Accepted_test

Alzheimer's disease (AD) is the most common neurodegenerative disease. Oxidative stress plays an important role in AD, and the major source of reactive oxygen species (ROS) in the brain is NADPH oxidase 2 (NOX2). We hypothesize that its inhibition may be an effective therapy for AD. There are selective NOX2 inhibitors - GSK2795039 and its analogues. Our aim was to determine the binding site of these inhibitors, their binding affinity and selectivity towards the NAD(P)H oxidase family, and their ability to inhibit oxidative stress in human microglial cells. We used methods of molecular dynamics, molecular modelling and docking (using the GROMACS and MOE programs) and flow cytometry to assess the ROS level in the cells. We found that the tested inhibitors bind to NOX2 with the highest affinity compared to other NAD(P)H oxidases. The inhibitors’ binding site coincides with the NADPH binding site and includes the amino acids Arg73, Leu76, Asp95, Asn97, Leu98, Phe202, Tyr324, Phe326, Trp337, Pro339, Thr341, Gly392, Thr393, Asn569, Phe570. Interactions with Asp95, Pro339, Phe340, Asn569, and Phe570 enhance the binding affinity. We identified GSK1, 14, 15, 26 as high-affinity inhibitors of NOX2. However, they have lower selectivity than other inhibitors. GSK18 reduced oxidative stress in the cells with highest efficacy. It decreased the ROS level by 150% and 60% compared to the microglial cells with Aβ and control, respectively. Thus, binding selectivity rather than affinity to NOX2 determines the ability of inhibitors to prevent oxidative stress.