Anti-APP monoclonal antibody, clone DB911D12

beta-APP cleaving enzyme (BACE) is responsible for the first of two proteolytic cleavages of the APP protein that together lead to the generation of the Alzheimer's disease-associated A beta peptide. It is widely believed that halting the production of A beta peptide, by inhibition of BACE, is an attractive therapeutic modality for the treatment of Alzheimer's disease. BACE is an aspartyl protease, and there is significant effort in the pharmaceutical community to apply traditional design methods to the development of active site-directed inhibitors of this enzyme. We report here the discovery of a ligand binding pocket within the catalytic domain of BACE that is distinct from the enzymatic active site (i.e., an exosite). Peptides, initially identified from combinatorial phage peptide libraries, contain the sequence YPYF(I/ L)P(L/I) and bind specifically to this exosite, even in the presence of saturating concentrations of active site-directed inhibitors. Binding of peptides to the BACE exosite leads to a concentration-dependent inhibition of proteolysis for APP-related, protein-based substrates of BACE. The discovery of this exosite opens new opportunities for the identification and development of novel and potentially selective small molecule inhibitors of BACE that act through exosite, rather than active site, binding interactions.

Presenilin-dependent intramembranous proteolysis mediates the dual cleavage of the Notch-1 protein (S4 and S3) as well as the beta amyloid precursor protein (beta APP) (gamma 40 and epsilon-site). beta APP has a valine residue just before the gamma 40 (amyloid beta [A beta] numbering) site and after the epsilon-site. Both gamma 40 and epsilon have multiple cleavage sites, and the varieties of gamma 40 cleavage are associated with Alzheimer's disease (AD). These lines of evidence suggest that valine plays a role in the intramembranous proteolysis. S4 cleavage in the middle of the Notch-1 transmembrane domain (TMD) corresponds to the gamma 40 cleavage of beta APP. The cleavage site is in the center of four sequential alanine residues between Ala1731 and Ala1732, neither of which has a valine residue. To investigate the effects of valine on presenilin-dependent intramembranous proteolysis, we replaced the transmembrane domain residue of Notch-1 with valine and analyzed the efficiency and precision at S4 and S3. We observed that all valine-mutated Notch-1 proteins have a dominant cleavage site (S4) between Ala1731 and Ala1732 with some variations of cleavage precision, suggesting that valine is not indispensable for determining the cleavage site of the Notch-1 transmembrane domain, but affects the efficiency and precision at S4 cleavage of the Notch-1 transmembrane domain. (c) 2006 Wiley-Liss, Inc.