PROJECT SUMMARY: The principle goal of this research is to develop selective and potent human 12/15-LOX inhibitors, which will probe the role of inflammation in Alzheimer's disease (AD), leading to drug candidates for this devastating human disease. The lack of any therapeutic for AD highlights the need for new approaches to its treatment. Among the novel targets, 12/15-lipoxygenase (12/15-LOX or 15-LOX-1) stands out for numerous reasons. 12/15-LOX is widely expressed in the central nervous system, and its protein levels and enzymatic activity are significantly elevated in patients with AD and those with a clinical diagnosis of mild cognitive impairment, suggesting an early involvement of the pathway in AD pathogenesis. Our laboratories have shown that genetic absence or the early pharmacological blockade of 12/15-LOX prevents cognitive impairment and the development of AD-like neuropathology in transgenic mouse models of the disease. In addition, we have also shown that the 12/15-LOX pharmacological blockade is beneficial after pathological phenotype of the disease has developed. PD146176, a first-generation, off-patent 12/15-LOX inhibitor, rescues learning and memory deficits, reduces A? levels and deposition, facilitates tau clearance and improves synaptic integrity in aged 3xTg mice. However, PD146176 is not a selective 12/15-LOX inhibitor and it has poor activity against the mouse 12/15-LOX relative to the human 12/15-LOX. Therefore, discovering more selective next-generation 12/15-LOX inhibitors that are patentable may provide better pleiotropic benefits. The three objectives of the current proposal are to first test our well-characterized, patented, potent, selective 12/15-LOX inhibitor, ML351, against our mouse AD model. ML351 has excellent ADME/PK properties so we will first reproduce the PD146176 results found previously in our laboratory with ML351 to confirm it as a viable AD therapeutic against 12/15-LOX. Second, we will discover additional candidate molecules toward therapeutic development against AD, utilizing our assays to identify novel, selective inhibitors for the human 12/15-LOX. We have already performed a successful 12/15-LOX high-throughput (HTP) screen in collaboration with the NIH of their new 500,000 compound library. We will screen the top 1000 compounds of this screen, test the top potent/selective hits against in vitro human 12/15-LOX, and determine their in vivo effectiveness against cultured mouse HT-22 neuronal cells. Third, we will optimize our newly discovered 12/15- LOX inhibitors for their ADME and PK properties and confirm their LOX/COX selectivity. Considering that ML351 has already been shown to cross the blood-brain barrier and protect against stroke damage, we are confident these studies will show that ML351 protects against AD symptoms and thus have an excellent chance of becoming an effective therapeutic tool against AD.