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

PSEN is the molecular target of the γ-secretase modulator photo-probe AR243 in living cells.

<p>(<b>A</b>) Schematic illustration of the photo-affinity labeling strategy in living cells. The active GSM photo-probe contains a diazirine photo-reactive group and a biotin moiety for affinity purification. Live cells were treated with the photo-probe and subsequently irradiated in two steps at 365 and 302 nm. Note that the use of long-wave UV light ≥300 nm did not induce acute cellular toxicity. Upon UV irradiation, the photo-probe forms a stable adduct with its specific target. Cellular membranes were prepared and solubilized, and target proteins of the photo-probe were captured and affinity purified using streptavidin-functionalized beads. After extensive washing, the purified material was eluted and resolved on SDS gels and probed by Western blotting. (<b>B</b>) N2a-ANPP cells were incubated with 200 nM of AR243 or DMSO vehicle, and target proteins of the GSM photo-probe were purified as described above. Western blotting with the antibody PSN2 demonstrated binding of the photo-probe to PSEN1-NTF. Incubation of cells with 20 µM of compound AR80 that lacked the biotin moiety, or omission of UV irradiation did not result in a labeling signal. Consistent with the <i>in vitro</i> labeling results, Western blotting with an antibody against the PSEN1-CTF did not demonstrate binding of the photo-probe confirming that the binding site is located within the PSEN1-NTF.</p

Synthesis and bioactivity of the γ-secretase modulator photo-probe AR243.

<p>(<b>A</b>) The γ-secretase modulator (GSM) photo-probe AR243 was synthesized by a copper(I)-catalyzed three-component coupling between piperidine-derivative <b>1</b>, diazirinyl-benzaldehyde <b>2</b>, and biotin-alkyne <b>3</b>. The intermediate was then de-protected to yield the free acidic photo-probe. i: CuBr, molecular sieves, Tol. 60°C, 2 d. ii: TBAF, THF, r.t., 2 h. (<b>B</b>) CHO cells with stable co-expression of wild type APP and wild type PSEN1 were treated with increasing concentrations of the photo-probe AR243 or DMSO vehicle, and Aβ levels in conditioned media were quantified by sandwich immunoassay. AR243 caused a dose-dependent decrease in Aβ42 levels with a concomitant increase in Aβ38 levels, confirming its bioactivity as a potent GSM with an IC₅₀ for Aβ42 reduction of 290 nM.</p

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

Discovery of γ-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 γ-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 <i>in vitro</i> pharmacological characterization. GSMs are promising agents for treatment or prevention of Alzheimer’s disease. They modulate the γ-secretase product spectrum (<i>i.e</i>., amyloid-β (Aβ) peptides of different length) and induce a shift from toxic Aβ42 to shorter Aβ species such as Aβ38 with no or minimal effect on the overall rate of γ-secretase cleavage. We describe the identification of a series of 4-hydroxypyridin-2-one derivatives, which display a novel type of γ-secretase modulation with equipotent inhibition of Aβ42 and Aβ38 peptide species