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Inhibitors of ?-secretase stabilize the complex and differentially affect processing of amyloid precursor protein and other substrates.

ABSTRACT: ?-Secretase inhibitors (GSIs) are drugs used in research to inhibit production of A? and in clinical trials to treat Alzheimer's disease (AD). They inhibit proteolytic activities of ?-secretase noncompetitively by unknown mechanisms. Here, we used cortical neuronal cultures expressing endogenous levels of enzymes and substrates to study the effects of GSIs on the structure and function of ?-secretase. We show that GSIs stabilize the interactions between the C-terminal fragment of presenilin (PS-CTF), the central component of the ?-secretase complex, and its partners the APH-1/nicastrin and PS1-NTF/PEN-2 subcomplexes. This stabilization dose-dependently correlates with inhibition of N-cadherin cleavage, a process limited by enzyme availability. In contrast, production of amyloid precursor protein (APP) intracellular domain (AICD) is insensitive to low concentrations of GSIs and is limited by substrate availability. Interestingly, APP is processed by both PS1- and PS2-containing ?-secretase complexes, while N-cadherin and ephrinB1 are processed only by PS1-containing complexes. Paradoxically, low concentrations of GSIs specifically increased the levels of A? without affecting its catabolism, indicating increased A? production. Our data reveal a mechanism of ?-secretase inhibition by GSIs and provide evidence that distinct ?-secretase complexes process specific substrates. Furthermore, our observations have implications for GSIs as therapeutics because processing of functionally important substrates may be inhibited at lower concentrations than A?.

SUBMITTER: Barthet G

PROVIDER: S-EPMC3157694 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Inhibitors of γ-secretase stabilize the complex and differentially affect processing of amyloid precursor protein and other substrates.

Barthet Gael G Shioi Junichi J Shao Zhiping Z Ren Yimin Y Georgakopoulos Anastasios A Robakis Nikolaos K NK

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20110519 9

γ-Secretase inhibitors (GSIs) are drugs used in research to inhibit production of Aβ and in clinical trials to treat Alzheimer's disease (AD). They inhibit proteolytic activities of γ-secretase noncompetitively by unknown mechanisms. Here, we used cortical neuronal cultures expressing endogenous levels of enzymes and substrates to study the effects of GSIs on the structure and function of γ-secretase. We show that GSIs stabilize the interactions between the C-terminal fragment of presenilin (PS- ...[more]

PMID: 21597003

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Project description:INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering A? production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP ?-site cleavage (equivalent to A?40), and Notch ?-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of A? production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain A? was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain A? reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting A? production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain A? in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain A? was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.

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Inhibitors of ?-secretase stabilize the complex and differentially affect processing of amyloid precursor protein and other substrates.

Publications

Inhibitors of γ-secretase stabilize the complex and differentially affect processing of amyloid precursor protein and other substrates.

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