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

描述(申请人提供)：肌萎缩侧索硬化症(ALS或Lou Gherig病)是一种成人起病的运动神经元病，特征是进行性运动功能丧失和呼吸功能不全死亡，通常在确诊后五年内。不存在适当的治疗方法。大多数肌萎缩侧索硬化症是散发性的；然而，有几个遗传位点与这种疾病的家族性形式有关，最常见的(FALS1)是编码铜/锌超氧化物歧化酶(SOD1)的基因的主要遗传突变。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。鉴于肌萎缩侧索硬化症可以由多种原因不明的侮辱引起，常见的疾病进展机制和一般神经保护途径是干预的目标。目的/假设：长期目标是开发一种基于一系列新化合物的ALS和其他运动神经元疾病的治疗方法，该化合物抑制Hsp90，Hsp90是一种热休克蛋白(HSP)，在调节构象异常的蛋白质的折叠和周转方面发挥关键作用。基本的假设是，这些化合物将通过两种有助于蛋白质质量控制的机制有效：(1)上调多个具有蛋白质伴侣和复性功能的热休克蛋白(HSPs)，从而防止受损的蛋白质(由基因突变或表观遗传修饰引起)采用有毒构象和聚集，以及(2)促进Hsp90客户蛋白的降解，包括突变和错误折叠的蛋白质，从而减少毒性负担。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。我们目标的实现将有助于推进NINDS的使命，减轻神经系统疾病的负担。具体目的1：检测NXD30001在已有的ALS体外原代培养模型中的作用。通过(1)在分离培养的胚胎小鼠脊髓-背根神经节(DRG)运动神经元中表达导致ALS的突变SOD1蛋白，以及(2)从突变的SOD1转基因小鼠胚胎制备脊髓-DRG培养物，建立测试模型。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在预防导致FALS的SOD1突变体毒性方面的有效性。具体目的2.在FALS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。将评估两种给药方案对延缓运动神经元病的发生和进展的作用，将热休克蛋白的诱导和Hsp90客户蛋白水平的降低作为NXD30001在脑和脊髓中生物活性的标志。 公共卫生相关性：唯一被批准用于治疗肌萎缩侧索硬化症(ALS)的药物利鲁唑对这种致命疾病的益处微乎其微。ALS的发病率为每10万人中有1-2人，这是一种严重的未得到满足的医疗需求，随着人口老龄化，这种需求只会增加。这项研究可能为抑制热休克蛋白90(Hsp90)作为治疗ALS的潜在方法提供原则证据，并可能加速发现和开发治疗ALS和其他运动神经元疾病的新疗法的进程。

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.