The main objective of the proposed project is to develop assays that we will use to discover small-molecule inhibitors of p300, an acetyltransferase enzyme implicated in Alzheimer's disease (AD). We have discovered that p300 acetylates tau in the early stages of AD and developed antibodies that uniquely allow us to probe this activity. We will build a high-throughput screening (HTS) assay that will, for the first time, enable identification of novel compounds that inhibit p300-mediated acetylation of tau. We will also utilize a logical series of assays to validate p300 inhibitors discovered from the HTS and prioritize these compounds for further studies in AD. Tau is a critical protein involved in th early pathogenesis of AD, and its hyperphosphorylated form (p-tau) is a major component of the neurofibrillary tangles (NFTs) found in the brains of AD patients. Evidence also suggests that tau, especially p-tau, causes neurodegeneration in AD. We recently discovered that tau is acetylated (ac-tau) by p300 and that hyperacetylation impairs tau's degradation, resulting in accumulation of pathogenic p-tau. Furthermore inhibiting p300 reduces ac-tau and leads to degradation of tau. We hypothesize that small-molecule inhibitors of p300 will thus clear tau's pathogenic forms and reduce neurodegeneration. Selective drug-like inhibitors of p300 with good in vivo properties are needed to further explore the role of ac-tau in AD. We have shown that C646, an inhibitor of p300, reduces tau acetylation in primary neurons and diminishes p-tau expression after short-term treatment. However, C646's use is limited for in vivo applications, and the few other published p300 inhibitors have low affinity and/or selectivity for this enzyme. Additionally, p300 regulates transcription through histone acetylation, which may play a role in cancer. Selective chemical probes will allow us to dissect the functions of p300 and assess the therapeutic utility of p300 inhibitors. We propose to meet the imperative need for such chemical probes by developing a new HTS assay to measure ac-tau using full-length tau and p300. Ac-tau will be detected by homogenous time-resolved fluorescence (HTRF) using the antibody against ac-tau developed in our lab. To date, no HTS have been published in PubChem for p300, and robust, scalable assays are just beginning to be reported for histone acetylation. In Aim 1, we will develop the HTRF assay for HTS and run a 31,600-compound pilot screen of diverse molecules. In Aim 2, we define a series of assays to prioritize inhibitors based on their mechanisms of action. In addition, we will characterize compounds' selectivity for p300 over related acetyltransferases. Finally, we will determine compounds' inhibition of acetylation in cell lines and primary cortical neurons. Significant and innovative outcomes from this work will be a) development of a suite of assays to screen and thoroughly characterize inhibitors of ac-tau; b) probes of p300 published in PubChem; c) a new HTRF format readily adaptable to other acetyltransferases. Lead compounds will probe the role of ac-tau in AD and other tauopathies, and will be a boon for researchers seeking to understand the biological functions of p300.