Novel small molecules up-regulators of NF-kB activity: effect on MnSOD expression

描述（由申请人提供）：头部和脊柱钝性创伤引起的脑损伤，包括创伤性脑损伤（TBI）、脑震荡、脊髓损伤，伴有脑内氧化应激增加。增加的氧化剂产生被细胞酶如锰超氧化物歧化酶（MnSOD）的活性抵消。MnSOD是参与危险的自由基氧物种（ROS），特别是超氧化物歧化的主要线粒体酶。由于ROS增加导致MnSOD失活，随后是酶的去除，并通过硝化发出信号。ROS的过度产生与所有脑创伤性疾病有关，如TBI，脊髓损伤和脑震荡。此外，MnSOD失效与散发性和遗传决定的神经退行性疾病有关。有趣的是，MnSOD是由NF-kB如p65和Rel在脑中非常丰富的NF-kB的激活特异性诱导的。考虑到这一有趣的背景以及TBI和其他与ROS增加相关的类似疾病的治疗选择有限且大多是支持性的事实，我们建议研究通过小分子诱导更高水平的MnSOD的可能性，作为治疗此类疾病的可能机制。这种小分子的开发可能与未来的药物开发相关，不仅可以使创伤性脑损伤患者受益，还可以使ROS损伤相关的神经退行性疾病患者受益。 我们已经建立了一个人类神经元系统，以发现上调NF-κ B表达的小分子，并通过压倒性的抑制机制引起其激活。我们已经成功地实施并小型化了该测定，以在高密度板中进行机器人液体处理，这对于执行大的小分子文库的筛选是理想的（参见初步数据）。我们已经进行了30万个化合物筛选，部分由NIH路线图计划和NIMH以及校内SRI研究计划资助。我们鉴定了约1647种命中化合物。对这些化合物的聚类分析揭示了14种感兴趣的化学支架。属于这14类的原型化合物和4种高效力单元素化合物已在脑细胞的二级测定中进行了测试。8种化合物已显示增加星形胶质细胞和神经元中NF-κ B p65的长期核存在，并引起神经元MnSOD增加。另外5种化合物（迄今为止都进行了测试）在星形胶质细胞中无活性，已显示出极大地增加神经元中的MnSOD活性。后者中的两种已被证明是强大的神经保护剂。我们建议研究这些化合物和其他从他们的发展，以确定其对原代神经元中MnSOD表达的影响，并评估其在创伤性脑损伤和神经退行性变的体外模型中的有益潜力。到两年结束时，我们可以在动物研究中进行后续研究，旨在验证TBI，脊髓损伤和ROS依赖性神经退行性疾病模型的结局改善。从这些研究中收集的证据可以衍生出一个更大的药物发现计划。 公共卫生相关性：由头部和脊柱的钝性创伤引起的脑痛苦，包括创伤性脑损伤（TBI）、脑震荡、脊髓损伤，伴有脑中氧化应激增加。增加的氧化剂产生被细胞酶如锰超氧化物歧化酶（MnSOD）的活性抵消。MnSOD是参与危险的自由基氧物种（ROS），特别是超氧化物歧化的主要线粒体酶。由于ROS增加导致MnSOD失活，随后是酶的去除，并通过硝化发出信号。ROS的过度产生与所有脑创伤性疾病有关，如TBI，脊髓损伤和脑震荡。MnSOD是通过激活NF-κ B特异性诱导的。我们已经发现了能够增加神经元中NF-κ B活性的化合物。 我们建议研究这些化合物和其他从他们的发展，以确定其对原代神经元中MnSOD表达的影响，并评估其在创伤性脑损伤和神经退行性变的体外模型中的有益潜力。到两年结束时，我们可以在动物研究中进行后续研究，旨在验证TBI，脊髓损伤和ROS依赖性神经退行性疾病模型的结局改善。从这些研究中收集的证据可以衍生出一个更大的药物发现计划。

DESCRIPTION (provided by applicant): Brain sufferance caused by blunt trauma to the head and to the spine, which includes traumatic brain injury (TBI), concussions, spinal cord injury is accompanied by increased oxidative stress in the brain. Increased oxidant generation is counteracted by the activity of cellular enzymes such manganese superoxide dismutase (MnSOD). MnSOD is major mitochondrial enzyme involved in the dismutation of dangerous radical oxygen species (ROS), specifically superoxide. Inactivation of MnSOD due to increased ROS is followed by the removal of the enzyme and is signaled by nitration. ROS overproduction has been associated with all brain traumatic conditions such as TBI, spinal cord injury and concussions. In addition MnSOD failure has been associated with neurodegenerative diseases both sporadic and genetically determined. Interestingly, MnSOD is specifically induced by activation of NF-kBs such as p65 and Rel very abundant NF-kB in the brain. Given this interesting background and the fact that treatment options for TBI and other similar conditions associated with increase ROS are limited and mostly supportive, we are proposing to study the possibility to induce higher levels of MnSOD via small molecules, as a possible mechanism of treating such conditions. The development of such small molecules could be relevant for future drug development which could benefit not only patients with traumatic brain injuries but also patients with ROS damage-associated neurodegenerative diseases. We have set up a human neuronal system to discover small molecules that up regulate NF-kB expression and causes its activation by overwhelming inhibitory mechanisms. We have successfully implemented and miniaturized this assay to robotic liquid handling in high density plates ideal to execute screening of large small molecules libraries (see preliminary data). We have run a 300,000 compound screening, partially funded by the NIH Roadmap program and NIMH, and by intramural SRI research program. We identified about 1647 hit compounds. Clustering analysis of these compounds has revealed 14 chemical scaffolds of interest. Prototype compounds belonging to these 14 classes and 4 high potency singletons have been tested in secondary assay in brain cells. 8 compounds have been shown to increase long term nuclear presence of NF-kB p65 in astrocytes and in neurons, and have caused neuronal MnSOD increase. Additional 5 compounds (all tested so far), inactive in astrocytes, have shown to greatly increase MnSOD activity in neurons. Two of the latter have shown to be powerful neuroprotectants. We propose to study these compounds and others obtained from their development to ascertain their effect on MnSOD expression in primary neurons and assess their beneficial potential in in vitro models of traumatic brain injury and neurodegeneration. By the end of the two years we could have leads to follow up in animal studies, aimed to verify improvement of the outcome in TBI, spinal cord injury and ROS dependent neurodegenerative models of disease. Evidence collected from these studies could spin off a larger drug discovery program. PUBLIC HEALTH RELEVANCE: Brain sufferance caused by blunt trauma to the head and to the spine, which includes traumatic brain injury (TBI), concussions, spinal cord injury is accompanied by increased oxidative stress in the brain. Increased oxidant generation is counteracted by the activity of cellular enzymes such manganese superoxide dismutase (MnSOD). MnSOD is major mitochondrial enzyme involved in the dismutation of dangerous radical oxygen species (ROS), specifically superoxide. Inactivation of MnSOD due to increased ROS is followed by the removal of the enzyme and is signaled by nitration. ROS overproduction has been associated with all brain traumatic conditions such as TBI, spinal cord injury and concussions. MnSOD is specifically induced by activation of NF- kBs. We have discovered compounds able to increase NF-kB activity in neurons. We propose to study these compounds and others obtained from their development to ascertain their effect on MnSOD expression in primary neurons and assess their beneficial potential in in vitro models of traumatic brain injury and neurodegeneration. By the end of the two years we could have leads to follow up in animal studies, aimed to verify improvement of the outcome in TBI, spinal cord injury and ROS dependent neurodegenerative models of disease. Evidence collected from these studies could spin off a larger drug discovery program.