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

基于人类神经元细胞的 HTS 测定法来鉴定小分子激活剂

• 批准号: 7364350
• 负责人: Maurizio Grimaldi
• 金额: $ 2.5万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7364350
• 关键词: Ablation; Accounting; Age; Aging; Alzheimer' s Disease; Amyloid; Animal Model; Animals; Apoptosis; Apoptotic; Assisted Living Facilities; Biological Assay; Boxing; Brain; Brain Diseases; Cell model; Cells; Chemistry; Cognitive; Condition; Data; Deterioration; Development; Disease; Disease Management; Disease Progression; Early Diagnosis; Economics; Effectiveness; Evolution; Excitatory Amino Acids; Failure; Family; Goals; Health; Healthcare; Healthcare Systems; Housing; Human; Immune System Diseases; Impaired cognition; In Vitro; Inflammatory; Institution; Intervention; Investments; Knock-out; Knowledge; Laboratories; Lead; Learning; Libraries; Life; Link; Long-Term Care; Long-Term Depression; Long-Term Potentiation; Love; Luciferases; Malignant Neoplasms; Marketing; Medical Surveillance; Memory; Memory Loss; Mental Depression; Mental Health; Modeling; Molecular; NF-kappa B; Nerve Degeneration; Nervous System Physiology; Neuraxis; Neurobiology; Neurodegenerative Disorders; Neurons; Neuropharmacology; Nuclear; Numbers; Pathogenesis; Pathology; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacological Treatment; Play; Population; Positioning Attribute; Reporter; Reporting; Research; Research Institute; Research Personnel; Resources; Role; Running; Screening procedure; Seizures; Self Care; Signal Pathway; Signal Transduction; Stimulus; Stroke; Symptoms; System; Testing; Thinking; Time; Toxic effect; Transcriptional Activation; Trauma; United States National Institutes of Health; Up-Regulation; Validation; base; cost; drug development; excitotoxicity; experience; follow-up; high throughput screening; improved; inhibitor/antagonist; interest; nervous system disorder; neuron apoptosis; neurotoxicity; novel; prototype; psychologic; repository; resilience; response; small molecule; small molecule libraries; social; statistics; success; tool

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.

描述(申请人提供)：目前仍缺乏对阿尔茨海默氏症等神经退行性疾病的有效治疗。只有边际对症治疗才能获得数据。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，管理阿尔茨海默病患者的经济成本就高达数十亿美元。此外，患者家属的心理和物质负担也是这种毁灭性疾病造成的额外和持续的损害。最近的统计数据计算出，仅仅将疾病延缓几年，就会对这种巨大疾病的经济和社会后果产生巨大影响。虽然已经投入了大量的精力和资源来研究病因和有效的治疗方法，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方法。在这方面，我们的建议是完全新颖的，可以开辟一个新的领域，以促进知识和接近这些条件的可能性。有多个迹象表明，核因子-kB系统在神经元弹性和细胞学习引起的变化中发挥作用，如长时程增强和抑制。一些报告表明，敲除大脑中的核因子-kB活性会导致对有毒刺激，如淀粉样蛋白、兴奋性氨基酸和创伤的敏感化。此外，核因子-kB的激活也是一种已知的抗细胞凋亡机制。基于这些前提，我们认为有空间来研究核因子-kB上调在减轻神经退行性现象和学习记忆衰竭方面的价值的可能性，这是一种独特的可能性。我们已经开发了一种原创的基于神经细胞的分析方法，它将使用荧光素酶报告来评估核因子-kB的上调。我们建议使用这种方法对我们可用的小分子文库进行高通量筛选。我们将对这些文库进行筛选，以确定能够增加核因子-kB表达的小分子(通常占总化合物的2-5%，最终将成为HITS)。我们计划在筛选的基础上，对选定的化合物进行一系列参数的体外验证，包括核因子-kB的表达、功能、体外神经毒性范例，以验证已识别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，由于拟议的研究，我们将确定1)可用作研究工具的新型小分子，以研究核因子-kB对信号通路的影响；2)可能的药物先导，可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。它们通常是有症状的，而不是治愈的，总体来说，它们的有效性很差。阿尔茨海默氏症协会和美国国立卫生研究院的统计数据估计，管理这些患者的成本高达数十亿美元。这些疾病的社会衰弱效应的一个重要部分是对家庭的经济和心理后果。这既是因为昂贵的长期护理，也是因为人们目睹了这些毁灭性的疾病对他们所爱的人造成的影响。据计算，如果阿尔茨海默病的进展即使推迟几年，在经济和心理层面上的影响都将是巨大的。最近的证据表明，核因子-kB信号系统参与了神经元的弹性，以及它们在不同的损伤中生存的能力，并参与了神经元学习和记忆的分子相关性。核因子-kB分子消融表明，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伤害更敏感。此外，神经退行性变的非凋亡和凋亡模型的直接神经保护作用被归因于直接或间接激活核因子-kB信号。此外，核因子-kB信号参与了长时程增强和长时程抑制的建立，这是发生在细胞水平上的两种学习和记忆模型。因此，神经元中核因子-kB的上调可能有助于同时攻击与阿尔茨海默病所见的学习和记忆丧失相关的神经元退化和神经元功能恶化。为了追求这一宏大范围，我们开发了一种原创的基于神经细胞的分析方法，该方法将使用荧光素酶报告程序评估核因子-kB的上调。我们建议使用这种方法对我们可用的小分子文库进行高通量筛选。我们将对这个文库进行筛选，以确定能够增加核因子-kB表达的小分子(通常是总化合物的2-5%，最终会成为HITS)。我们将继续进行筛选，对选定的化合物进行一系列参数的体外测试，包括核因子-kB的表达、功能、体外神经毒性范例，以验证已识别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，由于拟议的研究，我们将确定1)可用作研究工具的新型小分子，以研究核因子-kB对信号通路的影响；2)可能的药物先导，可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。

项目成果

Identification of novel small molecule activators of nuclear factor-κB with neuroprotective action via high-throughput screening.

• DOI: 10.1002/jnr.22526
• 发表时间: 2011-01
• 期刊: JOURNAL OF NEUROSCIENCE RESEARCH
• 影响因子: 4.2
• 作者: Manuvakhova, Marina S.;Johnson, Guyla G.;White, Misti C.;Ananthan, Subramaniam;Sosa, Melinda;Maddox, Clinton;McKellip, Sara;Rasmussen, Lynn;Wennerberg, Krister;Hobrath, Judith V.;White, E. Lucile;Maddry, Joseph A.;Grimaldi, Maurizio
• 通讯作者: Grimaldi, Maurizio