High-throughput screening for NAD+-replenishing neuroprotective compounds

产品说明：NAD+耗竭导致脑缺血/再灌注损伤、沃勒变性、多发性硬化的啮齿动物模型中的神经元死亡，并且发生在兴奋性毒性损伤和氧化应激之后。我们最近发现，NAD+耗竭也是由错误折叠和毒性形式的淀粉样蛋白原朊蛋白（TPrP）诱导的神经元死亡的主要原因，首次显示了NAD+耗竭在蛋白质错误折叠神经退行性疾病（PMND）的发病机制中的作用，PMND是一种疾病家族，包括阿尔茨海默病、帕金森病和肌萎缩性侧索硬化症等。我们发现，NAD+补充逆转了培养物中TPrP损伤的退化神经元的命运，并改善了朊病毒病小鼠模型的运动功能。我们的假设是，NAD+补充是一种新的治疗策略，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，我们开发了一种高通量筛选（HTS）方法来发现通过NAD+补充恢复神经元活力的小分子。在384孔板格式中优化的初步和验证性测定满足HTS就绪性的标准（Z '> 0.5，再现性，DMSO耐受性）。我们已经证明了初步试验的操作稳健性，使用一个小的结构多样的小分子（LOPAC 1280 TM）的集合中试筛选。我们建议将检测转移到位于佛罗里达斯克里普斯的分子库生产中心网络（MLPCN）实验室，以进行小型化和筛选斯克里普斯药物发现库的160 K子集。然后，我们将基于细胞内NAD+恢复的特异性、EC 50/TC 50和在培养的原代神经元和体内的活性来选择命中。我们还将根据化学易处理性，选择性潜力和药物相似性优先考虑命中。在我们计划的药物化学部分中，我们还将研究类似物以改善效果。作用模式研究将确定选定化合物的细胞靶点，并提供与NAD+耗竭相关的退行性途径的见解。我们协调的多学科计划解决了NIH计划公告PAR-12-058（“高通量筛选（HTS）发现化学探针的试验征集”）的所有目标。我们的团队将提供一组部分优化的，新颖的，靶向特异性和易处理的小分子，适合开发成用于多种脑部疾病神经保护治疗的新型先导化合物。此外，这些化合物将广泛用作分子探针，用于鉴定治疗机会和用于研究与NAD+代谢失败相关的脑病症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.