Abstract Alzheimer’s disease (AD) is a devastating neurodegenerative disorder which currently affects 5.8 million Americans. However, there are currently no FDA-approved treatments capable of delaying or stopping disease progression. Amyloid-beta (Aβ) plaques are believed to be integral to AD pathogenesis through their role in the “amyloid cascade hypothesis,” in which the accumulation of Aβ peptides initiates a chain of pathological events leading to neurodegeneration and ultimately AD. γ-secretase is an aspartyl protease responsible for processing a wide range of substrates. It is considered an attractive drug target because it cleaves amyloid precursor protein (APP) in the final step of proteolysis to generate Aβ peptides of varying lengths; moreover, mutations in APP and presenilin, the catalytic subunit of γ-secretase, have been linked to familial AD and shown to alter Aβ production. Unfortunately, all clinical trials of γ-secretase inhibitors proved unsuccessful due to off target effects on other γ-secretase substrates, such as Notch. The failures of γ- secretase inhibitors spurred the discovery and development of a new series of compounds known as γ- secretase modulators (GSMs). A subset of NSAIDs were initially discovered to selectively reduce levels of the pathogenic Aβ42 in favor of the less pathogenic Aβ38 without inhibition of Notch. Improvements on these compounds led to second generation GSMs. This project will focus on two classes of second generation GSMs: acid GSMs derived from NSAIDs and imidazole GSMs. Acid and imidazole GSMs have been characterized by multiple groups and showed promise in decreasing pathogenic Aβ species while increasing the less pathogenic species. However, they still demonstrated limitations in clinical trials regarding Aβ selectivity, potency, and toxicity. Our objective is to define the molecular mechanism of GSMs in order to improve them for AD drug development. Using GSM-based photoprobes to label γ-secretase, our laboratory has found that acid and imidazole GSMs bind to distinct sites on the presenilin subunit of γ-secretase. Based on this information, we hypothesize that acid and imidazole GSMs bind to distinct, allosteric sites of γ- secretase and work in synergy to effectively modulate Aβ production. Using approaches at the interface of biology and chemistry, this project aims to: 1) Determine the mechanism of binding of GSMs on γ-secretase and 2) Determine the effects of GSM combination on Aβ production, toxicity, and cognition. To investigate the molecular mechanisms of GSMs, we will use chemical probes combined with mass spectrometry and structural modeling to identify the precise binding sites. We will also evaluate pairs of acid and imidazole GSMs in Aβ- secreting cell lines and use the most effective concentrations to analyze toxicity and cognitive performance in an AD mouse model. Overall, the proposed studies will enable us to decode the mechanism of GSMs and, ultimately, develop GSMs with t...