A Sensitive and Versatile Fluorescent Activity Assay for ABHD12.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• The human metabolic serine hydrolase (mSH) family comprises numerous enzymes with unclear fundamental roles.
• α/β-hydrolase domain containing 12 (ABHD12) is an mSH enzyme with lysophosphatidylserine (LPS) as its apparent in vivo substrate.
• ABHD12, along with MAGL and ABHD6, also hydrolyzes monoacylglycerols (MAGs) like 2-arachidonoyl glycerol (2-AG) in vitro.

Purpose of the Study:

• To develop a sensitive, high-throughput fluorescent assay for measuring ABHD12 enzyme activity.
• To establish a method for screening inhibitors selective for ABHD12 over other mSH enzymes.
• To facilitate the study of ABHD12 function and the identification of novel therapeutic modulators.

Main Methods:

• A 96-well-plate fluorescent assay was developed using lysates from HEK293 cells overexpressing human ABHD12.
• The assay monitors glycerol liberation from the hydrolysis of 1(3)-acylglycerol (1(3)-AG), the preferred MAG substrate for ABHD12.
• Glycerol detection is coupled to an enzymatic cascade producing the fluorescent product resorufin, enabling kinetic monitoring.

Main Results:

• The developed assay allows for the simultaneous testing of inhibitor activities for up to 40 compounds.
• This methodology successfully identified the first class of inhibitors exhibiting selectivity for ABHD12 compared to other mSHs.
• The assay provides a sensitive and efficient means to study ABHD12 enzyme kinetics and inhibition.

Conclusions:

• A novel, sensitive fluorescent assay for ABHD12 activity has been established, suitable for high-throughput screening.
• This assay facilitates the discovery of selective ABHD12 inhibitors, advancing research into mSH enzyme functions.
• The findings pave the way for further investigation into the physiological roles of ABHD12 and the development of targeted therapeutics.