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...

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A complete 331,776-member library of tetrapeptides made of 24 amino acid building blocks was synthesized robotically on solid phase and subjected to a deconvolution based on the inhibitory potency of the sublibraries in a HPLC assay of the S-farnesyltransferase activity in vitro. One of the non-natural peptide and noncysteine-containing leads Nip-Trp-Phe-His (Nip=p-nitrophenyl-L-alanine) was optimized chemically to give a proteolytically stable pseudopeptide with a 200-fold potency compared with the original lead. The final compound was converted to the C-terminal ethyl ester: p-F-C6H4-CO(CH2)2-CO-Bta-D-Phepsi[CH2NH]His-OEt (Bta = benzothienyl-L-alanine) and shown to behave as a prodrug which was hydrolyzed back to the C-terminal acid following cell penetration. The method confirmed that several structurally original leads can be discovered in large libraries when deconvolution relies upon a highly specific assay and that these leads can be optimized by chemical modification to impart the final compound the desired pharmacological and pharmacokinetic properties.

Dipyrrolo[3,carbazole-1,3,4,6-tetraones can be considered as granulatimide analogues. In view of structure-activity relationship studies in these series, a parallel liquid-phase microwave-assisted synthesis was developed to generate a small library of compounds bearing various substituents at positions 8, 9, 10, or 11 on the aromatic framework.