Selected Non-steroidal Anti-inflammatory Drugs and Their Derivatives Target γ-Secretase at a Novel Site

Abstract

␥-Secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-␤ (A␤) peptides from processing intermediates of the ␤-amyloid precursor protein. Because A␤ peptides are thought to be causative for Alzheimer's disease, inhibiting ␥-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of ␥-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic A␤(1-42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of A␤(1-42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective ␥-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the ␥-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of ␥ (42)-cleavage over alternative cleavages in the ␤-amyloid precursor protein and, more importantly, alternative substrates.According to the "amyloid cascade hypothesis" an enhanced production or decreased clearance of toxic amyloid-␤ (A␤) 1 peptides is thought to be the cause of Alzheimer's disease (AD) (1). A␤ peptides are processing products (2) of the type I transmembrane protein ␤-amyloid precursor protein (␤APP) (3), which has undergone sequential cleavages by ␤-and ␥-secretase enzymes. A common denominator (reviewed by Hardy (4)) for mutations causative of familial AD (FAD) has been revealed, being abnormalities in the metabolism of ␤APP that appear to lead to an elevation of the production of the A␤(1-42) peptide species. This C-terminally elongated A␤ peptide is more prone to aggregation than the shorter and more abundant A␤(1-40) species. Consequently, the prevention of A␤ production by inhibiting either of the proteases required for processing of ␤APP is currently viewed as a promising approach toward a therapy for AD. The membrane-bound aspartyl protease ␤-site ␤APP-cleaving enzyme 1 (5, 6) is the major ␤-secretase required for the generation of A␤ peptides. ␤-Site ␤APP-cleaving enzyme 1 has been shown to cleave within th...

Signal Peptide Peptidase and γ-Secretase Share Equivalent Inhibitor Binding Pharmacology

“…NSAIDs modulate the cleavage site positions of SPP and ␥-secretase on their substrates without inhibiting enzyme activity, and it therefore seems likely that they affect the active site indirectly, if at all. Consistent with this, it was reported that displacement of [ 3 H]L-685,458 by NSAIDs from ␥-secretase is noncompetitive (18,37). We showed that noncompetitive displacement of [ 3 H]L-685,458 by sulindac sulfide also occurred for SPP (Fig.…”

“…If so, the presence of SPP might account for the incomplete displacement of [ 3 H]L-685,458 observed for selective ␥-secretase inhibitors (18), thus calling into question the conclusion that the catalytic active site and the allosteric binding site are present in a 2:1 molar ratio (18). Furthermore, it seems likely that earlier studies of this radioligand in whole cell extracts may have detected predominantly SPP binding rather than ␥-secretase binding (17,37 From the drug discovery perspective, similar pharmacology between ␥-secretase and SPP suggests the potential for off-target liabilities. Additional studies would be needed to understand to what extent inhibitor binding affects enzyme function in vivo and to determine what consequences, if any, would arise from dosing a nonselective drug.…”

“…To address this question, we utilized an active site-directed ␥-secretase radioligand (17,18,37) and took advantage of our finding that it binds with high affinity to both SPP and ␥-secretase. In the course of this study, we found that SPP family protein binding is abundant in cell homogenates, presenting a challenge to the evaluation of ␥-secretase and SPP binding under these conditions.…”

Random Mutagenesis of Presenilin-1 Identifies Novel Mutants Exclusively Generating Long Amyloid β-Peptides

“…DFK167 and sulindac sulfide are thought to shift the predominant cleavage site, probably through allosteric effects induced by direct PS binding (38,47,50,51). Several of our observations suggest that the effects of PS1 mutations and the allosteric effects of ␥-secretase inhibitors are likely to be tightly related, and we speculate that these effects might even be mediated by a shared mechanism.…”

An alternative interpretation of the amyloid Aβ hypothesis with regard to the pathogenesis of Alzheimer's disease

“…Compounds, like the sterols that naturally intercalate within the lipid bilayer, are known to affect many membrane functions. Recently, many investigators have reported that a class of polycyclic compounds known as nonsteroidal antiinflammatory drugs (NSAIDS) block the production of A␤ peptides in animals models of AD and cultured cells (37)(38)(39). It has been claimed that NSAIDS block A␤ production by affecting the conformation of PS1 (40), but it is also conceivable that NSAIDS might in some way modify interactions between intramembranous peptide segments.…”