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

Area of Science:

• Pharmacology
• Biochemistry
• Drug Discovery

Background:

• Selective chemical inhibitors are essential for reaction phenotyping to identify drug-metabolizing enzymes responsible for drug elimination.
• Limited availability of selective inhibitors for less common cytochrome P450 (CYP) and non-CYP enzymes poses a challenge in drug development.

Purpose of the Study:

• To develop and validate a multiplexed high throughput screening (HTS) assay for simultaneously monitoring the inhibition of multiple drug-metabolizing enzymes.
• To address the gap in selective inhibitors for less common drug-metabolizing enzymes.

Main Methods:

• Development of a multiplexed HTS assay utilizing 20 substrate reactions in a 384-well format for simultaneous enzyme inhibition monitoring.
• Integration of robotic systems and LC-MS/MS instrumentation for high throughput analysis.
• Virtual screening employed to identify potential inhibitors based on existing data and enzyme structures.

Main Results:

• The assay successfully screened approximately 4600 compounds, yielding numerous hits for various drug-metabolizing enzymes.
• Identified two time-dependent and selective aldehyde oxidase inhibitors: erlotinib and dibenzothiophene.
• Achieved a significantly higher hit rate compared to traditional HTS for biological targets, attributed to enzyme promiscuity and compound selection bias.

Conclusions:

• The developed multiplexed HTS assay is the first of its kind for drug-metabolizing enzymes, offering enhanced throughput and efficiency.
• The assay effectively identified novel selective inhibitors, including for aldehyde oxidase.
• Future work will focus on identifying inhibitors for enzymes lacking quality hits and thorough characterization of newly discovered inhibitors.