Compound E inhibited the production of all 3 -amyloid species with equal potency (Fig. di-coumarin compounds, we reveal a mechanism by which -secretase specificity is usually regulated and provide insights into the molecular basis by which familial presenilin mutations may impact the active site and specificity of -secretase. Furthermore, this class of selective inhibitors provides the basis for development of Alzheimer disease therapeutic Lasmiditan brokers. Keywords:affinity labeling, Alzheimer disease, allosteric regulation, di-coumarin -Secretase is usually a multi-protein membrane-bound complex that is currently at the front line of basic and translational research. It is composed of at least 4 proteins that include presenilin, nicastrin, Aph-1, and Pen-2 (1). Presenilin is usually believed to contain the active site of -secretase (24). It represents a novel class of protease that catalyzes peptide bond hydrolysis within the transmembrane hydrophobic environment and plays an essential role in a newly emerged signaling pathway known as regulated intramembrane proteolysis (5). -Secretase cleaves a variety of type I membrane proteins that include the amyloid precursor protein (APP) and the Notch family of proteins despite limited main sequence homology across targeted substrates (6). Elucidation of the mechanisms that control the specificity of -secretase for these substrates has been hindered by technical difficulties associated with intramembrane Lasmiditan enzymology. Determining the factors that contribute to -secretase specificity is critical to understanding the biology of this unique protease and targeting it for therapeutic purposes. -Secretase has become an appealing drug target for Alzheimer disease (AD) and malignancy because of its central role in the processing of APP and Notch (6). -Secretase cleaves Lasmiditan APP to generate neurotoxic A peptides, ranging from 37 to 46 aa in length (7). Among them, A40 and A42 have been extensively investigated for their association with AD (7). Additionally, disease-causing familial AD mutations within APP, presenilin-1 (PS-1), and presenilin-2 (PS-2) proteins result in an increase in the ratio Lasmiditan of A42 to A40 (examined in ref.7). Clearly, mutations in Pax1 both enzyme and substrate can influence the specificity of -secretase and lead to pathological effects. Non-selective inhibition of -secretase activity has been explored as an AD and malignancy therapeutic approach, but the abrogation of all activities of -secretase results in toxicity in the gastrointestinal tract as a result of the blockage of Notch1 signaling (8). Therefore, the development of selective inhibitors is necessary to investigate -secretase specificity and provide candidates for drug development. Recent studies have indicated that this ratio of A42 to A40, rather than the total amount of -amyloid, correlates with the amount of characteristic AD plaques in mouse models (9,10) as well as with the age of onset of familial AD (11). Furthermore, new evidence suggests that A40 may even play a neuroprotective role against AD progression, whereas A42 is usually more hydrophobic and more readily aggregates to form harmful oligomers and fibrils (10). Rationally, the discovery and development of selective -secretase inhibitors (GSIs) that specifically abrogate A42 production over A40 and Notch cleavage is usually a promising strategy for AD therapy. Weggen et al. discovered that a subset of nonsteroidal antiinflammatory drugs, referred to as -secretase modulators (GSMs), were able to selectively decrease -secretase-mediated production of A42 with a concomitant increase in A38, and experienced no effect on A40 or Notch1 cleavage (12). Conversely, other GSMs were decided to stimulate the production of A42 while reducing A38 cleavage. Subsequent studies have shown that these GSMs alter -secretase cleavage preference by binding directly to the APP substrate (13). Other compounds that target -secretase and preferentially inhibit A40 and A42 production over Notch1 processing have been reported (14,15), although the precise action mechanism of these molecules has not been established. Therefore, it is critical to develop a better understanding of the molecular basis of -secretase specificity to facilitate the development of selective GSIs for the treatment of AD and other human disorders. In the present study, we describe a class of allosteric GSI (AGSI) that contain a di-coumarin core and modulate -secretase specificity for A42 production Lasmiditan over A38, A40, and Notch cleavages. We have demonstrated that these inhibitors regulate -secretase activity by binding to an allosteric site within the -secretase complex. Furthermore, we have developed a multiple photo-affinity probe strategy using transition-state inhibitors.