Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles
Heather Ewing1, Virneliz Fernández-Vega2, Timothy P Spicer2
1Department of Chemistry, University of Washington, Seattle, WA, USA.
Journal of Biomolecular Screening
|May 6, 2016
Summary
Researchers developed a novel high-throughput screening assay to identify inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2), an enzyme crucial for mast cell maturation. This new method successfully screened over 370,000 compounds, discovering several potential inhibitors.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Human group III secreted phospholipase A2 (hGIII-sPLA2) is implicated in mast cell maturation.
- Currently, no potent inhibitors for hGIII-sPLA2 are available.
- Developing selective inhibitors is of therapeutic interest.
Purpose of the Study:
- To establish a high-throughput screening (HTS) assay for identifying hGIII-sPLA2 inhibitors.
- To utilize a fluorescence-based method mimicking the enzyme's natural substrate environment.
- To screen a large chemical library for novel hGIII-sPLA2 inhibitors.
Main Methods:
- Adapted a fluorescence-based enzyme activity monitoring method into an HTS format.
- Employed phospholipid vesicles containing a BODIPY-labeled phospholipid analogue as substrate.
- Utilized optical detection in a 1536-well plate format with non-UV excitation wavelengths.
- Screened a library of 370,276 small molecules.
Main Results:
- Successfully developed and implemented the first HTS optical screening assay for secreted phospholipase A2 inhibitors using a phospholipid vesicle substrate.
- Identified several hit compounds from the screened library.
- Uploaded screening data to PubChem for public access.
Conclusions:
- The developed HTS assay is effective for discovering hGIII-sPLA2 inhibitors.
- The assay's design, using phospholipid vesicles, provides a more natural substrate environment.
- This work represents a significant advancement in the search for therapeutic agents targeting hGIII-sPLA2.
