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

J Biomol Screen. 2016 Aug;21(7):713-21. doi: 10.1177/1087057116646742. Epub 2016 May 4.


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.

Keywords: automation or robotics; chemistry; high-content screening; lipids or lipid metabolism.

MeSH terms

  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / isolation & purification*
  • Fluorescent Dyes / chemistry
  • Fluorometry / methods*
  • High-Throughput Screening Assays / methods*
  • Humans
  • Kinetics
  • Mast Cells / chemistry
  • Mast Cells / metabolism
  • Phospholipases A2, Secretory / antagonists & inhibitors*
  • Phospholipases A2, Secretory / chemistry
  • Phospholipids / chemistry
  • Small Molecule Libraries / analysis


  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Phospholipids
  • Small Molecule Libraries
  • Phospholipases A2, Secretory