Abstract
Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages. A new diagnostic marker product for ONOO(-)is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO(-)formed from Nox2-derived O2 (̇̄)and nitric oxide synthase-derived nitric oxide.
Highlights
NADPH oxidase (Nox)4 enzymes (Nox1–5 and Duox1/2) have been proposed as potential therapeutic targets in the treatment of a variety of inflammatory and fibrotic diseases, including cancer [1,2,3]
Overview of high throughput screening (HTS)/reactive oxygen species (ROS) Assays Used in the Identification of Nox Inhibitors—To reliably identify inhibitors of Nox isoforms, we developed the following primary and secondary assays for HTS/ROS analysis (Fig. 2), as described recently [8]
coumarin boronic acid (CBA) reacts with H2O2 considerably slower (k ϭ 1.5 MϪ1sϪ1), as compared with its catalase-insensitive reaction with ONOOϪ (k ϭ 1.1 ϫ 106 MϪ1 sϪ1), to form a highly fluorescent product, COH [33]
Summary
NADPH oxidase (Nox) enzymes (Nox and Duox1/2) have been proposed as potential therapeutic targets in the treatment of a variety of inflammatory and fibrotic diseases, including cancer [1,2,3]. By-product of their primary catalytic function, the only known function of Nox enzymes is generation of ROS A major primarily H2O2 impediment to with little or no advancing Nox research is the paucity of selective inhibitors of Nox isoforms, including Nox and -2 [6]. This is due in part to the lack of reliable and high throughput-compatible detection probes and assays that are specific for O2. One of the objectives of this study is to identify small molecule inhibitors of the Nox isoform using the high throughput screening (HTS)/ROS-based assay(s) that largely eliminate false positives. The chemiluminescent probe, L-012, has been used in Nox assay [9]
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