An HTS Assay Based on Neuronal Human Cells to Identify Small Molecule Activators

描述（由申请人提供）：仍然缺乏对神经退行性疾病（如阿尔茨海默氏症）的有效治疗。只有边缘性肿瘤学治疗的数据可用。阿尔茨海默氏症协会和包括NIH在内的其他卫生机构估计，仅在美国，阿尔茨海默氏症患者管理的经济成本就高达数十亿美元。此外，病人家属的心理和物质负担也是这一毁灭性疾病造成的额外和持续损害的原因。最近的统计数字表明，只要将这一疾病的速度减缓几年，就会对这一巨大疾病的经济和社会后果产生巨大影响。虽然，已经在病因和有效治疗的研究中部署了相当大的努力和资源，但这种疾病的治愈方法还远远没有被发现。确实需要新的想法和办法。在这种情况下，我们的建议是完全新颖的，可以打开一个新的领域，以推进知识和接近这些条件的可能性。有几个迹象表明，NF-κ B系统在神经元的弹性和细胞学习引起的变化，如长时程增强和抑郁症中发挥作用。一些报告表明，敲除脑中的NF-kB活性会导致对毒性刺激（如淀粉样蛋白和兴奋性氨基酸）和创伤的敏感性。此外，NF-κ B的活化是已知的抗凋亡机制。基于这些前提，我们相信有空间来研究NF-kB上调可能在减轻神经退行性现象和学习记忆障碍方面具有价值的可能性，这是一种独特的可能性。我们已经开发了一个原始的神经元细胞为基础的测定，将评估NF-κ B的上调使用荧光素酶报告。我们建议使用该测定法对我们可用的小分子文库进行高通量筛选。我们将筛选这些文库以鉴定能够增加NF-kB表达的小分子（通常占总化合物的2-5%，将成为命中物）。我们计划在筛选之后对所选化合物进行体外验证，这些参数包括NF-κ B表达、功能、体外神经毒性范例，以验证所鉴定的化合物实际上具有神经保护和/或神经营养作用。我们相信，作为拟议研究的结果，我们将确定1）可用作研究工具的新型小分子，以研究NF-kB对信号通路的影响; 2）可能的药物线索，可以作为更大努力的发射台，旨在开发和测试活性和安全的化合物，以帮助对抗神经退行性疾病。阿尔茨海默氏症和其他神经退行性疾病的治疗选择相当有限。它们通常是症状性的，而不是治疗性的，而且一般来说，它们的效果很差。阿尔茨海默氏症协会和NIH的统计数据估计，管理这些患者的成本上升到数十亿美元。这些疾病对社会的破坏性影响的一个重要部分是对家庭的经济和心理后果。这是由于昂贵的长期护理和目睹这些毁灭性的疾病对他们所爱的人的影响。据计算，如果阿尔茨海默病的进展可以推迟几年，在经济和心理层面的影响将是巨大的。最近的证据指出，NF-κ B信号系统参与神经元的恢复力和它们在不同的损伤中存活的能力以及学习和记忆的神经元分子相关性。NF-kB分子消融已经表明，来自这些动物的神经元对包括创伤、淀粉样蛋白毒性和兴奋性毒性在内的损伤更敏感。来自神经变性的非凋亡和凋亡模型的直接神经保护作用也归因于NF-κ B信号传导的直接或间接激活。此外，NF-kB信号通路参与了长时程增强和长时程抑制的建立，这两种学习和记忆模型发生在细胞水平。因此，神经元中的NF-κ B上调可用于同时攻击与阿尔茨海默病中所见的学习和记忆丧失相关的神经元变性和神经元功能恶化。为了追求这个宏伟的范围，我们已经开发了一个原始的神经元细胞为基础的测定，将评估NF-κ B上调使用荧光素酶报告。我们建议使用该测定法对我们可用的小分子文库进行高通量筛选。我们将筛选该文库以鉴定能够增加NF-kB表达的小分子（通常占总化合物的2-5%，将成为命中物）。我们将在筛选之后，对所选化合物进行体外测试，测试的参数包括NF-κ B表达、功能、体外神经毒性范例，以验证所鉴定的化合物实际上具有神经保护和/或神经营养作用。我们相信，作为拟议研究的结果，我们将确定1）可用作研究工具的新型小分子，以研究NF-kB对信号通路的影响; 2）可能的药物线索，可以作为更大努力的发射台，旨在开发和测试活性和安全的化合物，以帮助对抗神经退行性疾病。

DESCRIPTION (provided by applicant): Effective treatment for neurodegenerative diseases, such as Alzheimer's, is still lacking. Only, marginal symptomatologic treatment is available to data. Alzheimer's association and other Health agencies, including NIH, have estimated that the economic cost of Alzheimer's disease patients management in the sole USA amounts to billions. Also the psychological and material burden on the patients' families accounts for additional and consistent damage done by this devastating disease. Recent statistics have calculated that just slowing down the disease by a few years will have an enormous impact on the economics and social consequences of this tremendous disease. Although, considerable efforts and resources have been deployed in the research for the causes and for an effective treatment, a cure for this disease is far from being discovered. New ideas and approaches are needed, indeed. In this context our proposal is completely novel and can open a new field to advance both knowledge and possibilities to approach these conditions. There are several indications that NF-kB system plays a role in neuronal resilience and in the changes induced by cellular learning such as long term potentiation and depression. Several reports have shown that knocking out NF-kB activity in the brain causes sensitization to toxic stimuli, such as ¿-amyloid, and excitatory aminoacids and to trauma. In addition, activation of NF-kB is a known anti-apoptosis mechanism. Based on these premises, we believe there is room to investigate the possibility that NF-kB up regulation could be of value in mitigating both neurodegenerative phenomenon and learning and memory failure, a unique possibility. We have developed an original neuronal cell-based assay that will assess NF-kB up-regulation using a luciferase reporter. We propose to use this assay to perform a high throughput screen of small molecules libraries available to us. We will screen these library to identify small molecules able to increase NF-kB expression (usually 2-5% of the total compounds, will turn out as hits). We plan to follow up the screening with in vitro validation of the selected compounds on a number of parameters which will include NF-kB expression, function, in vitro neurotoxicity paradigms to verify that the identified compounds have, in fact, a neuroprotective and/or a neurotrophic effect. We believe that as a result of the proposed studies we will have identified 1) novel small molecules that can be used as research tools to study the effects of NF-kB on signaling pathways; 2) possible drug leads that can serve as a launch pad for a larger effort aiming to develop and test active and safe compounds to help in the battle against neurodegenerative disorders. Treatment options for Alzheimer's and other neurodegenerative disorders are fairly limited. They are usually symptomatic rather than curative, and in general their effectiveness is poor. Statistics from the Alzheimer's association and from the NIH have assessed that the cost for the management of this patients rises to billions. An important part of the socially debilitating effect of these disorders is the consequences, both economic and psychological, on the families. This is due to both the expensive long term care and to the witnessing of the effect that these devastating disorders have on their loved one. It has been calculated that if the progression of Alzheimer disease could be delayed even by a few years the repercussions both at economic and psychological level will be enormous. Recent evidence have pointed out that NF-kB signaling system is involved in the resilience of neurons and in their ability to survive disparate insults and in neuronal molecular correlates of learning and memory. NF-kB molecular ablation has shown that neurons from these animals are more sensitive to insults including trauma, ¿-amyloid toxicity and excitotoxicity. Also a direct neuroprotective effect from both non apoptotic and apoptotic models of neurodegeneration has been attributed to direct or indirect activation of NF-kB signaling. In addition, NF-kB signaling has been involved in the establishment of long term potentiation and long term depression, two models of learning and memory that take place at cellular level. Therefore, NF-kB up regulation in neurons could be useful in attacking at the same time neuronal degeneration and deterioration of neuronal functions associated with loss of learning and memory as seen in Alzheimer's disease. To pursue this grand scope, we have developed an original neuronal cell-based assay that will assess NF-kB up-regulation using a lucipherase reporter. We propose to use this assay to perform a high throughput screen of small molecules libraries available to us. We will screen this library to identify small molecules able to increase NF-kB expression (usually 2-5% of the total compounds, will turn out as hits). We will follow up the screening with in vitro testing of the selected compounds on a number of parameters which will include NF-kB expression, function, in vitro neurotoxicity paradigms to verify that the identified compounds have, in fact, a neuroprotective and/or a neurotrophic effect. We believe that as a result of the proposed studies we will have identified 1) novel small molecules that can be used as research tools to study the effects of NF-kB on signaling pathways; 2) possible drug leads that can serve as a launch pad for a larger effort aiming to develop and test active and safe compounds to help in the battle against neurodegenerative disorders.