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 IC50 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

Synthesis of a potent photoreactive acidic gamma-secretase modulator for target identification in cells

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimers disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases. Compounds named gamma-secretase modulators (GSMs) can shift the substrate cleavage specificity of gamma-secretase toward the production of non-amyloidogenic, shorter A beta fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of gamma-secretase), supporting a mode of action involving binding to gamma-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in gamma-secretase proteolytic specificity should pave the way for the development of improved drugs against AD. (C) 2012 Elsevier Ltd. All rights reserved

The metalloprotease meprin beta generates amino terminal-truncated amyloid beta peptide species

The amyloid beta (A beta) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin beta cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin beta can also process APP in a manner reminiscent of beta-secretase. We identified cleavage sites of meprin beta in the amyloid beta sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating A beta variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin beta than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin beta on APP and A beta generation was also observed in the absence of BACE1/2 activity using a beta-secretase inhibitor and BACE knock-out cells, indicating that meprin beta acts independently of beta-secretase

The γ-secretase modulator photo-probe AR243 targets endogenous PSEN1 in human HEK293T cells.

<p>Photo-affinity labeling studies were performed with membrane preparations from human HEK293T cells as described in Fig. 1. Western blotting of purified target proteins demonstrated labeling of endogenous PSEN1-NFT but not PSEN1-CTF by AR243. Co-incubation with an excess of parent compound BB25 (100 µM) caused displacement of the photo-probe, demonstrating specificity of the binding to PSEN1-NTF. Input represents 0.02% of the total membrane material.</p

Discovery of gamma-secretase modulators with a novel activity profile by text-based virtual screening

We present an integrated approach to identify and optimize a novel class of gamma-secretase modulators (GSMs) with a unique pharmacological profile. Our strategy included (i) virtual screening through application of a recently developed protocol (PhAST), (ii) synthetic chemistry to discover structure-activity relationships, and (iii) detailed in vitro pharmacological characterization. GSMs are promising agents for treatment or prevention of Alzheimer&#039;s disease. They modulate the gamma-secretase product spectrum (i.e., amyloid-beta (A beta) peptides of different length) and induce a shift from toxic A beta 42 to shorter A beta species such as A beta 38 with no or minimal effect on the overall rate of gamma-secretase cleavage. We describe the identification of a series of 4-hydroxypyridin-2-one derivatives, which display a novel type of gamma-secretase modulation with equipotent inhibition of A beta 42 and A beta 38 peptide species

Chemical structures and Aβ42-lowering activities of γ-secretase modulators used in this study.

<p>IC₅₀ values were determined after treatment of CHO cells with stable co-expression of wild type human APP and PSEN1 as described in the Experimental Procedures. Note that AR366 is an inverse γ-secretase modulator with Aβ42-raising activity.</p

Structurally divergent acidic and non-acidic γ-secretase modulators compete binding of the photo-probe AR243 to the N-terminal fragment of PSEN1.

<p>Competition experiments were performed under cell-free conditions with membrane preparations from N2a-ANPP cells. Membranes were co-incubated with 0.5 µM of the photo-probe AR243 and 100 µM of acidic or non-acidic GSM competitor compounds. (A) The acidic GSM JNJ-40418677 abolished binding of the photo-probe to PSEN1-NTF. (B) Three non-acidic GSMs competed binding of the photo-probe to variable degrees. Compound 1 reduced binding of the photo-probe as effectively as the acidic compound JNJ-40418677. Compound 2 and E-2012 only partially competed binding of the photo-probe to PSEN1-NTF. Input represents 0.02% of the total membrane material.</p