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Development of an efficient structure‐based drug discovery platform for BACE1 Inhibitors for the treatment of Alzheimer's Disease
Author(s) -
Yen YuChen,
Kammeyer Annalissa,
Jensen Katherine C.,
Ghosh Arun K.,
Mesecar Andrew D.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.607.9
Subject(s) - enzyme , chemistry , drug discovery , combinatorial chemistry , biochemistry
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 tight‐binding inhibitors well described. Therefore, we have developed a platform for expression and purification of BACE1, including protein refolding from inclusion bodies, from E. coli in addition to 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 inhibitor behavior. Our methods allow for the purification of 16 mg of BACE1 enzyme from 1 L of culture scale, which is higher than reported yields in the current literature. For treatment of inhibitory data, we have identified binding affinity ranges where fitting kinetic data using the Morrison equation and plot and running a standard inhibitor to accurately determine the active enzyme concentration, is favored over classical models. To evaluate our platform from purification to X‐ray crystallography, we have tested known potent inhibitors previously reported by the Ghosh lab. The inhibitory constants and 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.