title: Inhibition of beta-secretase in vivo via antibody binding to unique loops (D and F) of BACE1.
abstract: β-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ∼0.76 nm), in primary neurons (EC(50) ∼1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ∼0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.
authors.affiliation: From the Department of Molecular and Developmental Genetics, VIB and; the Center for Human Genetics, KULeuven, 3000 Leuven, Belgium.
authors.forename: Lujia
authors.initials: L
authors.lastname: Zhou
authors.affiliation: From the Department of Molecular and Developmental Genetics, VIB and; the Center for Human Genetics, KULeuven, 3000 Leuven, Belgium.
authors.forename: Lucia
authors.initials: L
authors.lastname: Chávez-Gutiérrez
authors.affiliation: From the Department of Molecular and Developmental Genetics, VIB and; the Center for Human Genetics, KULeuven, 3000 Leuven, Belgium.
authors.forename: Katrijn
authors.initials: K
authors.lastname: Bockstael
authors.affiliation: From the Department of Molecular and Developmental Genetics, VIB and; the Center for Human Genetics, KULeuven, 3000 Leuven, Belgium.
authors.forename: Ragna
authors.initials: R
authors.lastname: Sannerud
authors.affiliation: From the Department of Molecular and Developmental Genetics, VIB and; the Center for Human Genetics, KULeuven, 3000 Leuven, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Annaert
authors.affiliation: the Neuroscience Discovery Research, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285, and.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: May
authors.affiliation: Johnson and Johnson Pharmaceutical Research and Development, 2340 Beerse, Belgium.
authors.forename: Eric
authors.initials: E
authors.lastname: Karran
authors.affiliation: From the Department of Molecular and Developmental Genetics, VIB and; the Center for Human Genetics, KULeuven, 3000 Leuven, Belgium,. Electronic address: Bart.DeStrooper@cme.vib-kuleuven.be.
authors.forename: Bart
authors.initials: B
authors.lastname: De Strooper
mesh_terms: D000544:Alzheimer Disease / Q000188:drug therapy / Q000201:enzymology / Q000276:immunology; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology / Q000494:pharmacology*; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000494:pharmacology*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D001921:Brain / Q000201:enzymology* / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D016296:Mutagenesis; D009474:Neurons / Q000201:enzymology*; D011480:Protease Inhibitors / Q000276:immunology / Q000494:pharmacology*; D017433:Protein Structure, Secondary
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D057134:Antibodies, Neutralizing; D011480:Protease Inhibitors; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse