High-throughput screening for NAD+-replenishing neuroprotective compounds

描述：NAD+耗竭导致脑缺血/再灌注损伤、沃勒变性、多发性硬化症等啮齿动物模型中的神经元死亡，并发生在兴奋性毒性侮辱和氧化应激之后。我们最近发现，NAD+缺失也是由一种错误折叠和有毒形式的淀粉样蛋白(TPrP)诱导的神经元死亡的主要原因，首次表明NAD+缺失在蛋白质错误折叠神经退行性疾病(PMND)的发病机制中所起的作用，PMND是一个包括阿尔茨海默病、帕金森病和肌萎缩侧索硬化症等疾病的家族。我们发现，NAD+的补充逆转了培养中TPrP损伤的退化神经元的命运，并改善了PrP病小鼠的运动功能。我们的假设是，NAD+补充是一种新的治疗策略，可以为许多衰弱的大脑疾病提供神经保护和神经恢复的好处。以TPrP诱导的神经元死亡为模型，我们发展了一种高通量筛选(HTS)方法，以发现通过NAD+补充来恢复神经元活力的小分子。在384孔板格式中优化的初级和确认性分析满足HTS准备标准(Z‘&gt；0.5，重复性，DMSO耐受性)。我们已经使用带有一小部分结构不同的小分子(LOPAC1280TM)的试点屏幕证明了初级分析的操作稳定性。我们建议将化验转移到位于佛罗里达州斯克里普斯的分子图书馆生产中心网络(MLPCN)实验室，用于斯克里普斯药物发现文库160K子集的小型化和筛选。然后，我们将根据细胞内NAD+恢复的特异性、EC50/TC50以及培养的原代神经元和体内的活性来选择HITS。我们还将根据化学物质的易操纵性、选择性的潜力和药物的相似性来确定命中的优先顺序。在我们计划的这一药物化学部分，我们还将研究类似物以提高效果。作用模式研究将确定选定化合物的细胞靶点，并提供与NAD+耗竭有关的退化途径的见解。我们协调的多学科计划满足了美国国立卫生研究院项目公告PAR-12-058(“高通量筛查(HTS)发现化学探针的化验征集”)的所有目标。我们的团队将提供一套部分优化的、新颖的、靶向特异的和易处理的小分子，适合开发成新的先导化合物，用于多种大脑疾病的神经保护治疗。此外，这些化合物将广泛用作分子探针，用于确定治疗机会，并研究与NAD+代谢失败有关的大脑疾病中神经元死亡的机制。

DESCRIPTION: NAD+ depletion causes neuronal death in rodent models of brain ischemia/reperfusion injury, Wallerian degeneration, multiple sclerosis and occurs after excitotoxic insults and oxidative stress. We have recently discovered that NAD+ depletion is also the primary cause of neuronal death induced by a misfolded and toxic form of the amyloidogenic prion protein (TPrP), showing for the first time the role of NAD+ depletion in the pathogenesis of a protein misfolding neurodegenerative disease (PMND), a family of diseases that comprise, among others, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. We showed that NAD+ replenishment reverses the fate of TPrP-injured degenerating neurons in culture and improves motor function in a mouse model of prion disease. Our hypothesis is that NAD+ replenishment is a novel therapeutic strategy for providing neuroprotective and neurorestorative benefits for many debilitating brain conditions. With TPrP-induced neuronal death as a model, we developed a high throughput screening (HTS) approach to discover small molecules restoring neuronal viability by NAD+ replenishment. The primary and confirmatory assays, optimized in the 384-well plate format, fulfill the criteria of HTS-readiness (Z'>0.5, reproducibility, DMSO tolerance). We have demonstrated the operational robustness of the primary assay using a pilot screen with a small collection of structurally diverse small molecules (LOPAC1280TM). We propose to transfer the assays to the Molecular Libraries Production Centers Network (MLPCN) laboratory at Scripps Florida for miniaturization and screening of a 160K subset of the Scripps Drug Discovery Library. We will then select hits based on the specificity of intracellular NAD+ restoration, EC50/TC50 and activity in cultured primary neurons and in vivo. We will also prioritize hits based upon chemical tractability, potential for selectivity, and drug-likeness. In this medicinal chemistry component of our plan we will also study analogs to improve effects. Mode of action studies will identify cellular targets o selected compounds and provide insights into degenerative pathways linked to NAD+ depletion. Our coordinated multidisciplinary plan addresses all objectives of the NIH program announcement PAR-12-058 ("Solicitation of Assays for High Throughput Screening (HTS) to Discover Chemical Probes"). Our team will deliver a set of partially optimized, novel, target-specific and tractable small molecules suitable for development into novel lead compounds for neuroprotective therapy in a number of brain conditions. Moreover, these compounds will be widely useful as molecular probes for identifying therapeutic opportunities and for studying the mechanisms of neuronal death in brain conditions linked to a failure of NAD+ metabolism.