Amyloid beta 42 peptide (Abeta42)-lowering compounds directly bind to Abeta and interfere with amyloid precursor protein (APP) transmembrane dimerization

Abstract

Following ectodomain shedding by beta-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by gamma-secretase result in the release of amyloid-beta (Abeta) peptides of variable length. Abeta peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer's disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent gamma-secretase modulator (GSM), selectively reduces Abeta42 production in favor of shorter Abeta species, such as Abeta38. By studying APP-TMS dimerization we previously showed that an attenuated interaction similarly decreased Abeta42 levels and concomitantly increased Abeta38 levels. However, the precise molecular mechanism by which GSMs modulate Abeta production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP-TMS dimers are destabilized by sulindac sulfide and related Abeta42-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the Abeta sequence. Strikingly, the attenuated APP-TMS interaction by GSMs correlated strongly with Abeta42-lowering activity and binding strength to the Abeta sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP-TMS. We conclude that these GSMs decrease Abeta42 levels by modulating APP-TMS interactions. This effect specifically emphasizes the importance of the dimeric APP-TMS as a promising drug target in Alzheimer's disease.

Fig. 1.

Chemical structures of sulindac sulfide (A), sulindac sulfone (B), sulindac (C), and indomethacin (D).

Fig. 2.

Interaction of compounds with Aβ42 determined by SPR analysis. Overlays of representative SPR sensorgrams obtained from injections of sulindac sulfide (A), sulindac sulfone (B), sulindac (C), or indomethacin (D). Synthetic Aβ42 peptide was immobilized yielding 1,700 response units (RUs). Compounds at indicated concentrations were injected for 60 s at a flow rate of 30 μL/ min. All binding curves were double-reference subtracted from DMSO buffer blank and the reference flow cell and adjusted to the molecular weight of the compounds.

Fig. 3.

Interaction between Aβ42 and sulindac sulfide monitored by solution-state NMR spectroscopy. two-dimensional ¹H, ¹⁵N correlation spectrum obtained for a 100 μM solution of Aβ42 in the presence and absence of sulindac sulfide. Chemical shifts are highlighted by dashed circles.

Fig. 4.

Dimerization of the APP–TMS in the presence of compounds. (A) Aβ residues 23–54. The TMS is highlighted by the light gray box. Glycine residues of the GxxxG motifs are shown in dark gray. The indicated sequence Aβ 29–42 comprises the amino acids which were inserted into the ToxR fusion protein. (B–E) ToxR assays. Dimerization in the presence of sulindac sulfide (B), sulindac sulfone (C), sulindac (D), and indomethacin (E) measured as β-Gal activity. DMSO control was set as 100% (mean ± SEM, n = 8–15). Asterisks indicate significant difference from DMSO control (^∗P < 0.0001, Student’s t test).

Fig. 5.

Sulindac derivatives and their effects on Aβ production, binding to Aβ42, and APP–TMS dimerization. (A) Unique scaffold from which sulindac-derived agents (A–H) have been evolved. R¹, R²: substituents. (B) Overview of unique sulindac derivatives. Structural features are listed. Compounds with Aβ42-lowering activity are classified as active, otherwise as inactive. (C) ELISA: Aβ40 and Aβ42 levels detected upon treatment of CHO cells with 60 μM compound. DMSO-treated cells served as control (set as 100%, mean ± SEM, n = 3, ^∗P < 0.05, Student’s t test). Sixty micromolar sulindac sulfide reduced Aβ42 by about 60% (gray line) without affecting Aβ40 levels (dashed black line) (7). (D) SPR analysis: Compounds at 40 μM were injected, the association phase was monitored for 30 s. Double-reference subtracted sensorgrams were adjusted to the respective molecular weight of the compounds. Response unit (RU) values at the end of the injection were displayed as percentage of RUs obtained upon injections of 40 μM sulindac sulfide in the same run (set as 100%, indicated by the horizontal gray line, mean ± SEM, n = 3, ^∗P < 0.01, Student’s t test). For sensorgrams, see Fig. S5. (E) ToxR assay: Compounds were tested at 10 μM. At this concentration, sulindac sulfide reduced dimerization strength by 33% as marked by the horizontal gray line. DMSO control was set as 100% (mean ± SEM, n = 3, ^∗P < 0.01, Student’s t test).

Fig. 6.

Sulindac sulfide (A), sulindac sulfone (B), sulindac (C), and indomethacin (D) flexibly docked to the APP–TMS. All compounds cluster at the smooth surface provided by glycines arranged in GxxxG motifs. Oxygen is depicted in red, nitrogen in blue, sulfur in dark yellow, fluorine in deep purple, and chlorine in lime green. Potential hydrogen bonds are indicated as black dashed lines.