Development of an HTRF Assay for the Detection and Characterization of Inhibitors of Catechol-O-Methyltransferase

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Use SAH to screen COMT inhibitors for Parkinson’s disease

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Are you looking for a suitable HTS assay to screen Catechol-O-Methyltransferase (COMT) inhibitors without being hampered by the fluorescence of catechol-type compounds? You’ll find an effective method for that in this article by Martha Kimos from LIBD.COMT’s role in dopamine metabolism in the brain has made this enzyme an attractive target to identify potential new treatments for Parkinson’s disease. Mechanistically speaking, COMT deactivates catecholamine neurotransmitters by transferring a methyl-group from S-adenosyl methionine (SAM). Many catechol-type compounds, i.e. COMT inhibitors, interfere with the fluorescence readouts commonly used in HTS assays. A nightmare for researchers that has limited the development of suitable HTS assays for a long time.Be smart and get back to enzymology basics to overcome these drawbacks! COMT, like many enzymes, needs substrates and co-substrates to function. Enzymatic activity means substrate modification and release of by-product.In the case of COMT, the transfer of a methyl group to the substrate results in the production of S-adenosyl-L-homocysteine (SAH). If you can measure SAH production, you can monitor COMT activity and assess the potency of inhibitory compounds. Discover step-by-step how Martha Kimos optimized and used SAH quantification in an HTS/MTS environment to determine potencies and kinetic features of COMT inhibitory compounds.Always remember your enzymatic basics!


Catechol-O-methyltransferase (COMT) plays an important role in the deactivation of catecholamine neurotransmitters and hormones. Inhibitors of COMT, such as tolcapone and entacapone, are used clinically in the treatment of Parkinson’s disease. Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening (HTS). Although assays using esculetin have been developed, these are affected by fluorescence, a common property of catechol-type compounds.
We have therefore evaluated a new homogenous time-resolved fluorescence (HTRF)-based assay from Cisbio (Codolet, France), which measures the production of S-adenosyl-L-homocysteine (SAH). The assay has been run in both HTS and medium-throughput screening (MTS) modes. The assay was established using membranes expressing human membrane-bound COMT and was optimized for protein and time to give an acceptable signal window, good potency for tolcapone, and a high degree of translation between data in fluorescence ratio and data in terms of [SAH] produced. pIC50 values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as Km,app determination.


Journal of Biomolecular Screening, 2016 June; 21(15):490-495.

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