Discovery of Potential Soluble Epoxide Hydrolase Inhibitors Using a High-Throughput Screening Assay on a 235 Compound Library

Significance of the Topic

Soluble epoxide hydrolase (sEH) plays a central role in the rapid hydrolysis of epoxyeicosatrienoic acids (EETs), lipid mediators with potent anti-inflammatory and cardioprotective properties. Inhibition of sEH represents a promising therapeutic approach to elevate EET levels and mitigate inflammatory and cardiovascular disorders. High-throughput screening (HTS) techniques can accelerate the discovery of novel sEH inhibitors by enabling rapid, reproducible evaluation of large compound collections.

Study Objectives and Overview

This study aimed to implement and validate an automated HTS assay to identify potential sEH inhibitors from a 235-compound anti-inflammatory library. The objectives were:

• Adapt a soluble epoxide hydrolase inhibitor screening assay for full automation and 384-well format.
• Screen all library compounds in duplicate within days rather than weeks.
• Evaluate assay robustness using the Z'-factor and identify hit compounds with IC50 values below 10 µM.

Methodology and Instrumentation

The automated HTS platform comprised three integrated Agilent instruments:

• Bravo automated liquid handling system with 384-channel pipetting head for reagent and compound transfers.
• BenchCel 4R microplate handler for plate stacking and automated movement between instruments.
• BioTek Synergy Neo2 hybrid multimode reader for fluorescence kinetic measurements.

Screening workflow:

• Reformat 235 anti-inflammatory compounds (10 mM stocks) into a 384-well source plate, then prepare dilution plates with seven ½-log concentration–response curves in duplicate.
• Dispense human recombinant sEH into assay plates, add test compounds (0.5 µL), preincubate 5 minutes at room temperature, then initiate reactions with the fluorogenic substrate PHOME (10 µL).
• Acquire kinetic fluorescence data every 3 minutes for 20 minutes at 360 nm/460 nm.
• Determine Z'-factor using DMSO (negative) and AUDA (positive) controls; calculate percent activity and IC50 values by four-parameter nonlinear regression.

Main Results and Discussion

The automated HTS delivered robust performance (average Z'-factor = 0.60) and a throughput of ~20 compounds per hour. Of the 235 compounds screened, 36 exhibited IC50 values below 10 µM and were designated as potential sEH inhibitors. Six compounds demonstrated particularly potent or well-defined concentration–response behavior:

• SC-75741 (NF-κB inhibitor): IC50 <10 nM
• trans-AUCB (benchmark sEH inhibitor): IC50 <10 nM
• Ciclesonide (glucocorticoid receptor prodrug): IC50 = 100 nM
• SCH 79797 (PAR1 antagonist): IC50 = 372 nM
• DuP-697 (COX-2 inhibitor): IC50 = 813 nM
• LCK Inhibitor (Src-family tyrosine kinase inhibitor): IC50 = 6 070 nM

These findings reveal unexpected dual-target activities, such as COX-2 and sEH inhibition by DuP-697, and suggest opportunities to explore synergistic anti-inflammatory mechanisms.

Benefits and Practical Applications

The automated HTS format offers:

• Rapid data generation in days versus weeks.
• High reproducibility and low inter- and intra-assay variability.
• Minimal reagent and sample consumption.
• Flexibility to adapt assay protocols for other enzyme targets and compound libraries.

This approach supports both early-stage drug discovery and basic research applications.

Future Trends and Potential Applications

Emerging directions include:

• Scaling to larger, more diverse compound libraries to uncover novel chemotypes.
• Integrating multiplexed enzyme assays or phenotypic screens in a single workflow.
• Combining HTS data with machine learning to predict hit quality and guide medicinal chemistry.
• Developing dual-target screening strategies to identify compounds with complementary activities (e.g., COX-2/sEH dual inhibitors).

Conclusion

An automated HTS assay for sEH inhibitors was successfully developed and validated, demonstrating robust performance and rapid throughput. Screening a 235-member anti-inflammatory library yielded 36 candidate inhibitors, including six high-potency hits. The platform’s versatility and efficiency make it a powerful tool for accelerating the discovery of enzyme inhibitors in both academic and industrial settings.

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