title: Development of an HTRF Assay for the Detection and Characterization of Inhibitors of Catechol-O-Methyltransferase.
abstract: 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.
authors.affiliation: Lieber Institute for Brain Development, Baltimore, MD, USA.
authors.forename: Martha
authors.initials: M
authors.lastname: Kimos
authors.affiliation: UCB Biopharma SPRL, Braine-l'Alleud, Belgium.
authors.forename: Maggi
authors.initials: M
authors.lastname: Burton
authors.affiliation: UCB Biopharma SPRL, Braine-l'Alleud, Belgium.
authors.forename: David
authors.initials: D
authors.lastname: Urbain
authors.affiliation: UCB Biopharma SPRL, Braine-l'Alleud, Belgium.
authors.forename: Didier
authors.initials: D
authors.lastname: Caudron
authors.affiliation: UCB Biopharma SPRL, Braine-l'Alleud, Belgium.
authors.forename: Murielle
authors.initials: M
authors.lastname: Martini
authors.affiliation: UCB Biopharma SPRL, Braine-l'Alleud, Belgium.
authors.forename: Michel
authors.initials: M
authors.lastname: Famelart
authors.affiliation: UCB Biopharma SPRL, Braine-l'Alleud, Belgium.
authors.forename: Michel
authors.initials: M
authors.lastname: Gillard
authors.affiliation: Lieber Institute for Brain Development, Baltimore, MD, USA james.barrow@libd.org.
authors.forename: James
authors.initials: J
authors.lastname: Barrow
authors.affiliation: UCB Biopharma SPRL, Braine-l'Alleud, Belgium.
authors.forename: Martyn
authors.initials: M
authors.lastname: Wood
mesh_terms: D001577:Benzophenones / Q000627:therapeutic use; D002394:Catechol O-Methyltransferase / Q000737:chemistry* / Q000187:drug effects; D065098:Catechol O-Methyltransferase Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000627:therapeutic use; D002396:Catechols / Q000627:therapeutic use; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009596:Nitrophenols / Q000627:therapeutic use; D010300:Parkinson Disease / Q000188:drug therapy; D012435:S-Adenosylhomocysteine / Q000737:chemistry; D054852:Small Molecule Libraries / Q000737:chemistry / Q000302:isolation & purification* / Q000627:therapeutic use; D000077867:Tolcapone
chemical_list: D001577:Benzophenones; D065098:Catechol O-Methyltransferase Inhibitors; D002396:Catechols; D009596:Nitrophenols; D054852:Small Molecule Libraries; D012435:S-Adenosylhomocysteine; D000077867:Tolcapone; D002394:Catechol O-Methyltransferase; C034221:catechol
keywords: COMT; HTRF; catechol-O-methyltransferase