Novel screening assay for antioxidant protection against peroxyl radical‐induced loss of protein function

Oxidative damage to proteins, implicated amongst other in the etiology and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), results in the loss of specific biological protein function. A simple, sensitive, and cost‐effective fluorimetric test to assess the antioxidant capacity of new chemical entities to protect proteins from loss of activity caused by reactive oxygen species (ROS) was developed using alkaline phosphatase (ALP) as model protein. Protein oxidation was induced by 2,2′‐azobis(2‐methylpropionamidine) dihydrochloride (AAPH) and the decrease in catalytic activity of ALP to hydrolyze 4‐methylumbelliferyl phosphate (4‐MUP) to fluorescent 4‐methylumbelliferone (4‐MU) was monitored as a marker of protein degradation. According to their capacity to protect ALP from peroxyl radical‐induced activity loss, ten reference antioxidants were divided into three classes, namely efficient (pIC50 > 5 for quercetin, chlorogenic acid, caffeic acid, mangiferin, and resveratrol), intermediate (4 < pIC50 ≤ 5 for melatonin, trolox, and ascorbic acid), and poor antioxidants (pIC50 < 4 for glutathione and D‐mannitol). Multifunctional drugs, having the ability to interact with several disease‐related targets are of interest in PD. Therefore, the capacity of three catechol‐O‐methyltransferase (COMT) inhibitors, entacapone, nitecapone, and tolcapone to protect ALP from oxidative damage was also investigated and found to be very similar to the most potent reference antioxidants. © 2007 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 2931-2944, 2007