Inhibition of β-Secretase in Vivo via Antibody Binding to Unique Loops (D and F) of BACE1

Abstract

␤-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 ϳ0.76 nM), in primary neurons (EC 50 ϳ1.8 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 ϳ0.47 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.

Arsenic Toxicity: The Effects on Plant Metabolism

“…The half-life of an AsIII-monothiol peptide complex is only about 1–2 s. The half-life increases to about 1.3 and 155 min when two or three intramolecular thiols are bound (Kitchin and Wallace, 2006b). The high stability of AsIII-trithiol complexes is supported by the finding that AsIII preferentially binds zinc-finger proteins containing three or more Cys residues in the zinc-finger motifs (Zhou et al, 2011). This study did not find AsIII binding to zinc-finger motifs with only two Cys residues, possibly due to the time needed to process the samples (Zhou et al, 2011).…”

ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1

“…Taken together, all our data strongly seem to support that sAPP accumulating in the lumen of RAB5-Q79L endosomes originates from local BACE1 activity in a subpool of RAB5-positive endosomes. Moreover, by using a BACE1 ectodomain-specific mAb 10B8 in an antibody uptake assay (25), we demonstrate that BACE1 is likely delivered to early endosomes via internalization of its surface-associated pool ( Fig. 1G and Fig.…”

Developing Therapeutic Antibodies for Neurodegenerative Disease

“…In an attempt to tackle BACE1 in a novel and selective way, we, and others, have recently developed antibodies that selectively bind and inhibit BACE1 activity [29,54]. This approach revealed several important aspects about antibody therapeutics directed against CNS targets.…”

“…To address this, we engineered a bispecific antibody that binds both TfR and BACE1 [123]. As discussed earlier, function-blocking antibodies to BACE1 have been shown to reduce Aβ levels in vivo by inhibiting APP cleavage [29,54]. Unfortunately, in order to have a modest reduction of Aβ in brain, very high and frequent dosing was required.…”