Development of clickable active site-directed photoaffinity probes for γ-secretase.

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin-1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.