The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer's disease.

Innate immunity is associated with Alzheimer's disease1, but the influence of immune activation on the production of amyloid-ß is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-ß. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-ß. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer's disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer's disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer's disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer's disease is thereby increased.

Development of Sulfonamide Photoaffinity Inhibitors for Probing Cellular γ-Secretase.

γ-Secretase is a multiprotein complex that catalyzes intramembrane proteolysis associated with Alzheimer's disease and cancer. Here, we have developed potent sulfonamide clickable photoaffinity probes that target γ-secretase in vitro and in cells by incorporating various photoreactive groups and walking the clickable alkyne handle to different positions around the molecule. We found that benzophenone is preferred over diazirine as a photoreactive group within the sulfonamide scaffold for labeling γ-secretase. Intriguingly, the placement of the alkyne at different positions has little effect on probe potency but has a significant impact on the efficiency of labeling of γ-secretase. Moreover, the optimized clickable photoprobe, 163-BP3, was utilized as a cellular probe to effectively assess the target engagement of inhibitors with γ-secretase in primary neuronal cells. In addition, biotinylated 163-BP3 probes were developed and used to capture the native γ-secretase complex in the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) solubilized state. Taken together, these next generation clickable and biotinylated sulfonamide probes offer new tools to study γ-secretase in biochemical and cellular systems. Finally, the data provide insights into structural features of the sulfonamide inhibitor binding site in relation to the active site and into the design of clickable photoaffinity probes.

Development and mechanism of γ-secretase modulators for Alzheimer's disease.

γ-Secretase is an aspartyl intramembranal protease composed of presenilin, Nicastrin, Aph1, and Pen2 with 19 transmembrane domains. γ-Secretase cleaves the amyloid precursor proteins (APP) to release Aß peptides that likely play a causative role in the pathogenesis of Alzheimer's disease (AD). In addition, γ-secretase cleaves Notch and other type I membrane proteins. γ-Secretase inhibitors (GSIs) have been developed and used for clinical studies. However, clinical trials have shown adverse effects of GSIs that are potentially linked with nondiscriminatory inhibition of Notch signaling, overall APP processing, and other substrate cleavages. Therefore, these findings call for the development of disease-modifying agents that target γ-secretase activity to lower levels of Aß42 production without blocking the overall processing of γ-secretase substrates. γ-Secretase modulators (GSMs) originally derived from nonsteroidal anti-inflammatory drugs (NSAIDs) display such characteristics and are the focus of this review. However, first-generation GSMs have limited potential because of the low potency and undesired neuropharmacokinetic properties. This generation of GSMs has been suggested to interact with the APP substrate, γ-secretase, or both. To improve the potency and brain availability, second-generation GSMs, including NSAID-derived carboxylic acid and non-NSAID-derived heterocyclic chemotypes, as well as natural product-derived GSMs have been developed. Animal studies of this generation of GSMs have shown encouraging preclinical profiles. Moreover, using potent GSM photoaffinity probes, multiple studies unambiguously have showed that both carboxylic acid and heterocyclic GSMs specifically target presenilin, the catalytic subunit of γ-secretase. In addition, two types of GSMs have distinct binding sites within the γ-secretase complex and exhibit different Aß profiles. GSMs induce a conformational change of γ-secretase to achieve modulation. Various models are proposed and discussed. Despite the progress of GSM research, many outstanding issues remain to be investigated to achieve the ultimate goal of developing GSMs as effective AD therapies.

γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.

γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic ß-amyloid peptides (i.e. Aß42) 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 Aß42 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.

BMS-708,163 targets presenilin and lacks notch-sparing activity.

The "Notch-sparing" γ-secretase inhibitor (GSI) BMS-708,163 (Avagacestat) is currently in phase II clinical trials for Alzheimer's disease. Unlike previously failed GSIs, BMS-708,163 is considered to be a promising drug candidate because of its reported Notch-sparing activity for the inhibition of Aß production over Notch cleavage. We now report that BMS-708,163 binds directly to the presenilin-1 N-terminal fragment and that binding can be challenged by other pan-GSIs, but not by γ-secretase modulators. Furthermore, BMS-708,163 blocks the binding of four different active site-directed GSI photoaffinity probes. We therefore report that this compound acts as a nonselective γ-secretase inhibitor.

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.

Familial Alzheimer disease presenilin-1 mutations alter the active site conformation of γ-secretase.

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase, and mutations in this protein cause familial Alzheimer Disease (FAD). However, little is known about how these mutations affect the active site of γ-secretase. Here, we show that PS1 mutations alter the S2 subsite within the active site of γ-secretase using a multiple photoaffinity probe approach called "photophore walking." Moreover, we developed a unique in vitro assay with a biotinylated recombinant Notch1 substrate and demonstrated that PS1 FAD mutations directly and significantly reduced γ-secretase activity for Notch1 cleavage. Substitution of the Notch Cys-1752 residue, which interacts with the S2 subsite, with Val, Met, or Ile has little effect on wild-type PS1 but leads to more efficient substrates for mutant PS1s. This study indicates that alteration of this S2 subsite plays an important role in determining the activity and specificity of γ-secretase for APP and Notch1 processing, which provides structural basis and insights on how certain PS1 FAD mutations lead to AD pathogenesis.

Piperidine acetic acid based γ-secretase modulators directly bind to Presenilin-1.

Aß42 is believed to play a causative role in Alzheimer's disease (AD) pathogenesis. γ-Secretase modulators (GSMs) are actively being pursued as potential AD therapeutics because they selectively alter the cleavage site of the amyloid precursor protein (APP) to reduce the formation of Aß42. However, the binding partner of acid based GSMs was unresolved until now. We have developed clickable photoaffinity probes based on piperidine acetic acid GSM-1 and identified PS1 as the target within the γ-secretase complex. Furthermore, we provide evidence that allosteric interaction of GSMs with PS1 results in a conformational change in the active site of the γ-secretase complex leading to the observed modulation of γ-secretase activity.

Dual role of alpha-secretase cleavage in the regulation of gamma-secretase activity for amyloid production.

Processing of the amyloid precursor protein (APP) by ß- and γ-secretases generates pathogenic ß-amyloid (Aß) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aß formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aß. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.