This research will identify the critical sites of interaction between novel pyridazine-derived soluble gamma secretase modulators (SGSMs) and their molecular target, as well as provide valuable information toward fostering an improved understanding of the mechanism by which these therapeutically relevant small molecules affect the production of specific Abeta peptide variants without inhibiting the enzyme’s activity. Despite the development of numerous potent SGSMs, the precise molecular target and the mechanism of action of this clinically relevant pyridazine series remain unknown. Our lab at the University of California, San Diego and the Tanzi lab at Massachusetts General Hospital have developed an extremely promising series of soluble pyridazine-derived γ-secretase modulators that inhibit the formation of the aggregation-prone Abeta42 peptide in favor of shorter, less pathogenic Abeta isoforms. We have synthesized an active pyridazine SGSM-photoprobe based on our SGSM currently under clinical consideration for cross-linking studies to demonstrate the binding site of these ligands within the γ-secretase enzyme. Additional experiments will be conducted using related γ-secretase modulator (GSM) compounds, structurally unrelated yet mechanistically related GSMs and gamma-secretase inhibitors (GSIs) in competitive labeling studies in order to further deconvolute the binding site within the γ-secretase enzyme. These experiments are critical for validating the mechanism of action of GSMs and differentiating this from gamma secretase inhibitors (GSIs), including the putative Notch-sparing GSIs.