Novel Multiplexed High Throughput Screening of Selective Inhibitors for Drug-Metabolizing Enzymes Using Human Hepatocytes.

Selective chemical inhibitors are critical for reaction phenotyping to identify drug-metabolizing enzymes that are involved in the elimination of drug candidates. Although relatively selective inhibitors are available for the major cytochrome P450 enzymes (CYP), they are quite limited for the less common CYPs and non-CYPs. To address this gap, we developed a multiplexed high throughput screening (HTS) assay using 20 substrate reactions of multiple enzymes to simultaneously monitor the inhibition of enzymes in a 384-well format. Four 384-well assay plates can be run at the same time to maximize throughput. This is the first multiplexed HTS assay for drug-metabolizing enzymes reported. The HTS assay is technologically enabled with state-of-the-art robotic systems and highly sensitive modern LC-MS/MS instrumentation. Virtual screening is utilized to identify inhibitors for HTS based on known inhibitors and enzyme structures. Screening of ~4600 compounds generated many hits for many drug-metabolizing enzymes including the two time-dependent and selective aldehyde oxidase inhibitors, erlotinib and dibenzothiophene. The hit rate is much higher than that for the traditional HTS for biological targets due to the promiscuous nature of the drug-metabolizing enzymes and the biased compound selection process. Future efforts will focus on using this method to identify selective inhibitors for enzymes that do not currently have quality hits and thoroughly characterizing the newly identified selective inhibitors from our screen. We encourage colleagues from other organizations to explore their proprietary libraries using a similar approach to identify better inhibitors that can be used across the industry.