Development and Characterization of a Novel Membrane Assay for Full-Length BACE-1 at pH 6.0

Abstract

β-Site amyloid precursor protein cleaving enzyme-1 (BACE-1) is a transmembrane aspartic protease that mediates the initial cleavage of the amyloid precursor protein (APP), leading to the generation of amyloid-β (Aβ) peptides that are thought to be causative of Alzheimer's disease (AD). Consequently, inhibition of BACE-1 is an attractive therapeutic approach for the treatment of AD. In general, in vitro biochemical assays to monitor BACE-1 activity have used the extracellular domain of the protein that contains the catalytic active site. This form of BACE-1 is catalytically active at acidic pH and cleaves APPbased peptide substrates at the β-site. However, this form of BACE-1 does not mimic the natural physiology of BACE-1 and shows minimal activity at pH 6.0, which is more representative of the pH within the intracellular compartments where BACE-1 resides. Moreover, high-throughput screens with recombinant BACE-1 at pH 4.5 have failed to identify tractable leads for drug discovery, and hence, BACE-1 inhibitor development has adopted a rational drug design approach. Here we describe the development and validation of a novel membrane assay comprising full-length BACE-1 with measurable activity at pH 6.0, which could be used for the identification of novel inhibitors of BACE-1.

Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted β-Secretase Using Single-Molecule Imaging

“…20 While the vast majority of drug development campaigns have utilized catalytically active, truncated versions of the protein, 21−23 recent reports suggest fundamental differences between the native, transmembrane enzyme and its truncated equivalent, such as homodimerization of the full-length construct but not the extracellular domain (ectodomain) 4,24 and differences in pH dependence on their activity. 25 Furthermore, it has also been shown that the proteolytic activity of BACE1 is modulated by the lipid environment surrounding the protein. 8 Taken together, this strongly motivates our development of a high-sensitivity assay compatible with membrane-embedded BACE1.…”

Development of a Novel β-Secretase Binding Assay Using the AlphaScreen Platform

“…Recently, an assay using full-length BACE1 screened at pH 6 was developed. 30 BACE1 activity could be regulated by the transmembrane and cytoplasmic domains, and BACE1 does reside in Golgi and endocytic compartments that have a pH ~6. 30 Thus, although the activity of the BACE1 extracellular domain is most active at pH 4.5, it may be that higher pH buffers better represent the physiological pH for BACE1.…”

“…30 BACE1 activity could be regulated by the transmembrane and cytoplasmic domains, and BACE1 does reside in Golgi and endocytic compartments that have a pH ~6. 30 Thus, although the activity of the BACE1 extracellular domain is most active at pH 4.5, it may be that higher pH buffers better represent the physiological pH for BACE1. A major advantage of the AlphaScreen assay described in this article compared with other assay formats is the enhanced sensitivity that is observed with this format, as shown in Supplementary Table 1.…”

“…30 Thus, although the activity of the BACE1 extracellular domain is most active at pH 4.5, it may be that higher pH buffers better represent the physiological pH for BACE1. Fig.…”

Emerging Alternative Proteinases in APP Metabolism and Alzheimer’s Disease Pathogenesis: A Focus on MT1-MMP and MT5-MMP

“…β-site APP cleaving enzyme 1 (BACE-1) is the main β-secretase. This type I transmembrane protein of the aspartyl proteinase family needs an acidic environment (optimum pH 4.5) to be enzymatically active (Saric et al, 2013). BACE-1 cleaves numerous substrates, which confers this enzyme a wide spectrum of physiological and pathological activities (Kuhn et al, 2012; Zhou et al, 2012; Vassar et al, 2014), but it is indisputably its ability to process APP that has attracted much attention, especially in relation to AD.…”