New substrate analogues of human serotonin N-acetyltransferase produce in situ specific and potent inhibitors

Melatonin is synthesized by an enzymatic pathway, in which arylalkylamine (serotonin) N-acetyltransferase catalyzes the rate-limiting step. A previous study [Khalil, E.M., De Angelis, J., Ishii, M. & Cole, P.A. (1999) Proc. Natl Acad. Sci. USA96, 12418-12423] reported the discovery of bromoacetyltryptamine (BAT), a new type of inhibitor of this enzyme. This compound is the precursor of a potent bifunctional inhibitor (analogue of the transition state), capable of interfering with both the substrate and the cosubstrate binding sites. This inhibitor is biosynthesized by the enzyme itself in the presence of free coenzyme A. In the present report, we describe the potency of new N-halogenoacetyl derivatives leading to a strong in situ inhibition of serotonin N-acetyltransferase. The new concept behind the mechanism of action of these precursors was studied by following the biosynthesis of the inhibitor from tritiated-BAT in a living cell. The fate of tritiated-phenylethylamine (PEA), a natural substrate of the enzyme, in the presence or absence of [H-3]BAT was also followed, leading to their incorporation into the reaction product or the inhibitor (N-acetyl[H-3]PEA and coenzyme A-S[H-3]acetyltryptamine, respectively). The biosynthesis of this bifunctional inhibitor derived from BAT was also followed by nuclear magnetic resonance during its catalytic production by the pure enzyme. In a similar manner we studied the production of another inhibitor generated from N-[2-(7-hydroxynaphth-1-yl)ethyl]bromoacetamide. New derivatives were also screened for their capacity to inhibit a purified enzyme, in addition to enzyme overexpressed in a cellular model. Some of these compounds proved to be extremely potent, with IC(50)s of approximate to 30 nM. As these compounds, by definition, closely resemble the natural substrates of arylalkylamine N-acetyltransferase, we also show that they are potent ligands at the melatonin receptors. Nevertheless, these inhibitors form a series of pharmacological tools that could be used to understand more closely the inhibition of pineal melatonin production in vivo.