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: 10391539
• 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/10391539
• 关键词: Active Sites; Adverse effects; Affinity; Autoimmune; Autoimmune Diseases; Bacterial Infections; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Cells; Chemicals; Chronic; Complement; Crystallization; 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; Lead; Liver Failure; Luciferases; Malignant Neoplasms; Mediating; Modeling; Mucosa- associated lymphoid tissue lymphoma translocation protein-1; Multiple Sclerosis; Mus; Neoplasm Metastasis; Neuraxis; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Pharmacology; 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; Ubiquitination; Work; analog; attenuation; base; 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; 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.

翻译后摘要：多发性硬化症，一种慢性自身免疫性炎症性疾病与脱髓鞘的 中枢神经系统（CNS）的疾病仍然是一个公共卫生问题。目前还没有已知的治疗方法， 硬化症虽然有几种疾病修饰治疗（DMT），但多发性硬化症的复发 DMTs经常发生，并且DMTs经常导致严重的副作用，如肝功能衰竭和胎儿结局。小说 需要治疗来减轻多发性硬化症患者的疾病负担。最近，我们发表了 Hectd 3是一种E3泛素连接酶，主要在免疫系统的T细胞中表达，其在免疫系统中起关键作用。 在实验性自身免疫性脑脊髓炎（EAE）致病性中的作用， 硬化症具体地说，我们发现Hectd 3通过调节EAE中的Th 17效应子应答来控制致病性Th 17效应子应答。 以非降解方式对Malt 1和Stat 3进行泛素化，导致Malt 1和Stat 3的稳定化。在 此外，Hectd 3介导的Stat 3的多聚泛素化促进Stat 3活化。此外，Hectd 3缺陷型 小鼠表现出EAE疾病评分、Th 17细胞致病性和效应Th 17细胞因子的降低。 此外，Hectd 3缺陷导致Th 17细胞致病性的细胞内在缺陷，其负责 Hectd 3 −/−小鼠中EAE的衰减。总的来说，我们的结果表明Hectd 3是一个关键的调节剂， EAE中的Malt 1和Stat 3信号转导。基于这些结果，我们假设消除Hectd 3-的化合物是有效的。 介导的底物泛素化可以降低EAE的严重程度。然而，尽管Hectd 3发挥着重要作用， 在多发性硬化症的发病机制中，目前还没有化学探针来进一步研究这些通路， 以及对多发性硬化症治疗的意义。因此，在本提案中，我们的目标是开发高通量 筛选分析，以确定和表征化学探针，以深入研究Hectd 3的生物化学， 介导的Malt 1和Stat 3信号通路及其在致病性Th 17细胞和EAE中的治疗潜力。 这项创新性的工作探索了Hectd 3在免疫调节中的新功能，特别是在致病性 Th 17细胞，鉴定Malt 1和Stat 3作为Hectd 3介导的泛素化的靶底物，以及 Hectd 3的新型化学探针的表征及其对EAE的影响。长期持续 这项工作的影响是鉴定调节Hectd 3对其靶底物的活性及其功能的化合物 在EAE中，为开发更特异和有效的免疫疗法以治疗多种 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。这些组合 这些方法将导致开发具有新抑制机制的独特Hectd 3抑制剂。这项工作 将通过推广治疗多发性硬化症的新的和有效的策略，在全球范围内推广。Hectd 3还 与促进乳腺癌耐药性、癌症转移有关（未发表的结果）， 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。