Abstract
The development of new methods that meet the demand of high-throughput, high-fidelity screening of hit compounds is important to researching 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 has been 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 noninhibitor, 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, suppression 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 faster (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 were in excellent agreement with values deduced from ELISA and mass spectrometry.