Development of Sulfonamide Photoaffinity Inhibitors for Probing Cellular γ-Secretase

gamma-Secretase is a multiprotein complex that catalyzes intramembrane proteolysis associated with Alzheimer's disease and cancer. Here, we have developed potent sulfonamide clickable photoaffinity probes that target gamma-secretase in vitro and in cells by incorporating various photoreactive groups and walking the clickable alkyne handle to different positions around the molecule. We found that benzophenone is preferred over diazirine as a photoreactive group within the sulfonamide scaffold for labeling gamma-secretase. Intriguingly, the placement of the alkyne at different positions has little effect on probe potency but has a significant impact on the efficiency of labeling of gamma-secretase. Moreover, the optimized clickable photoprobe, 163-BP3, was utilized as a cellular probe to effectively assess the target engagement of inhibitors with gamma-secretase in primary neuronal cells. In addition, biotinylated 163-BP3 probes were developed and used to capture the native gamma-secretase complex in the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) solubilized state. Taken together, these next generation clickable and biotinylated sulfonamide probes offer new tools to study gamma-secretase in biochemical and cellular systems. Finally, the data provide insights into structural features of the sulfonamide inhibitor binding site in relation to the active site and into the design of clickable photoaffinity probes.