Kinetics and Crystal Structure of Catechol-O-Methyltransferase Complex with Co-Substrate and a Novel Inhibitor with Potential Therapeutic Application MARIA JO ˜ AO BONIF ´ ACIO, MARGARIDA ARCHER, MARIA L. RODRIGUES, PEDRO M. MATIAS, DAVID A. LEARMONTH, MARIA ARM ´ ENIA CARRONDO, and PATR ´ ICIO SOARES-DA-SILVA Department of Research and Development, BIAL, S. Mamede do Coronado, Portugal (M.J.B., D.A.L., P.S.S.); Instituto de Tecnologia Quı´mica e Biolo ´ gica-Universidade Nova de Lisboa, Oeiras, Portugal (M.A., M.L.R., P.M.M., M.A.C.); and Instituto de Biologia Experimental e Tecnolo ´ gica, Oeiras, Portugal (M.A., M.L.R.) Received February 27, 2002; accepted June 27, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Catechol-O-methyltransferase (COMT; E.C. 2.1.1.6) is a ubiq- uitous enzyme in nature that plays an important role in the metabolism of catechol neurotransmitters and xenobiotics. In particular, inactivation of drugs such as L-3,4-dihydroxypheny- lalanine (L-DOPA) via O-methylation is of relevant pharmaco- logical importance, because L-DOPA is currently the most ef- fective drug used in the treatment of Parkinson’s disease. This justified the interest in developing COMT inhibitors as potential adjuncts to L-DOPA therapy. The kinetics of inhibition by BIA 3-335 (1-[3,4-dihydroxy-5-nitrophenyl]-3-(N-3'-trifluormethyl- phenyl)-piperazine-1-propanone dihydrochloride) were charac- terized using recombinant rat soluble COMT. BIA 3-335 was found to act as a potent, reversible, tight-binding inhibitor of COMT with a K i of 6.0  1.6 nM and displaying a competitive inhibition toward the substrate binding site and uncompetitive inhibition toward the S-adenosyl-L-methionine (SAM) binding site. The 2.0-Å resolution crystal structure of COMT in complex with its cosubstrate SAM and a novel inhibitor BIA 3-335 shows the atomic interactions between the important residues at the active site and the inhibitor. This is the first report of a three- dimensional structure determination of COMT complexed with a potent, reversible, and tight-binding inhibitor that is expected to have therapeutic applications. Catechol-O-methyltransferase (COMT; EC 2.1.1.6) cata- lyzes the methylation of small molecules containing a cate- chol structure, being responsible for the elimination of cate- chol-based neurotransmitters, catechol steroids, and xenobiotic catechols (Lautala et al., 2001). In humans and laboratory animals, COMT is present in all tissues studied (Lundstro ¨m et al., 1995); highest activities are found in liver, kidney, and gastrointestinal tract (Ma ¨ nnisto ¨ et al., 1992; Ma ¨ nnisto ¨ and Kaakkola, 1999). It is present as membrane- bound and soluble (S-COMT) forms, both encoded by a single gene using different promoters and translational regulation (Tenhunen and Ulmanen, 1993; Tenhunen et al., 1993, 1994). Both forms are present in practically all tissues in which S-COMT is the predominant form (Rivett et al., 1983; Kar- hunen et al., 1994; Ding et al., 1996), except in human brain, where membrane-bound COMT dominates (Tenhunen et al., 1993, 1994). Both the rat and human S-COMT have 221 amino acids with molecular masses of 24.7 and 24.4 kDa, respectively, and share 81% amino acid sequence identity (Salminen et al., 1990; Lundstro ¨m et al., 1991). The resolu- tion of the atomic structure and sequence comparisons re- vealed that all residues important for the binding of sub- strates and for the catalytic site are conserved in human and rat S-COMT (Vidgren et al., 1994; Lotta et al., 1995). The main clinical interest in COMT results from the pos- sibility of using COMT inhibitors as adjuncts in the therapy of Parkinson’s disease with L-3,4-dihydroxyphenylalanine (L- DOPA) (Ma ¨ nnisto ¨ and Kaakkola, 1989, 1990). This disease is characterized by progressive degeneration of the dopaminer- gic nigrostriatal pathways; at present, the most effective therapy is dopamine replacement with L-DOPA plus a pe- ripheral aromatic L-amino acid decarboxylase inhibitor. Un- der these circumstances, the methylation of L-DOPA becomes the major metabolization route in the periphery and only 5 to 10% of administered L-DOPA reaches the brain (Ma ¨ nnisto ¨ and Kaakkola, 1990). The inhibition of COMT would increase This work was supported in part by grant P003-P31B-02/97 BIAL-COMT from Age ˆncia de Inovac ¸a ˜ o and fellowships PRAXIS XXI/BIC/17185/98 (M.L.R) and PRAXIS XXI/BPD/17265/98 (M.A.). M.J.B. and M.A. contributed equally to the study. ABBREVIATIONS: COMT, catechol-O-methyltransferase; S-COMT, soluble catechol-O-methyltransferase; L-DOPA, L-3,4-dihydroxyphenylala- nine; BIA 3-335, 1-[3,4-dihydroxy-5-nitrophenyl]-3-[N-3'-trifluoromethyl-phenyl]-piperazine-1-propanone; SAM, S-adenosyl-L-methionine; MT, methyltransferases. 0026-895X/02/6204-795–805$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 4 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1278/1011898 Mol Pharmacol 62:795–805, 2002 Printed in U.S.A. 795 at ASPET Journals on October 13, 2017 molpharm.aspetjournals.org Downloaded from