Journal
JOURNAL OF BIOMOLECULAR SCREENING
Volume 21, Issue 5, Pages 490-495Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057115616793
Keywords
catechol-O-methyltransferase; COMT; HTRF
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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. pIC(50) values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as K-m,K-app determination.
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