Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetylCoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[ 3 H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.Melatonin (5-methoxy-N-acetyltryptamine) is a pineal hormone that modulates a variety of endocrinological, neurophysiological, and behavioral functions in vertebrates (1). It is involved in the regulation of circadian rhythms and in the reproduction of photoperiodic species (2). The chronobiotic effects of melatonin in humans have been mainly studied in circadian rhythm sleep disorders (3). Moreover, alterations of the melatonin profiles have been reported in other biological rhythm disorders (3). Melatonin exerts its effects through at least three targets: 2 receptor subtypes, mt 1 and MT 2 , and a binding site, MT 3 (4). The two first ones have been cloned (5-6) and their pharmacological effects largely studied, and several specific and potent ligands (7-9) discovered. The MT 3 subtype is still a putative binding site under intensive research from purification attempts to pharmacological characterizations (10 -11). Since melatonin is implicated in several types of mild to severe pathologies, including mood disorders (3, 12), it is considered a valuable therapeutic target. Beside the classical search for agonists and antagonists of the melatonin receptors, a series of programs was launched t...
: Since the discovery of the multi-drug resistance (MDR) phenotype, reversant agents of various origins and structures have been extensively studied. In the present work, two series of related 2,4,6-tris(amino)-striazines with di †erent MDR potential1 were studied by 15N NMR spectroscopy. The 15N nucleus allows an easy identiÐcation of two protonation sites and an estimation of the electronic e †ects. 15N was further found to be well suited to demonstrate the occurrence at room temperature of restricted rotation around the ArÈN bonds between the amino substituents and the s-triazine ring and to measure the rotational barriers. Crystal structures were determined by x-ray analysis of the compounds at various stages of protonation. The e †ects of the protonation at the sterically less hindered nitrogen of the triazine, detected by the NMR study, were conÐrmed in the solid-state structures. In the crystals, the orientation of the NÈH and NÈC bonds of the NHallyl substituent with respect to the triazine ring does not depend on the protonation state and corresponds to one of the conformations postulated in solution.
The standard method of peptide library synthesis involves coupling steps in which a single amino acid is reacted with a mixture of resin-bound amino acids. The more recently described positional scanning strategy (in which each position in the peptide sequence is occupied in turn by a single residue) is different since it involves the coupling of mixtures of amino acids to mixtures of resin-bound amino acids. In the present study, we analyze the compounds produced under these conditions measuring coupling rates and amounts of formed products, using mainly UV, HPLC, LC/MS and MS/MS techniques. Our data do not permit to conclude that the resulting libraries are complete. Indeed, our analytical data indicate that a large part of the di-, tri- and tetrapeptides synthesized with this method are not present in the final mixture. Although chemical compensation (in which poor coupling kinetics is compensated by a larger excess of the incoming amino acid) has been thought to counterbalance these biases, our experiments show that the compensation method does not take into account the crucial influence of the resin-bound amino acid and that even the dipeptide libraries obtained in this way are far from completeness. The present work provides strong evidence that the coupling of mixtures of amino acids to resin-bound residues, which is required by the positional scanning strategy, results in incomplete and/or non-equimolar libraries. It also clearly confirms that coupling rates in solid-phase peptide synthesis are dependent on the nature of both the incoming and the immobilized amino acid.
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