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

Abstract

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 (K i) obtained from the kinetic studies are in agreement with dissociation constants (K d) 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.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19

“…K i values, with the exception of MERS 3CL pro , were obtained by fitting the data to the Morrison equation (Eq. ( 1 )) for competitive inhibition 48 . For these fits, the V max was initially set to a value less than 1.2, the UIVT3 substrate concentration was fixed at 2 μM, and the K m value was fixed at a value of 250 μM.…”

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

“…Initial velocities were calculated by dividing the observed velocity in the absence of inhibitor (V o ) by the initial velocity at different inhibitor concentrations (V i ). K i values, with the exception of MERS 3CLpro, were obtained by fitting the data to the Morrison equation (Equation 1) for tight-binding inhibitors 45 . …”

Pepsin-like aspartic proteases (PAPs) as model systems for combining biomolecular simulation with biophysical experiments

“…Recently, macro-cyclic ligand bound structures of BACE1 were reported by Yen et al 88 (PDB IDs: 6NV7, 6NV9, 6NW3). The authors further reported experimental K i and K d values as well as several thermodynamic parameters (DH, TDS, and DG) of ligand binding (thermodynamic parameters were calculated using isothermal titration calorimetry (ITC) experiments).…”