Utility of Resazurin, Horseradish Peroxidase, and NMR Assays to Identify Redox-Related False-Positive Behavior in High-Throughput Screens.

Discerning false positives from true actives in high-throughput screening (HTS) output is fraught with difficulty as the reason of anomalous activity seen for compounds is often not clear-cut. In this study, we introduce a novel medium-throughput NMR assay for the identification of redox-cycling compounds (RCCs), which is based on detection of oxidation of a reducing agent. We compare its outcomes to those from horseradish peroxidase (HRP)/phenol red and resazurin (RZ)-based assays that are more commonly used for triaging HTS outputs. Data from NMR, RZ, and HRP redox assay are shown to correlate, with the NMR assay showing the greatest accuracy. In addition, historical data analysis was used to identify compounds frequently active in assays for redox-susceptible targets. We provide examples of compound classes found and conclude that the NMR redox assay offers a novel and reliable way of identifying RCCs at a medium throughput. The HRP and RZ assays are reasonable higher-throughput alternatives, with both showing similar sensitivity to redox-cycling and false-positive compounds. The RZ assay has a higher hit rate, reflecting its ability to pick up multiple modes of action.