A Potent Inhibitor of the Melatonin Rhythm Enzyme Ehab M. Khalil and Philip A. Cole* Laboratory of Bioorganic Chemistry The Rockefeller UniVersity 1230 York AVe., New York, New York 10021 ReceiVed April 21, 1998 Since the structural elucidation of melatonin (5-methoxy-N- acetyltryptamine) in 1959, 1 there has been growing interest in this “molecular pacemaker” hormone. 2 The biological roles of melatonin have been widely speculated on and include effects on aging, sleep, mood, immune response, cardiovascular fitness, and cancer. 3 The primary pharmacologic intervention that has been available to elucidate the actions of melatonin has been to administer the hormone to animals or people in large doses. Lacking from the arsenal of neuroendocrinologists has been a specific inhibitor of melatonin biosynthesis. Inhibitors of mela- tonin biosynthesis could not only help improve our understanding of circadian rhythm but they might also have therapeutic roles in mood and sleep disorders. The key enzyme to be targeted in this regard is serotonin N-acetyltransferase (arylalkylamine N- acetyltransferase, AANAT), also called the “melatonin rhythm enzyme”. 4 Here we report on the first potent and specific inhibitor of AANAT. AANAT catalyzes the transfer of the acetyl group from acetyl- CoA to the primary amine group of serotonin, affording N- acetylserotonin (Figure 1). The enzyme is found primarily in the pineal and regulates the circadian rhythm of melatonin production. 4 Like melatonin production itself, AANAT activity varies with diurnal periodicity with up to 100-fold increases at night. The pineal enzyme hydroxyindole O-methyltransferase catalyzes the conversion of N-acetylserotonin to melatonin, 5 and this step is primarily regulated by N-acetyl-5-hydroxytryptamine availability. AANATs constitute a family of 23 kDa proteins which share 80% amino acid identity and are members of a superfamily of proteins defined by the presence in tandem of two weakly conserved 15 amino acid sequences (motifs A and B). 4 Included in this superfamily are some histone N-acetyltransferases important in gene regulation 6 and aminoglycoside N-acetyltrans- ferases important in antibiotic resistance. 7 Recent steady-state kinetic studies on AANAT showed evi- dence for an ordered “BiBi” ternary complex kinetic mechanism in which acetyl-CoA binds first followed by serotonin (or the alternative substrate tryptamine). 8 In this mechanism, both substrates must bind to the enzyme prior to the release of either product, and the acetyl group is most likely transferred directly from acetyl-CoA to serotonin without the involvement of a covalent acetyl-enzyme intermediate. In this model, a “bisub- strate analog” containing the components of serotonin and acetyl- CoA covalently linked could potentially be a potent AANAT inhibitor. 9 We therefore synthesized compound 1 to evaluate this possibility (Scheme 1). Tryptamine 3 was reacted with bromoacetyl bromide to afford the bromoacetamide derivative 2. CoASH was alkylated with 2 in the presence of weakly basic conditions leading to thioether 1. Compound 1 was purified by reversed phase HPLC, demon- strated high purity (>98%) based on 1 H NMR, 31 P NMR, MS, and analytical HPLC, and was reasonably stable for extended periods in aqueous solution. A screening assay carried out with recombinant sheep AANAT 8 with varying concentrations of compound 1 and fixed substrate concentrations demonstrated that 1 was a very potent inhibitor with IC 50 ≈ 150 nM. This value is approximately 1000-fold lower than the substrate K m values or the IC 50 of any other reported inhibitor to date. 8 A time course demonstrated that in the presence of inhibitor the activity was still linear with time for at least 3 min, making less likely a “slow-binding” inhibition kinetic scheme. Steady-state kinetic inhibitor analysis of AANAT was carried out, and compound 1 was shown to be a linear competitive inhibitor versus the varied substrate acetyl-CoA (K i ) 90 nM) and a noncompetitive inhibitor versus tryptamine (Figure 2). This pattern of inhibition further supports an ordered BiBi model with acetyl-CoA binding before tryptamine (or serotonin). 10 A useful analysis of bisusbtrate inhibitory potency has been to compare the K i of the bisubstrate analogue to the product of the individual K m values of the substrates involved. 9 In this case, the (K m -acetyl-CoA)(K m -tryptamine)/K i ≈ 1 M. The entropic gain for linking the two substrates is comparable to the more potent examples of reported bisubstrate analogue enzyme inhibitors. 9 The potency of inhibition of AANAT by compound 1 is in a range that could potentially be useful for physiologic studies. We were next interested in exploring the specificity of compound 1 as an acetyltransferase inhibitor. In particular, we wanted to examine whether 1 could block the enzyme arylamine N-acetyltransferase. Arylamine N-acetyltransferase catalyzes the transfer of the acetyl group from acetyl-CoA to arylamines as well as arylalkylamines including serotonin. 11 Because it is also present in pineal, it can be a source of interference in assaying AANAT activity in crude extracts. 12 In contrast to AANAT, * Author to whom correspondence should be addressed: tel (212) 327- 7241, fax (212) 327-7243, email cole@rockvax.rockefeller.edu. (1) Lerner, A. B.; Case, J. D.; Heinzelman, R. V. J. Am. Chem. Soc. 1959, 81, 6084-6085. 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