Abstract
The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-β (Aβ) peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs), which are small molecules that selectively lower generation of the highly amyloidogenic Aβ42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP) and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aβ42 generation with an IC50 of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN), the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex.
Highlights
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease with an estimated 5.4 million patients in the USA [1]
GSIs that function as classical protease inhibitors and reduce enzymatic activity have displayed mechanism-based toxicity, which appears to be largely attributable to suppression of Notch processing [8]
Previous photo-affinity labeling studies have suggested that the binding site of non-steroidal anti-inflammatory drugs (NSAIDs)-type GSMs resides within the substrate amyloid precursor protein (APP) and not within PSEN or one of the three other c-secretase subunits [19]
Summary
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease with an estimated 5.4 million patients in the USA [1]. The intramembrane-cleaving protease c-secretase is responsible for the last step in the proteolytic release of Ab42 peptides from the amyloid precursor protein (APP), and is a principal therapeutic target in AD [3]. TMDs 6 and 7 of PSEN around the catalytic aspartate residues form a hydrophilic cavity within the membrane that may allow access for water molecules required to hydrolyze peptide bonds [5,6]. It appears that the substrate initially binds to a distinct substrate-binding (docking) site on the outer surface of the c-secretase complex and is subsequently transported into the active site and cleaved [7]
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