Molecular recognition at the active site of catechol-o-methyltransferase: energetically favorable replacement of a water molecule imported by a bisubstrate inhibitor.

Biologically active catechols, such as l-DOPA and the neurotransmitter dopamine, are inactivated by methylation. This reaction is catalyzed by the enzyme catechol-O-methyltransferase (COMT) in the presence of S-adenosylmethionine (SAM) and Mg ions. Small nitrocatechol-based inhibitors of COMT find application in the treatment of Parkinson disease by blocking unwanted methylation of the administered l-DOPA, thereby enhancing dopamine levels in the brain. 3] Recent studies have pointed towards additional therapeutic applications of COMT inhibition in other disorders of the central nervous system, such as schizophrenia and depression. We have developed a series of potent bisubstrate inhibitors for COMT which are competitive for both the catechol and the SAM binding sites. Based on the X-ray crystal structure of ligand 1 (IC50 = 9 nm) [7a] in a ternary complex with COMT and a Mg ion (PDB code: 1JR4), we started a detailed exploration of the molecular recognition properties of the entire active site of the enzyme. Importantly, we found that potentially hepatotoxic nitro groups, which are mandatory in catechol-based monosubstrate inhibitors, are not required for high-affinity bisubstrate inhibition. We substituted the nitro group in position 5 of 1 with appropriate lipophilic residues, such as the 4-fluorophenyl ring in 2 (IC50 = 31 nm), and found that the high, competitive inhibitory potency was maintained. Computer modeling studies suggested that the newly introduced lipophilic residue occupies a hydrophobic cleft near the surface of the enzyme. This initial proposal is validated here experimentally by X-ray crystallography. The crystal structure of 1 in a ternary complex with COMT and a Mg ion shows that the adenine moiety forms two hydrogen bonds, a moderately strong and a weak one (d(N···O): 3.0 and 3.4 , respectively), to a water molecule

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