High-throughput screening for reactive oxygen species scavengers targeting mitochondria

A high-throughput screening (HTS) method for reactive oxygen species (ROS) scavengers was established to explore compounds protecting mitochondria. Freshly prepared rat liver mitochondria and the ROS fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA), were coincubated for 90 min to establish the assay in 384-well plates. The fluorescence of DCF values was monitored with a microplate reader. The Z' factor value for HTS was 0.81. A sample library containing 75,230 compounds was screened, and 10 compounds with high ROS scavenging activity were identified, among which the compound, 5061, with the strongest ROS scavenging activity was reevaluated. 5061 showed potent free radical scavenging activity both in cell-free models and cell models, which were in good accordance with the HTS assay. The established fluorescent HTS assay was simple, sensitive, stable, highly reproducible and robust and suitable for the HTS assay for ROS scavengers targeting mitochondria.