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】：多发性硬化症是一种与大脑脱髓鞘相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，目前尚无已知的治疗方法。 尽管有多种疾病缓解治疗 (DMT)，但多发性硬化症会复发。 这种情况经常发生，DMT 常常会导致严重的不良反应，例如肝功能衰竭和胎儿结局。 需要治疗来减轻多发性硬化症患者的疾病负担。最近，我们发表了这一观点。 Hectd3 是一种 E3 泛素连接酶，主要在免疫系统的 T 细胞中表达，在免疫系统中发挥着关键作用。 实验性自身免疫性脑脊髓炎（EAE）致病性中的作用，这是一种人类多发性骨髓炎小鼠模型 具体来说，我们发现 Hectd3 通过调节 EAE 中的致病性 Th17 效应子反应。 以非降解方式泛素化 Malt1 和 Stat3，从而稳定 Malt1 和 Stat3。 此外，Hectd3 介导的 Stat3 多泛素化可促进 Stat3 激活。 小鼠的 EAE 疾病评分、Th17 细胞致病性和效应 Th17 细胞因子均降低。 此外，Hectd3 缺陷会导致 Th17 细胞致病性的细胞内在缺陷，从而导致 总体而言，我们的结果表明 Hectd3 是 EAE 的关键调节剂。 EAE 中的 Malt1 和 Stat3 信号传导 基于这些结果，我们努力寻找能够消除 Hectd3- 的化合物。 然而，尽管 Hectd3 发挥着重要作用，但底物介导的泛素化可以降低 EAE 的严重程度。 在多发性硬化症的发病机制中，目前还没有化学探针可以进一步研究其通路 因此，在本提案中，我们的目标是开发高通量。 筛选测定来识别和表征化学探针，以深入研究 Hectd3- 的生物化学 介导的 Malt1 和 Stat3 信号通路及其在致病性 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，特别是在致病性调节中 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 的长期持续影响。 这项工作的影响是确定调节 Hectd3 目标底物活性及其功能的化合物 EAE 领域的研究为开发更特异、更有效的免疫疗法来治疗多种疾病开辟了道路 鉴于当前的治疗挑战和有限的治疗选择，这一需求至关重要。 这项工作将导致开发具有新颖抑制机制的独特 Hectd3 抑制剂。 通过推广治疗多发性硬化症的新鲜有效策略，Hectd3 也将在全球范围内产生影响。 与促进乳腺癌耐药性、癌症转移（未发表的结果）有关 因此，该项目也可能对癌症和细菌防御产生重大影响。