title: Development of clickable active site-directed photoaffinity probes for γ-secretase.
abstract: 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.
authors.affiliation: Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Christina J
authors.initials: CJ
authors.lastname: Crump
authors.forename: Christopher W
authors.initials: CW
authors.lastname: am Ende
authors.forename: T Eric
authors.initials: TE
authors.lastname: Ballard
authors.forename: Nikolay
authors.initials: N
authors.lastname: Pozdnyakov
authors.forename: Martin
authors.initials: M
authors.lastname: Pettersson
authors.forename: De-Ming
authors.initials: DM
authors.lastname: Chau
authors.forename: Kelly R
authors.initials: KR
authors.lastname: Bales
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Li
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Johnson
mesh_terms: D000480:Alkynes / Q000737:chemistry; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry; D015153:Blotting, Western; D020134:Catalytic Domain; D057930:Click Chemistry; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D015394:Molecular Structure; D019920:Photoaffinity Labels / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D000480:Alkynes; D001577:Benzophenones; D019920:Photoaffinity Labels; C047723:benzophenone; D053829:Amyloid Precursor Protein Secretases