Design, synthesis and in vitro evaluation of novel benzo[b]thiophene derivatives as serotonin N-acetyltransferase (AANAT) inhibitors.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is the penultimate enzyme in melatonin (5-methoxy-N-acetyltryptamine) biosynthesis. It is the key-enzyme responsible of the nocturnal rhythm of melatonin production in the pineal gland. Specific AANAT inhibitors could be useful for treatment of different physiopathological disorders encountered in diseases such as seasonal affective disorders or obesity. On the basis of previous works and 3D-QSAR studies carried out in our laboratory, we have synthesized and evaluated four novel benzo[b]thiophene derivatives designed as AANAT inhibitors. Compound 13 exhibited high inhibitory activity (IC50 = 1.4 microM) and low affinities for both MT, (1100 nM) and MT2 (1400 nM) receptors.

Characterization and regulation of a CHO cell line stably expressing human serotonin N-acetyltransferase (EC 2.3.1.87).

Current melatonin research is essentially based on the finding of new molecular tools, including synthetic or natural agonists and antagonists for the melatonin receptors and synthetic inhibitors of the enzymes involved in its biosynthesis. Indeed, the use of these compounds will improve our understanding of some of the numerous mechanisms of action of melatonin. The present report deals with the establishment and description of a new cell line expressing in a stable manner human arylalkylamine-N-acetyltransferase (AANAT, E.C.2.3.1.87). This new cellular system permits one to check the capacity of newly discovered inhibitors to penetrate the cell and reach their target. Some emphasis is put on inhibitors of the bromoacetyltryptamine family since these precursor compounds form in situ bisubstrate inhibitors with strong affinity for the human enzyme. AANAT is known to undergo complex and rapid regulation by a subtle balance between extremely fast catabolism and protection against it, both due to serine phosphorylation. In the present report, this phosphorylation is shown to occur in vitro after incubation with several kinases (rho-kinase, chk-1, protein kinase A) but not with protein kinase C. Phosphorylation enhances the specific activity of the enzyme by a factor of two to five. This phosphorylation is also shown to occur after treatment of the cell with compounds such as forskolin and rolipram that enhance or protect the intracellular pool of cAMP or the cell-permeable cAMP analogue, dioctanoyl-cAMP. The specificity of the cellular model was assessed using a series of substrates and inhibitors of AANAT already described in the literature, and the characteristics of this cellular system are shown to correspond with those reported for the purified enzyme. This cell line was used to screen libraries of compounds in a living system and led to the discovery of several potent specific and non-toxic AANAT inhibitors.

Design, synthesis and in vitro evaluation of novel derivatives as serotonin N-acetyltransferase inhibitors.

Serotonin N-acetyltransferase (arylalkylamine N-acetyl-transferase, AANAT) is an enzyme that catalyses the first rate limiting step in the biosynthesis of melatonin (5-methoxy-N-acetyltryptamine). Different physiopathological disorders in human may be due to abnormal secretion of melatonin leading to an inappropriate exposure of melatonin receptors to melatonin. For that reason, we have designed, synthesized and evaluated as inhibitors of human serotonin N-acetyltransferase, a series of compounds that were able to react with coenzyme A to give a bisubstrate analog inhibitor. Compound 12d was found to be a potent AANAT inhibitor (IC50 = 0.18 microM).