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

高通量筛选分析，鉴定针对 Hectd3 的化学探针，Hectd3 是一种与多发性硬化症有关的 E3 泛素连接酶

• 批准号: 10597043
• 负责人: Dorina Avram
• 金额: $ 78.72万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597043
• 关键词: Active Sites; Adverse effects; Affinity; Autoimmune; Autoimmune Diseases; Bacterial Infections; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Cells; Central Nervous System; Chemicals; Chronic; Complement; Crystallography; Defect; Demyelinations; Development; Disease; Drug Kinetics; Ensure; Enzymes; Experimental Autoimmune Encephalomyelitis; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Immune system; Immunologics; Immunotherapy; In Vitro; Inflammatory; Informatics; Lead; Ligase; Liver Failure; Luciferases; Malignant Neoplasms; Mediating; Modeling; Mucosa- associated lymphoid tissue lymphoma translocation protein-1; Multiple Sclerosis; Mus; Neoplasm Metastasis; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Play; Polyubiquitination; Property; Protocols documentation; Public Health; Publishing; Relapse; Research; Role; Severities; Signal Transduction; Solid; Specificity; Stat3 Signaling Pathway; Structure; Structure-Activity Relationship; Substrate Interaction; T-Lymphocyte; Testing; Therapeutic; Toxic effect; Ubiquitin; Ubiquitination; Work; analog; attenuation; burden of illness; cancer drug resistance; cancer therapy; chemical synthesis; clinically relevant; cytokine; experimental study; fetal; high throughput screening; human model; immunoregulation; improved; in silico; in vivo; infancy; inhibitor; innovation; malignant breast neoplasm; mouse model; multiple sclerosis patient; multiple sclerosis treatment; novel; novel therapeutics; pharmacologic; programs; response; screening; side effect; small molecule libraries; therapeutic target; therapy development; ubiquitin-protein ligase

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.

摘要：多发性硬化症，一种与脱髓鞘有关的慢性自身免疫性炎症性疾病 中枢神经系统（CNS）仍然是一个公共卫生问题。目前尚无多种治疗方法 硬化症。尽管可以使用多种疾病改良治疗（DMT），但多发性硬化症的中继 经常发生，DMT经常会导致严重的不良反应，例如肝衰竭和胎儿结局。小说 需要疗法来减少多发性硬化症患者的疾病燃烧。最近，我们发表了 hectd3是E3泛素连接酶，主要在免疫系统的T细胞中表达 实验自身免疫性脑脊髓炎（EAE）的致病性作用，这是人类多重的小鼠模型 硬化。具体而言，我们发现HECTD3通过调节EAE中的致病性TH17效应子响应 MALT1和STAT3的泛素化以非降解的方式导致MALT1和STAT3的稳定。在 此外，hectd3介导的STAT3介导的多泛素化促进了STAT3激活。此外，hectd3缺乏效率 小鼠在EAE病评分，Th17细胞致病性和效应子Th17细胞因子中显示出降低。 此外，hectd3缺乏会导致Th17细胞致病性的细胞内部缺陷，该缺陷负责 hectd3 - / - 小鼠中EAE的衰减。总体而言，我们的结果表明Hectd3是 MALT1和STAT3信号传导。基于这些结果，我们假设化合物废除了hectd3- 介导的底物泛素化可以降低EAE的严重性。但是，尽管Hectd3发挥了重要作用 在多发性硬化症的发病机理中，目前尚无化学探针来进一步研究途径 以及对多发性硬化症治疗的影响。因此，在此提案中，我们旨在发展高吞吐量 筛选分析以识别和表征化学问题，以深入研究Hectd3-的生物化学 介导的MALT1和STAT3信号通路，以及它们在致病性TH17细胞和EAE中的治疗潜力。 这项创新的工作探讨了Hectd3在免疫调节中的新功能，特别是在致病性中 Th17细胞，MALT1和STAT3作为HECTD3介导的泛素化的靶标底物的鉴定，以及 hectd3的新化学问题的表征及其对EAE的影响。长期持续 这项工作的影响是识别化合物以调节其目标底物及其功能的HECTD3活动 在EAE中开放开发更具体和有效的免疫疗法以治疗多重疗法的途径 鉴于当前的治疗挑战和有限的治疗选择，硬化症，至关重要的需求。这些结合了 方法将导致具有新型抑制机制的独特HECTD3抑制剂的发展。这项工作 hectd3也有 暗示促进乳腺癌耐药性，癌症转移（未发表的结果）和 细菌感染。因此，该项目也可能对癌症和细菌防御产生重大影响。