Development and application of a high-throughput biophysical approach to study transmembrane protein interactions with small molecules
Alzheimer’s disease (AD) is the most common form of dementia in the U.S. with no disease-modifying therapies. A key hallmark of AD is the generation of amyloid-β (Aβ) peptides and plaques, which have long been thought to be drivers of AD pathogenesis. In this dissertation I use the immediate precursor to Aβ peptides, called C99, as a model single-pass transmembrane receptor (SPTMR) to investigate how small molecules interact with single-pass transmembrane domain proteins. SPTMRs are recalcitrant drug targets, often with minimal druggable pockets, so little is known pertaining to how strong or specifically small molecules will bind them in and around their transmembrane domains. Developing a method for drugging these types of targets may open a new pharmaceutical niche for treating diseases including neurodegeneration, cancers, and immune disorders. Using high-throughput nuclear magnetic resonance (NMR) spectroscopy, C99 was screened against libraries of small molecules, leading to identification of a molecule, verteporfin, that binds its tightly and specifically and inhibits Aβ formation. Inversely, several other small molecules that non-specifically bound both C99 and other membrane proteins were identified. Taken together, my work established a methodology to discover small molecule ligands for SPTMRs and how to validate them for specificity and activity.