Compounds That Bind APP and Inhibit Aβ Processing in Vitro Suggest a Novel Approach to Alzheimer Disease Therapeutics

Abstract

Extracellular deposits of aggregated amyloid-␤ (A␤)peptides are a hallmark of Alzheimer disease; thus, inhibition of A␤ production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of A␤ requires processing of the amyloid precursor protein (APP) by both ␤-and ␥-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for ␤-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and A␤ by BACE2 and selectively inhibit the production of A␤ 42 species by ␥-secretase in assays using CTF99. The compounds bind directly to APP, likely within the A␤ domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating A␤ production. Alzheimer disease (AD)1 is the leading cause of senile dementia. AD currently affects 4.5 million people in the United States and is anticipated to increase to more than 13 million by the year 2050 (1, 2). Histopathologically, AD is characterized by the prevalence of senile plaques, as well as neocortical atrophy, neuronal and synaptic loss, and the formation of neuritic tangles of the microtubule-associated protein tau (3). Senile plaques consist predominantly of fibrils of amyloid ␤ (A␤), a 39 -43-amino acid peptide generated by proteolysis of the amyloid precursor protein (APP) (4). Production of A␤ has been linked genetically to AD. Recent studies (5-7) have shown that A␤ multimerizes into neurotoxic oligomers that target the synapse, leading to disruption of long term potentiaton, cell death, and memory loss. These oligomers then coalesce into fibrils, ultimately forming the plaques originally used to characterize the disease (5-7).To produce A␤, APP, a type I single-transmembrane glycoprotein, is sequentially cleaved by two aspartyl proteases, ␤-and ␥-secretase. The initial cleavage is mediated by ␤-secretase (BACE1) on the luminal side of the endosomal membrane or at the cell surface (8). BACE1 cleavage generates a secreted Nterminal product (sAPP␤) and a transmembrane N-terminal product (CTF99). Processing of CTF99 within the transmembrane domain by the ␥-secretase enzyme complex generates the C-terminal end of A␤ and results in its release (9). The ␥-secretase enzyme is known to cleave CTF99 at any of several discrete positions, generating A␤ peptides that range in length from 38 to 42 amino acids. Although A␤ 40 is the predominant species produced both in vitro and in vivo, A␤ 42 is the more aggregenic species and the major component of amyloid plaques. The release of A␤ 42 and subsequent aggregation into insoluble fibrils are believed to lead to the formation of the dense plaques characteristic of AD (1...

Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage

“…BACE inhibitor IV completely eliminated BACE1 cleavage of the APP-C140 substrate, in contrast to Gleevec, which had no effect at all concentrations tested, thus again ruling out a direct inhibitory effect of Gleevec on BACE1 or an indirect effect through interaction with APP. Interestingly, it has been reported that a series of benzofuran-containing compounds selectively inhibit BACE processing of APP in vitro by binding directly to APP around the BACE cleavage site (17), and another study involving two flavonoid compounds reported similar results (18). It is unlikely that Gleevec inhibits BACE cleavage of APP by the same mechanism, however.…”

Development of Homogeneous 384-Well High-Throughput Screening Assays for Aβ1-40 and Aβ1-42 Using AlphaScreen™ Technology

“…Espeseth et al 11 describe an assay that is suitable for measuring β-secretase activity via measurement of sAPPβ. The same group also recently reported the development of assays that can measure sAPPα as well as genetically modified forms of Aβ peptides.…”

Survey of the year 2005 commercial optical biosensor literature

“…Also, among the 881 articles describing Biacore-based studies, 84% included information about which platform was used: most often studies were performed using the 3000 (38%), 2000 (22%), X (16%), and 1000 (4%) platforms, with the residual 4% using S51 [157,757,760,761,768,[779][780][781][782][783][789][790][791]798,918], Flexchip (formerly Applied Biosystems 8500 Affinity Analyzer) [138,338,434,435,583], Q [926,928,929,931,933,934,936,939,940], J [199,240,614,827,915], and Bialite [873].…”