title: γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.
abstract: γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic β-amyloid peptides (i.e. Aβ42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.
authors: Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139.
authors: Nikolay
authors: N
authors: Pozdnyakov
authors: Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139.
authors: Heather E
authors: HE
authors: Murrey
authors: Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065; Department of Pharmacology, Weill Graduate School of Medical Science of Cornell University, New York, New York 10021.
authors: Christina J
authors: CJ
authors: Crump
authors: Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139.
authors: Martin
authors: M
authors: Pettersson
authors: Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139.
authors: T Eric
authors: TE
authors: Ballard
authors: Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139.
authors: Christopher W
authors: CW
authors: Am Ende
authors: Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065; Department of Pharmacology, Weill Graduate School of Medical Science of Cornell University, New York, New York 10021.
authors: Kwangwook
authors: K
authors: Ahn
authors: Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065; Department of Pharmacology, Weill Graduate School of Medical Science of Cornell University, New York, New York 10021.
authors: Yue-Ming
authors: YM
authors: Li
authors: Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139. Electronic address: kelly.bales@pfizer.com.
authors: Kelly R
authors: KR
authors: Bales
authors: Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139. Electronic address: doug.johnson@pfizer.com.
authors: Douglas S
authors: DS
authors: Johnson
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D058227:Amyloidogenic Proteins / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002474:Cell-Free System; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008027:Light; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism; D019920:Photoaffinity Labels / Q000494:pharmacology; D010777:Photochemistry / Q000379:methods; D053763:Presenilins / Q000737:chemistry*; D011485:Protein Binding; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D058227:Amyloidogenic Proteins; D019920:Photoaffinity Labels; D053763:Presenilins; D011510:Proteolipids; C020485:proteoliposomes; D053829:Amyloid Precursor Protein Secretases
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