PROJECT SUMMARY Neurodegeneration in Alzheimer's disease (AD) is increasingly accepted to result from the toxicity of hyperphosphorylated tau aggregates, the major constituent of the neurofibrillary tangles (NFTs) in the brains of AD patients. The vast majority of AD drug development efforts have focused on inhibiting or clearing Aβ plaques. So far, these efforts have failed in clinical trials. In contrast to Aβ plaques, which poorly correlate with progression of clinical AD symptoms, the spatiotemporal distribution of NFTs cognitive impairment. Mounting evidence suggests that pre-tangle aggregates of hyperphosphorylated tau are toxic to neurons, and likely play a key role in AD neurodegeneration. Thanks to a technology for generating phosphorylated proteins called PIMAX, this project will be the first to screen drugs for inhibition of aggregation of hyperphosphorylated tau (hyper-p-tau). Until now, hyper-p-tau in sufficient quantities to screen a compound library has been unavailable. Previous projects screened compounds for binding either unphosphorylated tau, or tau's two cysteine groups, or tau with only 1 to 3 phosphates. As our PIMAX-generated hyper-p-tau drug target, we will use tau isoform 1N4R phosphorylated on up to 16 aa residues. In brains of AD patients, tau 1N4R is hyperphosphorylated, and 8 of these 16 residues are used to stage AD postmortem brains. Thus, due to our unique hyperphosphorylated target, this project does not duplicate previous drug projects targeting tau, and is instead a groundbreaking new approach to discovering inhibitors of tau aggregation into neurotoxic fibrils. Our preliminary data shows that our novel small molecules (JF compounds) can inhibit aggregation of hyper-p-tau 1N4R. Our most potent inhibitor to date, JF-19-73, dose- dependently antagonizes oligomer formation and neuronal cytotoxicity of hyper-p-tau 1N4R in vitro, suggesting its potential for neuroprotection. JF-19-73 is more potent than resveratrol in inhibiting hyper-p-tau 1NR4 aggregation. JF compounds have classic drug attributes (Rules of Five by Lipinski et al.), exhibit positive predictive scores to traverse the blood-brain barrier, and are non-toxic to cells. Aim 1 will optimize JF chemistry for solubility (e.g. reduction of ClogP), inhibition of formation of cytotoxic hyper- p-tau 1N4R oligomeric fibrils, and cytoprotection in vitro. Aim 2 will test the most potent hyper-p-tau aggregation inhibitors for ADME properties in vitro and identify JF lead compounds for future preclinical studies including animal and PK/PD studies. Overall Impact: Neurodegeneration in AD is increasingly accepted to result from the toxicity of hyperphosphorylated tau aggregates. This project will enable us to move into a lead development program with optimized JF derivatives that target hyperphosphorylated tau — the form of tau actually found in postmortem brains of AD patients. This project is unique because it is based on overcoming a longstanding critical barrier ...