Label-Free High-Throughput Screening of CYP3A4 Inhibitors Using Acoustic Ejection Mass Spectrometry.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Label-free high-throughput screening (HTS) accelerates drug discovery by measuring biochemical activity directly.
• Acoustic droplet ejection mass spectrometry (AEMS) is a next-generation platform for rapid data acquisition.
• Cytochrome P450 3A4 (CYP3A4) is a key enzyme in drug metabolism.

Purpose of the Study:

• To evaluate an AEMS workflow for label-free HTS of CYP3A4 inhibitors.
• To compare AEMS performance with a luminescence-based assay.
• To assess the impact of assay method and substrate on inhibitor identification.

Main Methods:

• Screening 9702 compounds using AEMS in a 384-well format for CYP3A4 inhibitors.
• Utilizing nifedipine as a substrate for initial AEMS screening.
• Retesting identified hits with AEMS using luciferin isopropyl acetal, the substrate for a luminescence assay.

Main Results:

• The first successful label-free HTS campaign for CYP3A4 inhibitors using AEMS was achieved.
• AEMS results with nifedipine were comparable to a luminescence assay but identified fewer inhibitors.
• Assay method and substrate significantly influenced inhibitor detection, confirming differences between methods.

Conclusions:

• AEMS is a robust and reliable tool for CYP3A4 inhibitor screening.
• AEMS offers high analytical precision, reduced false positives, and substrate flexibility.
• This platform enhances confidence in early drug discovery by validating hits and detecting metabolites.