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

项目摘要 亨廷顿病(HD)是由亨廷顿(HTT)基因突变引起的，该基因编码 突变的亨廷顿蛋白(MHTT)与扩展的聚谷氨酰胺链(PolyQ)。有毒物质的获得 MHTT功能异常是引起HD的主要原因。降低mHTT可能提供一种有效的治疗方法 通过改善其下游毒性来治疗HD。如果成功，这一策略可能会改变疾病 人类多动症的进展。事实上，通过各种遗传方法降低HTT水平一直是 显示能有效地减轻HD模型中的mHTT毒性。然而，遗传方法仍然面临着 在有效地向HD患者的受影响脑区提供服务方面面临重大挑战。因此，很小 可以降低mHTT水平的分子化合物是治疗HD的首选药物。细胞机制 促进mHTT清除对修改HD病理非常有意义，因为它们可以降低 MHTT蛋白和由此产生的有毒物种的水平，从而影响疾病的进展。泛素化 可以通过两条主要的蛋白质清除途径--泛素-蛋白酶体--引导mHTT清除 系统和自噬。NEMO样激酶(NLK)是一种进化上保守的丝氨酸/苏氨酸激酶 在中枢神经系统中的表达水平相对较高。我们发现NLK水平是 HD死后脑组织中的含量显著降低。我们进一步证明了NLK与HTT相互作用 并以一种依赖于激酶的方式降低人体细胞内源性mHTT水平。过度表达 小鼠纹状体中的NLK可减轻HD的病理，而NLK水平的遗传降低 加重HD小鼠的神经病理。此外，我们还发现NLK增强了mHTT 泛素化，表明NLK可能促进mHTT的降解。我们的临床前成果强劲 支持NLK在抑制疾病进展中具有神经保护作用，是一种潜在的新药 降低mHTT水平的药物靶点。在目标1中，我们将开发两个互补高通量 筛选(HTS)试验鉴定NLK激活剂，包括体外基于酶的HTRF试验 以及一种基于细胞的Bret试验来监测NLK同源二聚。目标2：我们将进行HTS试点 NLK激活剂活动，并使用9900个CNS焦点化合物库进行确认。

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.