Novel up-regulators of NF-kB activity: effect on MnSOD and TBI in vitro models

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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.