Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product.

Department of Neurology, Merck Research Laboratory, West Point, PA 19486, USA.
Protein & Cell (Impact Factor: 3.22). 07/2011; 2(7):573-84. DOI: 10.1007/s13238-011-1076-4
Source: PubMed

ABSTRACT We have previously described a novel artificial NFEV β-secretase (BACE1) cleavage site, which when introduced into the amyloid-β precursor protein (APP), significantly enhances APP cleavage by BACE1 in in vitro and cellular assays. In this study, we describe the identification and characterization of a single chain fragment of variable region (scFv), specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide, and its conversion to IgG1. Both the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage products. EV-IgG1 can detect as little as 0.3 nmol/L of the EV peptide. EV-IgG1 antibody was purified, conjugated with alkaline phosphatase and utilized in various biological assays. In the BACE1 enzymatic assay using NFEV substrate, a BACE1 inhibitor MRK-3 inhibited cleavage with an IC(50) of 2.4 nmol/L with excellent reproducibility. In an APP_NFEV stable SH-SY5Y cellular assay, the EC(50) for inhibition of EV-Aβ peptide secretion with MRK-3 was 236 nmol/L, consistent with values derived using an EV polyclonal antibody. In an APP_NFEV knock-in mouse model, both Aβ_EV40 and Aβ_EV42 peptides in brain homogenate showed excellent gene dosage dependence. In conclusion, the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro, cellular screening assays and in vivo biochemical studies. The methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.

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    ABSTRACT: Reduction in cerebrospinal fluid (CSF) amyloid β42 (Aβ42) and elevation in total tau and phospho-thr181 tau consistently differentiate between Alzheimer's disease (AD) and age-matched control subjects. In contrast, CSF β-site APP-cleaving enzyme activity (BACE1) and soluble amyloid precursor proteins α and β (sAPPα and sAPPβ) are without consistent patterns in AD subjects. Plasma sampling is much easier, with fewer side effects, and is readily applied in primary care centers, so we have developed and validated novel plasma BACE activity, sAPPβ, and sAPPα assays and investigated their ability to distinguish AD from age-matched controls. Plasma BACE activity assay was sensitive and specific, with signal being immunodepleted with a specific BACE1 antibody and inhibited with a BACE1-specific inhibitor. Plasma sAPPβ and sAPPα assays were specific, with signal diluting linearly, immunodepleted with specific antibodies, and at background levels in APP knockout mice. In rhesus monkeys, BACE1 but not γ-secretase inhibitor led to significant lowering of plasma sAPPβ with concurrent elevation of plasma sAPPα. AD subjects showed a significant increase in plasma BACE1 activity, sAPPβ, sAPPα, and Aβ42 (P < 0.001) compared with age-matched controls. In conclusion, plasma BACE activity and sAPP endpoints provide novel investigative biomarkers for AD diagnosis and potential pharmacodynamic biomarkers for secretase inhibitor studies. © 2012 Wiley Periodicals, Inc.
    Journal of Neuroscience Research 09/2012; 90(12):2247-58. · 2.73 Impact Factor


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May 29, 2014