Development of Label-Free, Enzymatic CYP17A1 Assays Using the Agilent 6150 Single Quadrupole LC/MS System

Significance of the Topic

This application note addresses the critical role of CYP17A1 in human steroid hormone biosynthesis and its implication in diseases such as prostate cancer, polycystic ovary syndrome, Cushing’s syndrome, and congenital adrenal hyperplasia. Reliable, high-throughput assays for CYP17A1 inhibition are essential for drug discovery targeting steroidogenic pathways.

Objectives and Overview of the Study

The main goal was to develop two label-free in vitro enzymatic assays for CYP17A1 activities (17α-hydroxylase and C17,20-lyase) in a 96-well, high-throughput format. The assays quantify conversion of natural steroid substrates to their products using a single quadrupole LC/MS platform.

Methodology and Instrumentation

Enzymatic assays were performed under physiologically relevant conditions:

• Enzyme source: Rat testicular microsomes (100 μg/mL for hydroxylase; 250 μg/mL for lyase).
• Substrates: Progesterone (1 μM) for hydroxylase; 17α-hydroxyprogesterone (1 μM) for lyase.
• Reaction: Incubate enzyme, substrate, NCEs (0.012–200 μM), and cofactors at 37 °C for 10 min; quench with acetonitrile; centrifuge; transfer supernatant for analysis.
• Detection: Agilent 1290 Infinity LC System coupled to Agilent 6150 Single Quadrupole LC/MS with Jet Stream ESI (positive mode).
• LC conditions: Zorbax Poroshell 120 EC C18 column (2.1 × 30 mm) at 55 °C; 0.8 mL/min; run times of 0.6–0.72 min per injection.
• MS settings: Drying and sheath gas at 350 °C; capillary voltage 3000 V; fragmentor 70 V; dwell time 24 ms per m/z.
• Data analysis: Monitor substrate depletion and product formation; determine IC50 values with XLfit by fitting dose-response curves.

Key Results and Discussion

Both assays demonstrated robust performance:

• Hydroxylase assay: Injection-to-injection time of 0.9 min; clear separation of substrate (m/z 331.2 [M+H]+) and product (m/z 287.2 [M+H]+). IC50 for metconazole was 8.8 μM (product formation) and 8.4 μM (substrate depletion).
• Lyase assay: Injection cycle of 0.8 min; substrate (m/z 331.2) and product androstenedione (m/z 289.2) resolved. IC50 for metconazole was 2.9 μM (product formation).
• Signal sensitivity and minimal interference allowed reliable quantitation at physiologically low substrate conversion (<10% for lyase, higher for hydroxylase).

Benefits and Practical Applications

The label-free LC/MS approach offers several advantages:

• High sensitivity and specificity without the need for fluorescent or radiolabelled substrates.
• Rapid run times enabling full 96-well plate analysis in under 1.5 hours.
• Applicability to screening of new chemical entities in drug discovery targeting CYP17A1.
• Scalability to other cytochrome P450 isoforms and assay formats.

Future Trends and Potential Applications

Upcoming developments may include automation of sample preparation, multiplexed detection of multiple CYP activities in parallel, miniaturization to 384-well formats, and integration of high-resolution MS to profile metabolic pathways and off-target effects.

Conclusion

Two efficient, label-free CYP17A1 enzymatic assays were established using an Agilent single quadrupole LC/MS system. They deliver fast, sensitive, and reliable measurements of both hydroxylase and lyase activities in a high-throughput format, supporting accelerated inhibitor screening in pharmaceutical research.

References

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