Presenilin is the molecular target of acidic γ-secretase modulators in living cells

The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-β (Aβ) peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs), which are small molecules that selectively lower generation of the highly amyloidogenic Aβ42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP) and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aβ42 generation with an IC(50) of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN), the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex.     

In silico identification of natural product inhibitors for γ-secretase activating protein,a therapeutic target for Alzheimer's disease

Alzheimer's disease (AD) is clinically characterized by the aggregation of neurotoxic amyloid-β (Aβ) peptides in the brain. γ-Secretase catalyzes the reaction of Aβ formation. Inhibition of γ-secretase activating protein (GSAP) reduces Aβ production without disrupting other molecular functions and serves as a promising therapeutic target for lowering Aβ and curing AD. Till date, no proven drug is available for curing AD because of the nonexistence of crystal/NMR structure of GSAP. Thus in the present study, for the first time, we adopted in silico method to predict the 3D structure of GSAP via comparative modeling and studied the architecture and function of GSAP through simulation studies. Docking studies with 4153 phytochemicals revealed that GSAP having a better binding affinity with macaflavanone C, (E)-1-[2,4-dihydroxy-3-(3-methylbut-2-enyl)phenyl]-3-(2,2-dimethyl-8-hydroxy-2H-benzopyran-6-yl)prop-2-en-1-one, and monachosorin B as compared with the standard drug, imatinib. Further, the molecular dynamics analysis suggested that only two phytochemicals, namely, macaflavanone C and (E)-1-[2,4-dihydroxy-3-(3-methylbut-2-enyl)phenyl]-3-(2,2-dimethyl-8-hydroxy-2H-benzopyran-6-yl)prop-2-en-1-one) significantly disrupt the original property of GSAP and also cleared the absorption, distribution, metabolism, and excretion test. These natural compounds may be utilized in future for curing AD after further investigations.     

Therapeutic effect of CHF5074, a new γ-secretase modulator,in a mouse model of scrapie

In transmissible spongiform encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrP^C is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ-secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 d longer than vehicle-treated mice (p < 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrP^Sc in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared with the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrP^Sc toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.Key words: TSE, prion, murine model, γ-secretase modulator, therapy     

Therapeutic effect of CHF5074, a new γ-secretase modulator,in a mouse model of scrapie

In transmissible spongiform encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrP^c is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ-secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 d longer than vehicle-treated mice (p &lt; 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrP^Sc in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared with the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrP^Sc toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.     

Design and synthesis of a novel series of cyanoindole derivatives as potent γ-secretase modulators

The discovery, design and synthesis of a new series of GSMs is described. The classical imidazole heterocycle has been replaced by a cyano group attached to an indole nucleus. The exploration of this series has led to compound 26-S which combined high in vitro and in vivo potency with an acceptable drug-like profile.     

SAR studies of acidic dual γ-secretase/PPARγ modulators

A novel set of dual γ-secretase/PPARγ modulators characterized by a 2-benzyl hexanoic acid scaffold is presented. Synthetic efforts were focused on the variation of the substitution pattern of the central benzene. Finally, we obtained a new class of 2,5-disubstituted 2-benzylidene hexanoic acid derivatives, which act as dual γ-secretase/PPARγ modulators in the low micromolar range. We have explored broad SAR and successfully improved the dual pharmacological activity and the selectivity profile against potential off-targets such as NOTCH and COX. Compound 17 showed an IC(50) Aβ42=2.4 μM and an EC(50) PPARγ=7.2 μM and could be a valuable tool to further evaluate the concept of dual γ-secretase/PPARγ modulators in animal models of Alzheimer's disease.     

Design and synthesis of bicyclic heterocycles as potent γ-secretase modulators

The evolution of amide 3 into conformationally restricted bicyclic triazolo-piperidine 14-S as a γ-secretase modulator is described. This is a potential disease modifying anti-Alzheimer’s drug which demonstrated high in vitro and in vivo potency against Aβ42 peptide, reduced lipophilicity and enhanced brain free fraction compared to the previous series.     

The phospholipid base-exchange system as a possible modulator of γ-aminobutyric acid transport in brain cells

Mixed cell suspensions from rabbit brain have been used to study the effect of base exchange in membrane phospholipids, on amino acid accumulation in vitro. -Aminobutyric acid (GABA), glutamic acid, and aminoisobutyric acid have been used. The accumulation of [³H]GABA, at concentrations employing the high-affinity uptake system, was measured after base-exchange reactions with ethanolamine, choline, orL-serine. Serine incorporation induced an increase of GABA uptake at all the concentrations used, while choline incorporation essentially led to inhibition of GABA accumulation. Ethanolamine exchange produced both stimulation and inhibition. The observed effects were not specific for GABA. Neuronal and glial cell perikarya and synaptosomes were studied in the same system in an attempt to resolve the complex type of response obtained with the mixed suspension. Cell specificity was found with respect to stimulation or inhibition of GABA transport after base exchange but, in some cases, the isolated fractions retained the multiphasic response observed with the mixed suspension.     

Synthesis and structure–activity relationship of a novel series of heterocyclic sulfonamide γ-secretase inhibitors

γ-Secretase inhibitors have been shown to reduce the production of β-amyloid, a component of the plaques that are found in brains of patients with Alzheimer’s disease. A novel series of heterocyclic sulfonamide γ-secretase inhibitors that reduce β-amyloid levels in cells is reported. Several examples of compounds within this series demonstrate a higher propensity to inhibit the processing of amyloid precursor protein compared to Notch, an alternative γ-secretase substrate.     

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.     

LY411575, a potent γ-secretase inhibitor,suppresses osteoclastogenesis in vitro and LPS-induced calvarial osteolysis in vivo

A series of osteolytic bone diseases are usually related to excessive bone resorption and osteoclast formation. Thus, agents or drugs which can target osteoclast development and attenuate bone loss are potentially considerable in preventing and treating of bone lytic diseases. In recent years, many studies have reported that Notch signaling has substantial impacts on the process of osteoclast differentiation, maturation, and bone destruction. In the present study, we showed that LY411575, a γ-secretase inhibitor, could potently suppress osteoclast differentiation, osteoclast-specific gene expression, and bone resorption via suppressing Notch/HES1/MAPK (ERK and p38)/Akt-mediated NFATc1 induction in vitro. Consistent with in vitro results, LY411575 exhibited protective effects in lipopolysaccharides-induced calvarial bone destruction in vivo. Collectively, these results indicate that LY411575 may have therapeutic potential in the treatment of osteoclast-mediated osteolytic bone diseases.     

Development of photoaffinity probes for γ-secretase equipped with a nitrobenzenesulfonamide-type cleavable linker

We have developed photoaffinity probes for γ-secretase with a nitrobenzenesulfonamide-type linker that can be cleaved with 2-mercaptoethanol under physiological conditions.     

Triazoloamides as potent γ-secretase modulators with reduced hERG liability

Synthesis and SAR studies of novel aryl triazoles as gamma secretase modulators (GSMs) are presented in this communication. Starting from our aryl triazole leads, optimization studies were continued and the series progressed towards novel amides and lactams. Triazole 57 was identified as the most potent analog in this series, displaying single-digit nanomolar Aβ42 IC(50) in cell-based assays and reduced affinity for the hERG channel.     

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.     

Structure and function of γ-secretase

The γ-secretase complex is a prime target for pharmacological intervention in Alzheimer’s disease and so far drug discovery efforts have yielded a large variety of potent and rather specific inhibitors of this enzymatic activity. However, as γ-secretase is able to cleave a wide variety of physiological important substrates, the real challenge is to develop substrate-specific compounds. Therefore, obtaining structural information about γ-secretase is indispensable. As crystal structures of the complex will be difficult to achieve, applied biochemical approaches need to be integrated with structural information obtained from other intramembrane-cleaving proteases. Here we review current knowledge about the structure and function of γ-secretase and discuss the value of these findings for the mechanistic understanding of this unusual protease.     

Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10h.     

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells

How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.