Skip to main content
SpringerLink
Log in

Synthesis and characterization of a new fluorogenic substrate for monoacylglycerol lipase and application to inhibition studies

  • Note
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Human monoacylglycerol lipase (MAGL), a soluble serine hydrolase that belongs to the α/β hydrolase fold superfamily, regulates 2-arachidonoyl glycerol level in the endocannabinoid system, which is implicated in a number of severe diseases, and therefore, inhibition of MAGL activity is crucial in the treatment of these diseases. We have synthesized a red fluorogenic substrate, 7-hydroxyresorufinyl-arachidonate (7-HRA), for a new MAGL assay. This assay is simple, sensitive, and reliable and useful for identifying compounds that modulate MAGL activity. In addition, the assay emits red fluorescence, which can significantly reduce interference due to compound fluorescence and dust or lint, all of which fluoresce in the blue wavelength. MAGL catalyzes the hydrolysis of 7-HRA to generate arachidonic acid and a highly red fluorescent resorufin, excitation at 571 nm and emission at 588 nm, with a Km of 0.87 μM and Vmax of 26 nmol min−1 mg protein−1. The known MAGL inhibitors URB602, methyl arachidonyl fluorophosphonate, and JZL184 were used to validate the test assay. The assay was highly reproducible with an overall average Z′ value of 0.80. This new red fluorogenic substrate and the resulting enzyme assay could be used in high-throughput screening to identify and develop new potential MAGL inhibitors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Matuszak N, Muccioli GG, Labar G, Lambert DM (2009) Synthesis and in vitro evaluation of N-substituted maleimide derivatives as selective monoglyceride lipase inhibitors. J Med Chem 52:7410–7420

    Article  CAS  Google Scholar 

  2. King AR, Lodola A, Carmi C, Fu J, Mor M, Piomelli D (2009) A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors. Br J Pharmacol 157:974–983

    Article  CAS  Google Scholar 

  3. Minkkilä A, Myllymäki MJ, Saario SM, Castillo-Melendez JA, Koskinen AMP, Fowler CJ, Leppänen J, Nevalainen T (2009) The synthesis and biological evaluation of para-substituted phenolic N-alkyl carbamates as endocannabinoid hydrolyzing enzyme inhibitors. Eur J Med Chem 44:2994–3008

    Article  Google Scholar 

  4. Muccioli GG, Labar G, Lambert DM (2008) CAY10499, a novel monoglyceride lipase inhibitor evidenced by an expeditious MGL assay. ChemBioChem 9:2704–2710

    Article  CAS  Google Scholar 

  5. Wang Y, Chanda P, Jones PG, Kennedy JD (2008) A fluorescence-based assay for monoacylglycerol lipase compatible with inhibitor screening. Assay Drug Dev Technol 6:387–393

    Article  CAS  Google Scholar 

  6. Holtfrerich A, Makharadze T, Lehr M (2010) High-performance liquid chromatography assay with fluorescence detection for the evaluation of inhibitors against human recombinant monoacylglycerol lipase. Anal Biochem 399:218–224

    Article  CAS  Google Scholar 

  7. Zhang J-H, Chung TDY, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73

    Article  Google Scholar 

  8. Beisson F, Ferté N, Nari J, Noat G, Arondel V, Verger R (1999) Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings. J Lipid Res 40:2313–2321

    CAS  Google Scholar 

  9. Fritzsche M, Mandenius C-F (2010) Fluorescent cell-based sensing approaches for toxicity testing. Anal Bioanal Chem 398:181–191

    Article  CAS  Google Scholar 

  10. Savinainen JR, Yoshino M, Minkkilä A, Nevalainen T, Laitinen JT (2010) Characterization of binding properties of monoglyceride lipase inhibitors by a versatile fluorescence-based technique. Anal Biochem 399:132–134

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank the University of Milan for financial support.

Conflict of interest

The authors declare that they have no conflict of interests.

Author information

Authors and Affiliations

  1. Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, Università degli Studi di Milano, via G. B. Grassi 74, 20157, Milan, Italy

    Simone Lauria, Silvana Casati & Pierangela Ciuffreda

Authors
  1. Simone Lauria
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Silvana Casati
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pierangela Ciuffreda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pierangela Ciuffreda.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 152 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lauria, S., Casati, S. & Ciuffreda, P. Synthesis and characterization of a new fluorogenic substrate for monoacylglycerol lipase and application to inhibition studies. Anal Bioanal Chem 407, 8163–8167 (2015). https://doi.org/10.1007/s00216-015-8991-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-015-8991-9

Keywords

Search

Navigation