PROJECT SUMMARY: During my career as a scientist, one of the many contributions I intend to make is to discover and/or develop compounds that can be used as chemical probes for membrane proteins involved in human diseases. In the context of this proposal, the disease of interest is Alzheimer’s disease (AD), and the protein target I intend to focus on is the Amyloid Precursor Protein (APP). AD is the 6th leading cause of death in the U.S., affecting one in three people over the age of 65 and is estimated to cost the nation $1.1 trillion in healthcare by the year 2050. The APP gene, which codes for the Amyloid Precursor Protein, was the first described to have genetic mutants that gave rise to autosomal dominant familial AD (fAD; also referred to as early onset AD). The amyloid cascade hypothesis makes the case that the molecular hallmark of AD and ultimately dementia is instigated by the accumulation of Aβ peptides, a product of APP proteolysis by gamma-secretase following β-secretase cleavage and a major component of the presenile plaques associated with AD. Further genetic evidence from the discoveries of dominant mutations in PSEN1 and PSEN2, which code for catalytic protein subunits of the gamma-secretase complex, support the involvement of the amyloidogenic pathway in AD. Clinical failures of the some of the early Aβ-centric therapeutics (such as gamma-secretase inhibitors [GSIs]) were due, at least in part, to the off-target effects of broad spectrum inhibition of the complex. A pharmacological target that has not been thoroughly explored is the C99 domain of APP, which has no available chemical probes, is the immediate precursor to Aβ, and has been shown to involved in several aspects of AD pathogenesis. The basic science questions this proposal aims to answer are: what types of affinities (Kd) can we expect to find between a small transmembrane protein (such as C99) and small-molecules; and how would a C99-small molecule complex affect gamma-secretase-mediated proteolysis of C99? My driving hypothesis is that by coupling nuclear magnetic resonance (NMR) spectroscopy-based high-throughput screening (HTS) with very careful compound validation, we can develop novel small molecules that can potentially perturb APP biology in a broad range of hydrophobic environments without introducing off-target effects on other γ- secretase cleavage targets. More specifically, I propose to screen for a compound that specifically binds to C99, leading to interference with its recognition by γ-secretase, leaving other off-target secretase substrates susceptible to normal (healthy) cleavage. To test this, I propose to validate my HTS hits using both NMR and biochemical assays, and to use medicinal chemistry (via SAR by catalog and by collaboration) to create high-affinity, C99-specific small-molecule probes. I will also screen a curated subset of the 20,000 most chemically diverse molecules in the Vanderbilt Discovery Library to discover new leads. The results ...