Abstract (Project Summary) Alzheimer's Disease (AD) is one of the major neurodegenerative disorders and leading cause of dementia, mostly prevalent in the aging population. Aging-associated impairments and cognitive decline due to AD and other neurodegenerative disorders severely affect the quality-of-life for elders and add significantly to related socioeconomic costs, which in the U.S. was estimated to total over $655 billion in 2020. Chronic systemic inflammation is an underlying driver for several disease states including cancer, autoimmune disorders, and cardiovascular disease. Neuroinflammation, defined as an inflammatory response in the brain, spinal cord, glia, or other neuronal tissue, contributes significantly to neurodegenerative signaling cascades which underlie AD and other diseases such as FTLD, PD, HD, MS, ALS, and TBI. Particularly in AD, initial neuroinflammatory response (M2 phenotype) develops as a defense mechanism within the CNS to rid itself of diverse noxious agents, cellular debris, and misfolded proteins such as Tau and Aβ. Despite their intended roles to attenuate tissue injury, cellular inflammatory processes in the CNS that become chronic and uncontrolled (M1 phenotype) inhibit regeneration and promote neurodegeneration. Overactivation of Kv1.3, a member of the super family of voltage-gated potassium channels, is heavily implicated in tipping the M1 (classical activation) / M2 (alternative activation) phenotype expression in microglia from a balanced steady-state (good neuronal health) to predominately the M1 phenotype mode of microglial activation associated with AD. Both peptidic and small molecule inhibitors of Kv1.3 have shown efficacy in numerous animal models of AD, but their further development has been hampered by poor drug-like properties. As such, using a fragment based pharmacophore analysis and design, Hager Biosciences has discovered differentiated scaffold classes small molecule Kv1.3 channel blockers which showed potent inhibitions in CHO cells stably expressing h Kv1.3 using electrophysiological whole-cell voltage-clamp format. Therefore, the goal of this project is to further develop these compounds as innovative potential treatments for AD. Thus, the 1st Specific Aim is to (a) complete the in vitro characterization of analog leads by generating Kv1.3 inhibitory IC50 values for compounds inhibiting Kv1.3 >50% at 1 uM - using whole cell voltage-clamp protocol, and (b) select representative leads for initial pharmaceutical profiling assays (solubility, permeability, microsomal stability, protein binding, and efflux ratio). The 2nd Specific Aim is to (a) initiate iterative multi-parameter lead optimization campaign to improve target potency and ADMET properties based on 2-4 structural classes which provided compounds with optimal overall profiles emerging from Specific Aim 1; and (b) select representative leads possessing inhibitory Kv1.3 IC50 values < 100 nM and perform selectivity screen against Kv1.4, ...