An Inhibitor Binding Pocket Distinct from the Catalytic Active Site on Human β-APP Cleaving Enzyme

Abstract

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 Abeta peptide. It is widely believed that halting the production of Abeta 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.

Inhibition of the Proteolytic Activity of Pregnancy-associated Plasma Protein-A by Targeting Substrate Exosite Binding

“…There are examples of proteolytic inhibition by exosite targeting in the literature. For example, matrix metalloproteinase-2 cleavage of type I gelatin and type IV collagen has been inhibited by targeting the collagen binding domain using a synthetic peptide (53), and a substrate binding pocket distinct from the catalytic site of ␤-amyloid precursor protein cleaving enzyme has been targeted by synthetic peptides (54). Also, many exosite inhibitors have been developed toward the coagulation enzyme factor VIIa (55), but although specific for this proteinase, all of its several biological substrates are targeted by such inhibitors (56).…”

Essential Dynamics on Different Biological Systems: Fis Protein, tvMyb1 Transcriptional Factor and BACE1 Enzyme

“…In addition to the active site, some proteolytic enzymes contain additional binding pockets, termed exosites, which engage substrates at locations distal to the active site (Krishnaswamy & Betz, 1997;Maun et al, 2003). These binding pockets on some proteolytic enzymes can act as allosteric regulators of the enzyme activity through conformational changes to the active site, in order to cause an augmentation or diminution of the enzyme's catalytic reactivity (Kornacker et al, 2005). While exosites were reported for serine and cysteine proteases, few examples of exosites have been reported for aspartyl proteases (Kornacker et al, 2005).…”

“…These binding pockets on some proteolytic enzymes can act as allosteric regulators of the enzyme activity through conformational changes to the active site, in order to cause an augmentation or diminution of the enzyme's catalytic reactivity (Kornacker et al, 2005). While exosites were reported for serine and cysteine proteases, few examples of exosites have been reported for aspartyl proteases (Kornacker et al, 2005). Recently, we have reported the location for the exosite of BACE1 enzyme (Gutierrez et al, 2010).…”

Structure and modeling in the design of β‐ and γ‐secretase inhibitors

“…Some examples of classical TSAs are shown in Figure 6. Some notable exceptions are: (1) a nonpeptide inhibitor based on a lead from an Automated Ligand Identification System (ALIS) technology developed at NeoGenesis [Annis et al, 2004], (2) spiropiperidine inhibitors identified via HTS screening [Barrow et al, 2008], (3) a tyramine inhibitor identified via fragment-based screening [Kuglstatter et al, 2008], and (4) exosite inhibitors containing the sequence YPYF(I/ L)P(L/I) [Kornacker et al, 2005] identified from combinatorial phage peptide libraries. The ''exosite'' is a ligand-binding pocket within the catalytic domain of BACE-1 that appears to be distinct from the catalytic site.…”