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.

项目摘要亨廷顿氏病（HD）是由亨廷顿（HTT）基因的突变引起的，该基因编码该基因突变的亨廷顿蛋白（MHTT），具有扩展的聚谷氨酰胺道（Polyq）。有毒的收益 MHTT的功能是HD的主要原因。降低MHTT可能会提供有效的方法通过改善其下游毒性来治疗高清。如果成功，该策略可能会改变疾病人类高清的进展。实际上，通过多种遗传方法降低HTT水平已经显示可有效减轻HD模型中的MHTT毒性。但是，遗传方法仍然面对有效地向受影响的HD患者大脑区域的有效分娩方面面临重大挑战。那很小可以降低MHTT水平的分子化合物非常需要治疗HD。细胞机制促进MHTT清除对修改HD病理具有极大的兴趣，因为它们可以降低 MHTT蛋白和产生的有毒物种的水平，从而影响疾病的进展。泛素化可以通过两种主要蛋白质清除途径引导MHTT清除 - 泛素 - 蛋白酶体系统和自噬。线性样激酶（NLK）是一种进化配置的序列/苏氨酸激酶并在中枢神经系统中以相对较高的水平表达。我们发现NLK级别是高清验尸大脑的高清大大改善。我们进一步证明了NLK与HTT相互作用并在人类细胞中以激酶依赖性方式降低内源性MHTT水平。过表达小鼠纹状体中的NLK减弱了HD病理，而NLK水平的遗传降低加剧了HD小鼠的神经病理学。此外，我们发现NLK增强了MHTT 泛素化，表明NLK可能有助于MHTT降解。我们的临床前结果强烈 NLK在改变疾病进展中具有神经保护作用的支持，并且是一种潜在的新颖降低MHTT水平的药物目标。在AIM 1中，我们将开发两个完整的高通量筛选（HTS）测定以识别NLK激活剂，包括基于体外酶促的HTRF测定法以及基于细胞的BRET测定法以监测NLK均二聚化。在AIM 2：我们将进行飞行员HTS 使用9900 CNS焦点化合物的库进行NLK激活剂的活动并命中确认。