细胞致病性的细胞固有缺陷，这是导致 Hectd3-−/−小鼠对EAE的减毒作用。总体而言，我们的结果表明Hectd3是一种关键的调节因子 EAE中的MALT1和STAT3信号转导。基于这些结果，我们假设取消Hectd3的化合物- 介导的底物泛素化可以降低EAE的严重程度。然而，尽管Hectd3扮演着重要的角色 在多发性硬化症的发病机制方面，目前尚无化学探针进一步研究其作用途径。 以及对多发性硬化症治疗的启示。因此，在这项提议中，我们的目标是发展高吞吐量 用于深入研究Hectd3-生物化学的鉴定和表征化学探针的筛选方法 介导的MALT1和STAT3信号通路及其在致病Th17细胞和EAE中的治疗潜力。 这项创新工作探索了Hectd3在免疫调节中的新功能，特别是在致病 Th17细胞，鉴定Malt1和STAT3作为Hectd3介导的泛素化的靶底物，以及 Hectd3新型化学探针的表征及其对EAE的影响。长期持续的 这项工作的影响是确定在其靶底物上调节Hectd3活性的化合物及其功能 在EAE中开辟途径，开发更特异和有效的免疫疗法来治疗多发性 硬化症，鉴于目前的治疗挑战和有限的治疗选择，这是一个至关重要的需求。这些加在一起 这些方法将导致具有新的抑制机制的独特的Hectd3抑制剂的开发。这部作品 通过推广治疗多发性硬化症的新的有效策略，将拥有全球影响力。Hectd3也有 与促进乳腺癌耐药性、癌症转移有关(未发表的结果)，以及 细菌感染。因此，该项目可能还会对癌症和细菌防御产生重大影响。

Abstract: Multiple sclerosis, a chronic autoimmune inflammatory disease associated with demyelination of the central nervous system (CNS), remains a public health issue. Currently there is no known cure for multiple sclerosis. Although several disease-modifying treatments (DMTs) are available, relapsing of multiple sclerosis occurs frequently and DMTs often result in severe adverse effects such as liver failure and fetal outcomes. Novel therapies are needed to reduce the disease burden for multiple sclerosis patients. Recently, we published that Hectd3, an E3 ubiquitin ligase, is expressed predominantly in T cells of the immune system, which play a critical role in pathogenicity of experimental autoimmune encephalomyelitis (EAE), a mouse model of human multiple sclerosis. Specifically, we found that Hectd3 controls pathogenic Th17 effector response in EAE by regulating ubiquitination of Malt1 and Stat3 in a non-degradative manner, resulting in stabilization of Malt1 and Stat3. In addition, Hectd3-mediated polyubiquitination of Stat3 promotes Stat3 activation. Moreover, Hectd3-deficient mice showed reduction in EAE disease scores, Th17 cell pathogenicity and effector Th17 cytokines. Furthermore, Hectd3 deficiency causes a cell-intrinsic defect in Th17 cell pathogenicity that is responsible for the attenuation of EAE in Hectd3−/− mice. Overall, our results demonstrate that Hectd3 is a critical modulator of Malt1 and Stat3 signaling in EAE. Based on these results, we hypothesize that compounds abolishing Hectd3- mediated ubiquitination of substrates can lower EAE severity. However, although Hectd3 plays significant roles in pathogenesis of multiple sclerosis, currently there is no chemical probe to further investigate the pathways and the implication in therapy of multiple sclerosis. Therefore, in this proposal, we aim to develop high throughput screening assays to identify and characterize chemical probes to investigate in depth the biochemistry of Hectd3- mediated Malt1 and Stat3 signaling pathways, and their therapeutic potential in pathogenic Th17 cells and EAE. This innovative work explores the novel function of Hectd3 in immune regulation, specifically in pathogenic Th17 cells, the identification of Malt1 and Stat3 as target substrates for Hectd3-mediated ubiquitination, and characterization of novel chemical probes for Hectd3, and their impact on EAE. The long-term sustained impact of this work is to identify compounds to modulate Hectd3 activity on its target substrates and its functions in EAE to open avenues for development of more specific and effective immune therapies to treat multiple sclerosis, a crucial need given current treatment challenges and limited therapeutic options. These combined approaches will lead to the development of unique Hectd3 inhibitors with novel inhibition mechanisms. This work will have a global reach by promoting fresh and effective strategies to treat multiple sclerosis. Hectd3 has also been implicated in promoting breast cancer drug resistance, cancer metastasis (unpublished results), and bacterial infections. Therefore, this project may also have significant impact on cancers and bacterial defense.