Tau Therapeutic Targeting Through Pathological PAD Interactions in ADRD

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder, accounting for 60-80% of dementia, for which there are no effective therapies. The hallmark neuropathologies are protein deposition of amyloid-β (Aβ) and tau neurofibrillary tangles (NFTs). Therapeutic targeting of tau has not been widely explored in clinical trials. Recent findings provide new insight into tau pathology: Exposure of a 17 amino acid N-terminal sequence of tau that is sequestered in normal brain appears to be necessary. This region comprises a Phosphatase Activation Domain (PAD) that is aberrantly displayed in all pathological forms of tau. Interestingly, PAD acts as biologically active motif that is sufficient to activate a signaling pathway involving protein phosphatase 1 (PP1) and glycogen synthase kinase 3β (GSK3β) that is associated with the inhibition of fast axonal transport and toxicity in neurons. The monoclonal antibody (TNT1), specific for the PAD-tau epitope, blocks toxic effects of pathogenic tau. Therefore, PAD provides a molecular basis for altered kinase activities in AD and tauopathies representing a novel therapeutic target to treat tauopathies and AD Related Disorders. Based on these observations, a HTS screen strategy was developed based on AlphaLisa technology with a goal of identifying small molecules that can inhibit the PAD/TNT1 protein-protein interaction (PPI). Eleven hits were identified from initial screens inhibit the PAD-TNT interaction with sub-micromolar IC50s. These compounds serve as a basis for developing candidate therapeutics in collaboration with the Thatcher laboratory at the University of Arizona. We have successfully designed and optimized cell-based assays utilizing NanoBRET technology for validation of target engagement in neurons. Synthetic test compounds developed from the initial screen were found to efficiently disrupt the pathological PAD-Tau-PP1γ interaction at nanomolar concentrations. Compounds were tested for toxicity in both HEK293t and primary cultured cortical neurons, where no toxicity was associated with tested compounds. Three developed compounds were tested for their ability to rescue neurodegeneration and axonal transport disruption using immunostaining and confocal imaging as well as assessing their ability to inhibit Tau-PP1γ interaction. This phenotypic validation is important for establishing therapeutic efficacy and is critical for optimization of vivo dose response for active candidate compounds.