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

点击翻译按钮获取中文摘要

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.