Development of an HPLC/UV assay for the evaluation of inhibitors of human recombinant monoacylglycerol lipase

Del Carlo, S; Manera, C; Chicca, Andrea; Arena, C; Bertini, S; Burgalassi, S; Tampucci, S; Gertsch, Jürg; Macchia, M; Saccomanni, G (2015). Development of an HPLC/UV assay for the evaluation of inhibitors of human recombinant monoacylglycerol lipase. Journal of pharmaceutical and biomedical analysis, 108, pp. 113-121. Pergamon Press 10.1016/j.jpba.2015.02.007

[img] Text
DelCarlo_JPharmaBiomedAnalysis_2015.pdf - Published Version
Restricted to registered users only
Available under License Publisher holds Copyright.

Download (1MB) | Request a copy

Monoacylglycerol lipase (MAGL) is a membrane-associated cytosolic serine hydrolase which catalyses the hydrolysis of the endocannabinoid 2-arachidonoylglycerol into arachidonic acid and glycerol. MAGL represents the link between the endocannabinoid and the eicosanoid system indeed its inhibition enhances endocannabinoid signalling and lowers eicosanoid production. Here we present a radioactive-free, sensitive and solid HPLC-UV based method to evaluate MAGL activity by using 4-nitrophenylacetate (4-NPA) as substrate. The enzymatic activity is measured by quantifying the 4-nitrophenol (PNP) (λ = 315 nm) formation on a C18 stationary phase. The method was validated by calculating IC50 values of the reference inhibitors JZL184, CAY10499 and JW642 and confirming the irreversible and non-competitive mechanism of inhibition for JZL184. Furthermore in order to resemble the catalytic conditions of MAGL at cell membrane level, the surfactant Triton X-100 was added, as a micelle forming agent and 4-nitrophenyldodecanoate (4-NPDo) was used as lipophilic substrate for MAGL. The data obtained confirmed that the HPLC method is an alternative, radioactive-free approach for the screening and characterization of new MAGL inhibitors. Finally this assay prevents, in an unequivocal manner, any interference related to the intrinsic absorbance of screened compounds or metabolites generated upon enzymatic cleavage which could seriously affect the assay readout.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Other Institutions > NCCR TransCure
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Chicca, Andrea and Gertsch, Jürg

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

0731-7085

Publisher:

Pergamon Press

Language:

English

Submitter:

Valentina Rossetti

Date Deposited:

06 Sep 2016 12:39

Last Modified:

06 Sep 2016 12:39

Publisher DOI:

10.1016/j.jpba.2015.02.007

PubMed ID:

25743577

Uncontrolled Keywords:

4-Nitrophenol; 4-Nitrophenylacetate; 4-Nitrophenyldodecanoate; HPLC–UV; MAGL

BORIS DOI:

10.7892/boris.87529

URI:

https://boris.unibe.ch/id/eprint/87529

Actions (login required)

Edit item Edit item
Provide Feedback