Developing HTS assays for identifying NLK activators to target Huntington's disease

开发 HTS 检测方法来鉴定 NLK 激活剂以靶向亨廷顿病

基本信息

批准号：
10783153
负责人：
Wenzhen Duan
金额：
$ 45.03万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-14 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10783153
关键词：
Adopted Affect Alleles Animals Applications Grants Atrophic Attenuated Autophagocytosis Autopsy Biological Assay Biology Bioluminescence Brain Brain region CRISPR/Cas technology Cells Central Nervous System Chemicals Chemosensitization Corpus striatum structure Data Development Disease Disease Progression Disease model Dose Drug Kinetics Energy Transfer Face Fluorescence Future Genes Genetic Heterozygote Homodimerization Human Huntington Disease Huntington gene Immunomodulators Impairment In Vitro Inhibition of Apoptosis Lead Libraries Life Ligands Luciferases Measures Monitor Mus Mutation N-terminal NGFR Protein Neuroblastoma Neurons Neurotrophic Tyrosine Kinase Receptor Type 1 Pathogenesis Pathogenicity Pathology Pathway interactions Patients Peripheral Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology and Toxicology Phosphorylation Phosphotransferases Play Protein-Serine-Threonine Kinases Proteins Research Personnel Role Safety Series Serine Signal Transduction System Time Tissues Toxic effect Traumatic Brain Injury Ubiquitin Ubiquitination Undifferentiated assay development cerebral atrophy drug development drug metabolism efficacy evaluation experience genetic approach high throughput screening illness length in vivo interest meter mouse model multicatalytic endopeptidase complex mutant nemo-like kinase neural neuropathology neuroprotection new therapeutic target novel overexpression polyglutamine pre-clinical protein aggregation protein degradation response screening small molecule stable cell line tool

项目摘要

Project Summary Huntington’s disease (HD) is caused by the mutation of the Huntingtin (HTT) gene, which encodes the mutant huntingtin protein (mHTT) with an expanded polyglutamine tract (polyQ). The gain of toxic function of mHTT is the major cause of HD. Lowering mHTT may provide an effective approach in treating HD by ameliorating its downstream toxicity. If successful, this strategy may modify disease progression in human HD. In fact, lowering of HTT levels by a variety of genetic approaches has been shown to effectively mitigate mHTT toxicity in HD models. However, genetic approaches still face significant challenges in effective delivery to the affected brain regions of HD patients. Thus, small molecule compounds that can lower mHTT levels are highly desired to treat HD. Cellular mechanisms promoting mHTT clearance are of great interest in modifying HD pathology because they can lower the levels of the mHTT protein and resultant toxic species, thus affecting disease progression. Ubiquitination can direct mHTT for clearance through two major protein clearance pathways - the ubiquitin-proteasome system and autophagy. Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase and expressed at relatively high levels in the central nervous system. We found that NLK levels are significantly decreased in HD postmortem brains. We further demonstrated that NLK interacts with HTT and lowers endogenous mHTT levels in a kinase-dependent manner in human cells. Overexpression of NLK in the mouse striatum attenuates HD pathology, whereas genetic reduction of NLK levels exacerbates the neuropathology in HD mice. Furthermore, we discovered that NLK enhanced mHTT ubiquitination, indicating that NLK may facilitate mHTT degradation. Our preclinical results strongly support that NLK has a neuroprotective role in modifying disease progression and is a potential novel drug target to lower mHTT levels. In Aim 1, we will develop two complementary high-throughput screening (HTS) assays to identify NLK activators, including an in vitro enzymatic-based HTRF assay and a cell-based BRET assay to monitor NLK homodimerization. In Aim 2: We will conduct a pilot HTS campaign for NLK activator and hit confirmation using a library of 9900 CNS focus compounds.

项目概要亨廷顿病 (HD) 是由亨廷顿 (HTT) 基因突变引起，该基因编码具有扩展的多聚谷氨酰胺束（polyQ）的突变亨廷顿蛋白（mHTT）获得毒性。 mHTT 的功能是 HD 的主要原因，降低 mHTT 可能为 HD 提供有效的方法。通过改善 HD 的下游毒性来治疗 HD 如果成功，该策略可能会改变疾病。事实上，通过多种遗传方法降低 HTT 水平已成为人类 HD 的进展。研究显示可有效减轻 HD 模型中的 mHTT 毒性，但仍面临遗传方法的挑战。因此，如何有效地将药物输送到 HD 患者受影响的大脑中是一项重大挑战。非常需要能够降低 mHTT 水平的分子化合物来治疗 HD 的细胞机制。促进 mHTT 清除对于改变 HD 病理学非常有意义，因为它们可以降低 mHTT 蛋白和由此产生的有毒物质的水平，从而影响疾病的进展。可以指导 mHTT 通过两个主要的蛋白质清除途径 - 泛素-蛋白酶体进行清除 Nemo 样激酶 (NLK) 是一种进化上保守的丝氨酸/苏氨酸激酶。我们发现 NLK 水平在中枢神经系统中表达相对较高。我们进一步证明了 NLK 与 HTT 相互作用并以激酶依赖性方式降低人体细胞内源性 mHTT 水平。小鼠纹状体中的 NLK 可以减轻 HD 病理学，而 NLK 水平的遗传降低此外，我们发现 NLK 增强了 mHTT 泛素化，表明 NLK 可能促进 mHTT 降解。支持 NLK 在改变疾病进展方面具有神经保护作用，并且是一种潜在的新型药物降低 mHTT 水平的药物靶点在目标 1 中，我们将开发两种互补的高通量药物。用于鉴定 NLK 激活剂的筛选 (HTS) 测定，包括体外基于酶的 HTRF 测定目标 2：我们将进行一项试点 HTS。 NLK 激活剂的活动，并使用 9900 种 CNS 焦点化合物库进行确认。