Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles

Author:

Ewing Heather1,Fernández-Vega Virneliz2,Spicer Timothy P.2,Chase Peter2,Brown Steven3,Scampavia Louis2,Roush William R.4,Riley Sean3,Rosen Hugh3,Hodder Peter2,Lambeau Gerard5,Gelb Michael H.1

Affiliation:

1. Department of Chemistry, University of Washington, Seattle, WA, USA

2. The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, FL, USA

3. The Scripps Research Institute, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA

4. The Scripps Research Institute, Dept. of Chemistry, Scripps Florida, Jupiter, FL, USA

5. Institut de Pharmacologie Moléculaire et Cellulaire, UMR7275, Centre National de la Recherche Scientifique et Université de Nice-Sophia-Antipolis, Valbonne, France

Abstract

There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate.

Publisher

Elsevier BV

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