A Structure-Based Discovery Platform for BACE2 and the Development of Selective BACE Inhibitors.

The ability to perform routine structure-guided drug design for selective BACE inhibitors has been limited because of the lack of robust platform for BACE2 expression, purification, and crystallization. To overcome this limitation, we developed a platform that produces 2-3 mg of pure BACE2 protein per liter of E. coli culture, and we used this protein to design macrocyclic compounds that potently and selectively inhibit BACE1 over BACE2. Compound 2 was found to potently inhibit BACE 1 (Ki = 5 nM) with a selectivity of 214-fold over BACE2. The X-ray crystal structures of unbound BACE2 (2.2 Å) and BACE2 bound to compound 3 (3.0 Å and Ki = 7 nM) were determined and compared to the X-ray structures of BACE1 revealing the S1-S3 subsite as a selectivity determinant. This platform should enable a more rapid development of new and selective BACE inhibitors for the treatment of Alzheimer's disease or type II diabetes.

Development of an Efficient Enzyme Production and Structure-Based Discovery Platform for BACE1 Inhibitors.

BACE1 (Beta-site Amyloid Precursor Protein (APP) Cleaving Enzyme 1) is a promising therapeutic target for Alzheimer's Disease (AD). However, efficient expression, purification, and crystallization systems are not well described or detailed in the literature nor are approaches for treatment of enzyme kinetic data for potent inhibitors well described. We therefore developed a platform for expression and purification of BACE1, including protein refolding from E.coli inclusion bodies, in addition to optimizing a reproducible crystallization procedure of BACE1 bound with inhibitors. We also report a detailed approach to the proper analysis of enzyme kinetic data for compounds that exhibit either rapid-equilibrium or tight-binding mechanisms. Our methods allow for the purification of ∼15 mg of BACE1 enzyme from 1 L of culture which is higher than reported yields in the current literature. To evaluate the data analysis approach developed here, a well-known potent inhibitor and two of its derivatives were tested, analyzed, and compared. The inhibitory constants (Ki) obtained from the kinetic studies are in agreement with dissociation constants (Kd) that were also determined using isothermal titration calorimetry (ITC) experiments. The X-ray structures of these three compounds in complex with BACE1 were readily obtained and provide important insight into the structure and thermodynamics of the BACE1-inhibitor interactions.