Novel Hsp90 Inhibitors: Proof-of-Principle for Treatment of Motor Neuron Diseases

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS或Lou Gherig's disease）是一种成人发病的运动神经元疾病，其特征是运动功能的进行性丧失和呼吸功能不全导致的死亡，通常在诊断后5年内发生。没有适当的治疗方法。大多数ALS病例是散发性的；然而，一些基因位点与家族性形式的疾病有关，最常见的（fALS1）是编码Cu/ zn超氧化物歧化酶（SOD1）基因的显性遗传突变。fALS1的实验模型用于化合物的临床前测试，最终用于治疗散发性和家族性ALS。鉴于ALS可由多种且大多不明的损伤引起，常见的疾病进展机制和一般的神经保护途径是干预的目标。目标/假设：长期目标是开发一种基于抑制Hsp90的新化合物的ALS和其他运动神经元疾病的治疗方法，Hsp90是一种热休克蛋白（HSP），在调节异常构象蛋白的折叠和转换中起关键作用。潜在的假设是，这些化合物将通过两种有助于蛋白质质量控制的机制发挥作用：(1)上调具有蛋白伴侣和重折叠功能的多种热休克蛋白（HSPs），从而阻止受损蛋白（由基因突变或表观遗传修饰引起）采用有毒构象和聚集；(2)促进Hsp90客户蛋白（包括突变蛋白和错误折叠蛋白）的降解，从而减轻毒性负担。短期目标是评估我们假定的先导化合物NXD30001在原代组织培养模型和fALS转基因小鼠模型中的应用。实现我们的目标将有助于进一步实现该研究所减轻神经系统疾病负担的使命。具体目的1：在现有的ALS体外原代培养模型中测试NXD30001。通过(1)在小鼠胚胎脊髓-背根神经节（DRG）解离培养的运动神经元中表达引起als的突变SOD1蛋白，以及(2)从突变SOD1转基因小鼠胚胎制备脊髓-DRG培养物，建立实验模型。这些实验将确定NXD30001在运动神经元中的生物活性，特别是HSPs的表达和Hsp90客户蛋白的增加，并将评估NXD30001在预防fals引起的SOD1突变体毒性方面的有效性。具体目标2。在als动物模型中进行概念验证研究，以确定NXD30001化合物的体内效用。NXD30001的疗效将在表达致病SOD1突变体SOD1G93A的转基因小鼠中进行评估。将评估两种剂量方案延缓运动神经元疾病的发病和进展，使用Hsp90客户蛋白水平的诱导和Hsp90客户蛋白水平的降低作为NXD30001脑和脊髓生物活性的标志物。

DESCRIPTION (provided by applicant): Amyotrophic Lateral Sclerosis (ALS or Lou Gherig's disease) is an adult onset motor neuron disease characterized by progressive loss of motor function and death from respiratory insufficiency, usually within five years of diagnosis. No adequate treatment exists. Most cases of ALS are sporadic; however, several genetic loci have been linked to familial forms of the disease, the most common (fALS1) being dominantly inherited mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1). Experimental models of fALS1 are used in preclinical testing of compounds for eventual use in treating sporadic as well as familial ALS. Given that ALS can be caused by multiple, mostly unidentified insults, common mechanisms of disease progression and general neuroprotective pathways are targets for intervention. Objective/Hypothesis: The long term objective is to develop a therapy for ALS and other motor neuron disorders based on a novel series of compounds that inhibit Hsp90, a heat shock protein (HSP) that plays a pivotal role in regulating folding and turnover of proteins with aberrant conformation. The underlying hypothesis is that these compounds will be efficacious through two mechanisms that contribute to protein quality control: (1) upregulating multiple heat shock proteins (HSPs) with protein chaperoning and refolding function, thereby preventing damaged proteins (arising from genetic mutation or epigenetic modification) from adopting toxic conformations and aggregating, and (2) promoting degradation of Hsp90 client proteins, including mutant and misfolded proteins, thereby reducing the toxic burden. The short term objective is to evaluate our presumptive lead compound, NXD30001 in primary tissue culture models and a transgenic mouse model of fALS. The realization of our goals will help further the mission of the NINDS, to reduce the burden of neurologic disease. Specific Aim 1: To test NXD30001 in existing in vitro primary culture models of ALS. Test-models are established by (1) expressing ALS-causing mutant SOD1 proteins in motor neurons of dissociated cultures of embryonic murine spinal cord-dorsal root ganglia (DRG), and (2) preparing spinal cord-DRG cultures from mutant SOD1 transgenic mouse embryos. These experiments will establish bioactivity of NXD30001 in motor neurons, specifically expression of HSPs and increased turnover of Hsp90 client proteins, and will evaluate the effectiveness of NXD30001 in preventing toxicity of fALS-causing SOD1 mutants. Specific Aim 2. Perform proof-of-concept studies in an fALS animal model to determine the in vivo utility of the NXD30001 compound. The efficacy of NXD30001 will be assessed in transgenic mice expressing the disease-causing SOD1 mutant, SOD1G93A. Two dosage regimens will be evaluated for delaying onset and progression of motor neuron disease, using induction of HSPs and reduction in levels of of Hsp90 client proteins as markers of NXD30001 bioactivity in brain and spinal cord. PUBLIC HEALTH RELEVANCE: The only approved drug for treating Amyotrophic Lateral Sclerosis (ALS), riluzole, provides minimal benefit for this fatal disease. With an incidence of 1-2 per 100,000, ALS represents a serious unmet medical need, which will only grow as the population ages. The proposed research may provide proof-of-principle evidence for the inhibition of heat shock protein 90 (Hsp90) as a potential approach for treating ALS, and will likely accelerate the process of discovery and development of novel therapeutics for ALS and other motor neuron disorders.