The Virtual Screening And Biological Test For β-secretase Modulators

In recent years, Alzheimer’s disease (AD) has been very popular among people over65years old. AD is a neurodegenerative disease and the accumulation of β-amyloid peptide in brain is the hallmark for the onset and progression of AD. B-secretase (BACE-1) is essential for the generation of8-amyloid peptide. Therefore, the research of BACE-1inhibitors is pivotal for the treatment of Alzheimer’s disease. Compared with traditional drug development, computer-aided drug screening could greatly accelerate the speed of drug design, saving a lot of money and becoming an increasingly important technology in the drug development campaign. In addition, computer virtual screening technology and biological experiments can complement each other.In this study, we used our newly developed in-house docking tool FIPSDock and molecular dynamics simulation tool Gromacs. Particularly, we have generated a receptor ensemble and two virtual ligand libraries from MICAD and DrugBank for virtual screening. Moreover, cytotoxicity and WesternBlot analysis were performed to assess the effect of Gefitinib. Specific contents and conclusions are as follows:(1) We constructed a multi-configuration BACE1receptor collection from the PDB. A collection of small molecule ligands were established from the MICAD and DrugBank. FIPSDock was used in the virtual screening process against the receptor ensemble. Hence, we could account for protein flexibility in virtual screening. Finally we obtained a total of8BACE1active site binding molecules such as Imatinib, Gefinitib etc. The binding free energy of the two moleculars are-12.70kcal/mol and-9.34kcal/mol respectively.(2) We used molecular dynamics simulation tool Gromacs to further analyze the docking results. We make6ns simulations for the complex structure derived from docking, including2QU2-Gefinitib,2VIZ-Imatinib etc. The structure comparison and energy value mapping display that Gefitinib and imatinib bind well with BACE1in the activity pocket. The interaction include hydrophobic interaction with Trp, Phe, Tyr, He residues and hydrogen bonding with Gln, Ser, Arg residues. These analysis fully proved the stable interaction between ligand molecules and BACE1structures.(3) Through enzymatic and cell activity assays, we determined20uM as the Gefitinib concentration at which normal neural cells remained viable. According to ELISA and Westernblot analysis Gefitinib could suppress the levels of extracellular Aβ40/42in dose-dependent manner and increased level of sAPPa and the decreased level of sAPPB. All of these results confirmed that Gefinitib can regulate the activity of BACE1and is a potential lead compound of BACE-1.The present study which coupled the computer virtual screening techniques and biological experiments has demonstrated that Gefitinib could be used as a potential BACE1lead compound.