ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 331 (2004) 198–200 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.04.026 Notes & Tips Microplate screening assay to identify inhibitors of human catechol-O-methyltransferase Mika Kurkela, a Antti Siiskonen, b Moshe Finel, a Päivi Tammela, a Jyrki Taskinen, b and Pia Vuorela a,¤ a Viikki Drug Discovery Technology Center, Faculty of Pharmacy, P.O. Box 56, University of Helsinki, FIN-00014 Helsinki, Finland b Division of Pharmaceutical Chemistry, Faculty of Pharmacy, P.O. Box 56, University of Helsinki, FIN-00014 Helsinki, Finland Received 24 February 2004 O-methylation by catechol-O-methyltransferase (COMT) 1 is an important metabolic pathway leading to inactivation of catecholamine neurotransmitters such as norepinephrine and dopamine [1] and elimination of cat- echol steroids and xenobiotic catechols [2]. Levodopa remains the most eVective drug for treatment of Parkin- son’s disease [1]. To enhance its availability in the brain, where it is decarboxylated, resulting in release of dopa- mine, it is usually given with a dopa-decarboxylase inhibitor, which blocks its peripheral breakdown. Under these conditions, levodopa is metabolized in the periph- ery via an alternative path involving COMT, which degrades it to 3-O-methyldopa. If COMT could be blocked in the periphery, more levodopa would there- fore be available in the brain. Two COMT inhibitors, entacapone, which acts mainly in the periphery, and tol- capone, which acts both centrally and peripherally, have been developed. Clinical use of tolcapone has, however, been restricted, because there have been reports of liver toxicity [3, and refs. within]. Although there has been a recent report concerning one potent COMT inhibitor [4], more are needed. A rapid, robust, and sensitive means of determining COMT activity would be valuable in rela- tion to the development of COMT inhibitors. Several methods of measuring COMT activity have been developed [5–7], but none is well suited to automa- tion and high throughputs. In this report we describe development of a 96-well microplate assay to identify inhibitors of human S-COMT, using aesculetin as substrate. Methylation of aesculetin to scopoletin is measured directly in a reaction mixture in which S-(5'- adenosyl)-L-methionine (AdoMet) serves as methyl- group donor. The reaction is followed Xuorometrically. The human gene segment encoding the water-soluble form of COMT, S-COMT, was ampliWed from total brain RNA (Invitrogen) and subcloned into the pBAD/ Myc-HisC plasmid (Invitrogen). Expression of recombi- nant S-COMT in Escherichia coli cells was induced by arabinose. The cells were collected by centrifugation (10 min, 3000g, 4 °C). The cells (the centrifugation pellet) were suspended in 1 ml of buVer solution (50 mM phos- phate, 300 mM NaCl, 5 mM MgCl 2 , pH 7.4) and dis- rupted by treatment with lysozyme and DNase I followed by sonication. The supernatant from the subse- quent centrifugation (the cytosolic fraction) was used as an S-COMT source in the studies. It was stored at ¡20 °C until use. Aesculetin was obtained from Extra- synthèse (d502); catechol (C-9510), 4-chlorocatechol (Library of Rare Chemicals), 4-nitrocatechol (N-7126), and 3,5-dinitrocatechol (D-131) were bought from Sigma; and 3,4-dihydroxybenzoic acid (D 10,980-0) and 2,3-dihydroxynaphthalene (D 11,600-9) were bought from Aldrich. The aesculetin and competing catechol substrates were dissolved in dimethyl sulfoxide (DMSO) and diluted with aqueous buVer solution (100 mM phos- phate, 5 mM MgCl 2 , 20 mM L-cysteine, pH 7.4) for a Wnal DMSO concentration of 2% in the 100 l of reac- tion mixture. All of the reagents were dissolved in the same buVer solution, since the presence of magnesium ions is essential for COMT-catalyzed methylation, and puriWed human S-COMT requires cysteine as a reducing agent to maintain its activity [8]. For each sample, inhib- itor and aesculetin solutions were placed in 3 wells of a ¤ Corresponding author. Fax: +358-9-19159138. E-mail address: pia.vuorela@helsinki.W (P. Vuorela). 1 Abbrevations used: COMT, catechol-O-methyltransferase; DM- SO, dimethyl sulfoxide.