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

Abstract

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 [3H]BAT was also followed, leading to their incorporation into the reaction product or the inhibitor (N‐acetyl[3H]PEA and coenzyme A‐S[3H]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 IC50s of ≈ 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.

Vanillin Analog – Vanillyl Mandelic Acid, a Novel Specific Inhibitor of Snake Venom 5′‐Nucleotidase

“…Numerous studies have used specific inhibitors of enzymes as a biochemical or pharmacological tool to characterize and establish the mechanism of action . On similar lines, we demonstrated the involvement of Naja naja venom 5′NUC in inducing anticoagulant effect by the use of specific inhibitor .…”

“…Although it is widely accepted that 5′NUC plays an important role in snake envenomation strategies, apart from its hemostatic functions modulation, its other pharmacological effects are not well characterized . Several studies have used specific inhibitors of enzymes as a biochemical or pharmacological tool to characterize/or establish its mechanism of action . The involvement of N. naja venom 5′NUC in inducing anticoagulant effect was demonstrated by the use of specific inhibitor, vanillyl acid (VA) .…”

“…Tannins are known to be general inhibitors of snake venom toxins . The reported substrate analog inhibitors , even though specific for NUC, are known to act as ligands for receptors, thus restricting its use for in vivo studies . Therefore, in this respect VMA is a non‐nucleoside in nature, a potent and specific inhibitor of therapeutic application.…”

Molecular evidence that melatonin is enzymatically oxidized in a different manner than tryptophan: investigations with both indoleamine 2,3-dioxygenase and myeloperoxidase

“…Grohmann et al [17]). Our laboratory group has been involved for several years in understanding the mechanisms of action of melatonin at the molecular level, including the enzymology of its biosynthesis [18][19][20][21] and its interaction with melatonin receptors [22][23][24][25]. In order to explore further the catabolism of melatonin, which to date is based on only a few reports, we expressed recombinant human IndoDO in bacteria, leading to expression of a protein tagged with a hexahistidine moiety [12].…”

On the Organization of a Drug Discovery Platform

“…Indeed, a feature of this method is of great interest for us: it has poor sensitivity but a high robustness [71] and can be used not only for screening purpose but also for monitoring poor-affinity target/compound interactions, as in the first step of the search for receptor ligands. In addition to its obvious use as an analysis technique, we have adapted this method in various frames, including a way to better trace enzymatic activity [72], use a fluorine-labeled spy molecule as a ligand for an enzyme (that screened compounds can chase) [73], or verify changes in a component of a reaction [74]. These techniques are all automatable and thus can be used with less staff power as long as the automate is running by itself-day and night-and the validation of the assay guarantees stability of the components over time.…”