A simple assay for detection of small‐molecule redox activity

In our research, we found that high throughput screens against certain targets, such as cysteine proteases and metal containing enzymes, often yield undevelopable hits because of their lack of specificity and irreversible nature. Weeding through these undesirable hits to get to those of interest can often be difficult, negatively impacting the chance of success in drug discovery. In order to better understand the mechanism of this inhibition, LC/MS/MS analysis was performed with cathepsin L (cat L), a cysteine protease. Incubation of these undesirable compounds with cat L resulted in specific oxidation of the active site cysteine. Based on this mechanism of inhibition, a surrogate high throughput fluorescence assay was developed to measure the tendency of compounds to oxidize enzyme targets. In this assay, reactive compounds catalyze two 1 electron transfers from DTT to resazurin, resulting in the formation of the fluorescent resorufin. Using this assay, we were able to show that a fluorescence increase was observed only with suspected redox inhibitors, and not with known tool compounds. Finally, we tested 400 compounds from a recent drug discovery project, and compared their reactivity in this assay against their potency with the target of interest. Compounds that clearly inhibit via this mechanism could easily be picked out from those that specifically inhibit the target. Hence this detection assay serves as a valuable tool for quickly triaging redox active compounds from HTS campaigns. GlaxoSmithKline Pharmaceuticals, Collegeville PA