Sensitive and Continuous Screening of Inhibitors of β-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) at Single SPR Chips

Abstract

Developments of new methods that meet the demand of high-throughput, high-fidelity screening of hit compounds are important to searching modalities of important diseases such as neurological disorders, HIV, and cancer. A surface plasmon resonance (SPR)-based method capable of continuously screening enzyme inhibitors at a single chip with antibody-amplified signal enhancement is developed. The proof of concept is demonstrated by monitoring the cleavage of chip-confined peptide substrates (a segment of the amyloid precursor protein (APP) with the Swiss mutation) by β-site APP cleaving enzyme 1 (BACE1). In the presence of a non- inhibitor, BACE1 clips the peptide substrate at the cleavage site, detaching a fragment that is homologous to the N-terminus of the amyloid beta (Aβ) peptide. Consequently, a subsequent injection of the Aβ antibody does not lead to any molecular recognition or SPR signal change at the chip. In contrast, abolishment of the BACE1 activity by a strong inhibitor leaves the peptide substrate intact, and the subsequent antibody attachment produces an easily detectable SPR signal. Compared to the widely used FRET (fluorescence resonance energy transfer) assay, the method reported here is more cost effective, as unlabeled peptide is used as the BACE1 substrate. Furthermore, the assay is more rapid (each screening cycle lasts for ca. 1.5 h) and can be continuously carried out at a single, regenerable SPR chip for more than 30 h. Consequently, excellent reproducibility (RSD% < 5%) and throughput can be attained. Two inhibitors were screened and their half maximal inhibitory concentrations (IC50) determined by the SPR method are in excellent agreements with values deduced from ELISA and mass spectrometry.

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of β‐secretase as the Alzheimer's disease drug target*

“…This is in contrast with our recent findings that the unlabelled BACE1 substrate with identical peptide sequence exhibits significantly different kinetic behaviour than the labelled substrates, by up to several orders of magnitude [24,25]. The exceptions which applied unlabelled substrate, to the best of our knowledge, used surface plasmon resonance [26], an electrochemical method [27] and HPLC-MS [5]. In the first two cases, the substrate needed to be immobilized onto a solid surface and the kinetic parameters of such assays were not reported.…”

Aptamer based surface plasmon resonance sensor for aflatoxin B1

“…Surface plasmon resonance (SPR) sensing is a powerful analytical technique for molecular interactions [19][20][21][22][23][24][25][26]. It is relatively challenging to develop SPR sensors for small [27], inhibition [28] or sandwich strategies [29].…”

Multidisciplinary approaches for targeting the secretase protein family as a therapeutic route for Alzheimer's disease

“…Recently, various other detection methodologies have been developed, for example, surface plasmon resonance (SPR) . Briefly, Yi et al used a single SPR chip to aid in the sensitive and continuous screening of BACE1 inhibitors. This method was able to reduce cost and enhance performance with the continuous use of multiple assays.…”

“…113 Recently, various other detection methodologies have been developed, for example, surface plasmon resonance (SPR). 174,175 Briefly, Yi et al 176…”