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

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.

Small-molecule inhibitors as potential therapeutics and as tools to understand the role of phospholipases A2

“…The assay provides rapid, selective and quantifiable measurement of GVIA iPLA 2 -specific activity, without requiring purification of the enzyme. Although PAPC is a common choice for the radiolabeled substrate A fluorometric high-throughput screening assay for sPLA 2 s using phospholipid vesicles was developed and used to identify human GIII sPLA 2 inhibitors on a library of 370,276 small molecules [145]. The substrate is present in phospholipid vesicles, because this matrix more closely resembles the natural substrate of human GIII sPLA 2 , as opposed to phospholipid/detergent mixed micelles.…”

A Robust Liposomal Platform for Direct Colorimetric Detection of Sphingomyelinase Enzyme and Inhibitors

“…However, these assays typically use enzyme activities of >1 U/mL and are orders of magnitude less sensitive than required for applications in quantifying biologically relevant SMase activities. Liposomes formulated from both natural − and synthetic lipids have been shown to release their cargo in the presence of a target protein, causing a colorimetric response. Release of encapsulated contents from liposomes can also be triggered by osmotic shock, as used in detecting pathogens with attomolar sensitivity .…”