Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to formN-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 acetyl-CoA) 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-[3H]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. Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to formN-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 acetyl-CoA) 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-[3H]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. Substrate specificity and inhibition studies of human serotonin N-acetyltransferase.Journal of Biological ChemistryVol. 275Issue 50PreviewThroughout the text, all of theVmax units read “μmol/min/mg protein,” whereas they should read “nmol/min/mg protein.” This mistake is especially evident in the following occurrences. Full-Text PDF Open Access arylalkylamine N-acetyltransferase human ovine dithiothreitol glutathione S-transferase polyacrylamide gel electrophoresis bisubstrate analog inhibitor high pressure liquid chromatography phenylethylamine N-(9-fluorenyl)methoxycarbonyl liquid chromatography-mass spectrometry nitrophenylalanine norleucine 4-fluorophenylalanine 4-chlorophenylalanine cyclohexylalanine benzothienylalanine single ion-monitored collision-induced dissociation naphthylalanine 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Melatonin (5-methoxy-N-acetyltryptamine) is a pineal hormone that modulates a variety of endocrinological, neurophysiological, and behavioral functions in vertebrates (1.Reiter R.J. Endocr. Rev. 1991; 12: 151-180Crossref PubMed Scopus (2063) Google Scholar). It is involved in the regulation of circadian rhythms and in the reproduction of photoperiodic species (2.Reiter R.J. Experientia (Basel). 1993; 49: 654-664Crossref PubMed Scopus (907) Google Scholar). The chronobiotic effects of melatonin in humans have been mainly studied in circadian rhythm sleep disorders (3.Delagrange P. Guardiola-Lemaitre B. Clin. Neuropharmacol. 1997; 20: 482-510Crossref PubMed Scopus (68) Google Scholar). Moreover, alterations of the melatonin profiles have been reported in other biological rhythm disorders (3.Delagrange P. Guardiola-Lemaitre B. Clin. Neuropharmacol. 1997; 20: 482-510Crossref PubMed Scopus (68) Google Scholar). Melatonin exerts its effects through at least three targets: 2 receptor subtypes, mt1and MT2, and a binding site, MT 3(4.Dubocovich M.L. Cardinali D.P. Guardiola-Lemaitre B. Hagan R.M. Krause D.N. Sugden B. Vanhoutte P.M. Yocca F.D. Melatonin Receptors: The IUPHAR Compendium of Receptor Characterization and Classification. IUPHAR Media, London1998: 187-193Google Scholar). The two first ones have been cloned (5.Reppert S.M. Weaver D.R. Ebisawa T. Neuron. 1994; 13: 1177-1185Abstract Full Text PDF PubMed Scopus (1035) Google Scholar, 6.Reppert S.M. Godson C. Mahle C.D. Weaver D.R. Slaugenhaup S.A. Gusella J.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8734-8738Crossref PubMed Scopus (828) Google Scholar) and their pharmacological effects largely studied, and several specific and potent ligands (7.Dubocovich M.L. Masana M.I. Iacob S. Sauri D.M. Arch. Pharmacol. 1997; 355: 365-375Crossref Scopus (290) Google Scholar, 8.Teh M.T. Sugden D. Arch. Pharmacol. 1998; 358: 522-528Crossref Scopus (44) Google Scholar, 9.Steinhilber D. Carlberg C. Exp. Opin. Ther. Patents. 1999; 9: 281-290Crossref Scopus (12) Google Scholar) discovered. The MT 3subtype is still a putative binding site under intensive research from purification attempts to pharmacological characterizations (10.Molinari E.J. North P.C. Dubocovich M.L. Eur. J. Pharmacol. 1996; 301: 159-168Crossref PubMed Scopus (99) Google Scholar, 11.Paul P. Lahaye C. Delagrange P. Nicolas J.P. Canet E. Boutin J.A. J. Pharmacol. Exp. Ther. 1999; 290: 334-340PubMed Google Scholar). Since melatonin is implicated in several types of mild to severe pathologies, including mood disorders (3.Delagrange P. Guardiola-Lemaitre B. Clin. Neuropharmacol. 1997; 20: 482-510Crossref PubMed Scopus (68) Google Scholar, 12.Mahle C.D. Watson A.J. Exp. Opin. Invest. Drugs. 1997; 6: 399-406Crossref PubMed Scopus (10) Google Scholar), 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 that was aimed at the control of the levels of circulating melatonin. Indeed, melatonin biosynthesis is catalyzed by a series of enzymes, the penultimate of which in the synthesis cascade, serotoninN-acetyltransferase (arylalkylamineN-acetyltransferase, AANAT,1 EC 2.3.1.87), catalyzes the rate-limiting step. It has been shown that large increases in its activity are responsible for large increases in circulating melatonin levels in vertebrates (13.Klein D.C. Weller J.L. Science. 1970; 169: 1093-1095Crossref PubMed Scopus (640) Google Scholar, 14.Arendt S. Melatonin and the Mammalian Pineal Gland. Chapman and Hall Ltd., London1995Google Scholar). AANAT regulates melatonin biosynthesis by controlling the production ofN-acetyl-5-hydroxytryptamine from serotonin (5-hydroxytryptamine) and acetyl-CoA (15.Voisin P. Namboodiri M.A.A. Klein D.C. J. Biol. Chem. 1984; 259: 10913-10918Abstract Full Text PDF PubMed Google Scholar). In the next step, the conversion of N-acetyl-5-hydroxytryptamine to melatonin is catalyzed by hydroxyindole-O-methyltransferase. However, this transferase is expressed at relatively constant levels, and the rate of this step is regulated by the availability ofN-acetyl-5-hydroxytryptamine. Furthermore, the level of AANAT is controlled by a catabolic system involving light, cAMP, and a proteasome multienzyme complex (16.Gastel J.A. Roseboom P.H. Rinaldi P.A. Weller J.L. Klein D.C. Science. 1998; 279: 1358-1360Crossref PubMed Scopus (254) Google Scholar). Therefore, AANAT appears to be a major target for the control of the pineal hormone circulation. Sheep AANAT (oAANAT) has been cloned and overproduced inEscherichia coli (17.Coon S.L. Roseboom P.H. Baler R. Weller J.L. Namboodiri M.A.A. Koonin E.V. Klein D.C. Science. 1995; 270: 1681-1683Crossref PubMed Scopus (311) Google Scholar). Once purified, this recombinant ovine enzyme has been crystallized and the crystal coordinates published (18.Hickman A.B. Klein D.C. Dyda F. Mol. Cell. 1999; 3: 23-32Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 19.Hickman A.B. Namboodiri M.A.A. Klein D.C. Dyda F. Cell. 1999; 97: 361-369Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Knowledge of the human enzyme has progressed more slowly, although Coon et al. (20.Coon S.L. Mazuruk K. Bernard B. Roseboom P.H. Klein D.C. Rodriguez I.R. Genomics. 1996; 34: 76-84Crossref PubMed Scopus (104) Google Scholar) have reported the structure of the human gene encoding this enzyme. Studies of the enzyme distribution outside the retina and pineal gland have shown its presence in different peripheral tissues such as gastrointestinal tract (21.Huether G. Experientia (Basel). 1993; 49: 665-670Crossref PubMed Scopus (280) Google Scholar), testes (22.Borjigen J. Wang M.M. Snyder S.H. Nature. 1995; 378: 783-785Crossref PubMed Scopus (240) Google Scholar), and ovaries (23.Itoh M.T. Ishizuka B. Kuribayashi Y. Amemiya A. Sumi Y. Mol. Hum. Reprod. 1999; 5: 402-408Crossref PubMed Scopus (151) Google Scholar) as well as in other limited brain regions (24.Uz T. Manev H. Biol. Psychiatry. 1999; 45: 175-179Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 25.Fleming J.V. Barrett P. Coon S.L. Klein D.C. Morgan P.J. Endocrinology. 1999; 140: 972-978Crossref PubMed Scopus (27) Google Scholar, 26.Hamada T. Ootomi M. Horikawa K. Niki T. Wakamatu H. Ishida N. Biochem. Biophys. Res. Commun. 1999; 258: 772-777Crossref PubMed Scopus (20) Google Scholar). Substrate and cosubstrate specificities of the ovine enzyme have been only marginally addressed so far (14.Arendt S. Melatonin and the Mammalian Pineal Gland. Chapman and Hall Ltd., London1995Google Scholar, 27.Deguchi T. J. Neurochem. 1975; 24: 1083-1085Crossref PubMed Scopus (35) Google Scholar, 28.Yang H.T. Neff N.H. Neuropharmacology. 1976; 15: 561-564Crossref PubMed Scopus (24) Google Scholar, 29.Shen S. Brémont B. Serraz I. Andrieux J. Poncet A. Mathé-Allainmat M. Chanut E. Trouvin J.H. Langlois M. Eur. J. Pharmacol. 1996; 307: 133-140Crossref PubMed Scopus (12) Google Scholar, 30.DeAngelis J. Gastel J. Klein D.C. Cole P.A. J. Biol. Chem. 1998; 273: 3045-3050Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 31.Khalil E.M. DeAngelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and served as the sole basis for the characterization of AANAT from other species, rat (32.Zhan-Poe X. Craft C.M. J. Pineal Res. 1999; 27: 49-58Crossref PubMed Scopus (14) Google Scholar), rabbit (33.Abe M. Itoh M.T. Miyata M. Ishikawa S. Sumi Y. Exp. Eye Res. 1999; 68: 255-262Crossref PubMed Scopus (64) Google Scholar), ox (34.Craft C.M. Murage J. Brown B. Zhan-Poe X. Mol. Brain Res. 1999; 65: 44-51Crossref PubMed Scopus (25) Google Scholar), and hamster (35.Gauer F. Poirel V.J. Garidou M.L. Simonneaux V. Pévet P. Mol. Brain Res. 1999; 71: 87-95Crossref PubMed Scopus (35) Google Scholar). In the present work, hAANAT (EC 2.3.1.87) was expressed as a fusion protein in a bacterial system, and the protein was purified and cleaved, leading to large amounts of partially pure biological material. The human enzyme was then characterized biochemically and compared with the ovine form. The search for potent substrates and inhibitors was carried out in a large screening process that included synthetic and natural ligands. Two techniques are available for the measurement of AANAT activity, the highly sensitive, extraction-based assay of Deguchi (27.Deguchi T. J. Neurochem. 1975; 24: 1083-1085Crossref PubMed Scopus (35) Google Scholar) and the chromatographic assay of Thomas et al. (36.Thomas K.B. Zawilska J. Iuvone P.M. Anal. Biochem. 1990; 184: 228-234Crossref PubMed Scopus (50) Google Scholar). The latter has been developed for and is limited to fluorescent compounds (37.Itoh M.T. Shinozawa T. Sumi Y. Brain Res. 1999; 830: 165-173Crossref PubMed Scopus (40) Google Scholar), whereas the former is difficult to automatize. Since our program required a relatively high throughput capacity, we developed an HPLC assay for substrate and inhibitor screening running less than 6 min per sample. By using labeled acetyl-CoA, it was possible to test any type of ethylamine derivative as potential substrate, regardless of its water/chloroform repartition, in contrast to the Deguchi extraction assay (27.Deguchi T. J. Neurochem. 1975; 24: 1083-1085Crossref PubMed Scopus (35) Google Scholar). Large tri-, tetra-, and pentapeptide combinatorial libraries were also screened as potential sources of inhibitors, leading to active leads in the micromolar range, displaying either pure or mixed competitive kinetic behavior. Molecular modeling and structure-activity relationship studies made it possible to pinpoint the amino acid residue of the pentapeptide inhibitor S 34461 that interacts with the cosubstrate-binding site. The present report is the first one on the biochemical description of the human enzyme. This study reveals some unexpected differences with the ovine enzyme. Finally, synthetic substrates of AANAT and new inhibitors are discovered and described. The human arylalkyl N-acetyltransferase cDNA coding region (kindly provided by Dr. D. C. Klein and Dr. S. L. Coon, National Institutes of Health, Bethesda) was inserted into the bacterial expression vector pGEX-4T (Amersham Pharmacia Biotech). TheE. coli strain BL21(DE3)pLysS was transformed with the resulting plasmid and grown overnight at 37 °C in Luria Broth supplemented with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml). The culture was diluted 1:25 in fresh medium and grown at 37 °C, until it reached an absorbance at 595 nm of 0.7. Isopropyl-1-thio-β-d-galactopyranoside was then added to a final concentration of 0.2 mm, and the culture was maintained at 24 °C for 6 h. The cells were harvested by centrifugation (5,000 × g, 4 °C, 10 min), frozen in dry ice, and stored at −80 °C until further use. All the procedures were performed at 0–4 °C. Approximately 10 g of a frozen bacteria pellet expressing GST-hAANAT was thawed in 40 ml of 2× phosphate-buffered saline containing 10 mmdithiothreitol (DTT), a mixture of protease inhibitors (Complete, Roche Molecular Biochemicals, 1 tablet/50 ml), and with or without the detergent Tween 85 (at 1% v/v). The thawed bacterial suspension was sonicated (setting, 70/100, probe 1 cm diameter) eight times for 1 min duration, with 1-min interval between each sonication. The preparation was immediately centrifuged (20,000 × g, 20 min), and the supernatant was slowly (40 ml/h) passed through a glutathione-Sepharose column (Amersham Pharmacia Biotech, 10 ml packed volume), equilibrated with buffer A (2× phosphate-buffered saline, pH 6.9, containing 10 mm DTT). The column was initially washed with 40 ml of buffer A and then with 150 ml of buffer B (50 mm Tris/HCl, pH 8.0, containing 100 mm sodium citrate, 10 mm DTT, and 10% (v/v) glycerol). The GST-AANAT was eluted sequentially with 40 ml of 10 mm glutathione in buffer B. Eight-ml fractions were collected and immediately assayed for AANAT activity. The fractions containing the AANAT activity were pooled and stored frozen at −80 °C. For comparison purposes, the ovine enzyme has been purified after expression in bacteria as a fusion GST-oAANAT protein as reported (30.DeAngelis J. Gastel J. Klein D.C. Cole P.A. J. Biol. Chem. 1998; 273: 3045-3050Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The GST-AANAT was cleaved by thrombin treatment, and the active fractions were pooled and dialyzed against 10 liters of buffer C (20 mm Tris/HCl, pH 8.0, containing 1 mm EDTA, 2 mm DTT, 500 mm NaCl, and 10% glycerol) overnight using Slide-A-Lyser (Pierce) to remove the free glutathione. The dialyzed sample was then incubated (0–4 °C, 12 h) with thrombin (Roche Molecular Biochemicals, 1 unit/ml). The sample was passed over a mixed column bed containing 5 ml of glutathione-Sepharose and 3 ml of benzamidine-Sepharose (Amersham Pharmacia Biotech), previously equilibrated with the dialysis buffer. The unbound fractions were collected, and the column was washed with three additional column volumes. The collected fractions were assayed for AANAT activity, and those containing AANAT activity were pooled and stored at −80 °C. The protein concentration was determined by the Bradford assay (Protassay, Bio-Rad) with bovine serum albumin as standard. Purity was assessed by densitometry (GS710, Bio-Rad) on Coomassie Blue-stained polyacrylamide electrophoresis gels. This new assay for the AANAT activity was based upon reverse-phase HPLC using either absorbance or radiochemical detection of N-acetylserotonin. The reaction mixture contained 10 μl of enzyme (1 μg for the human enzyme, but 10–50 times less for the ovine enzyme), in a phosphate buffer (50 mm sodium phosphate, pH 6.8, containing 500 mmNaCl and 2 mm EDTA), 10 μl of [3H]acetyl-CoA (129GBq/mmol), 1 mmacetyl-CoA, 4 mm serotonin, in a final volume of 100 μl. After incubation of 30 min at 37 °C, it was stopped by the addition of 50 μl of 10% trichloroacetic acid solution. Thirty μl of this solution were analyzed by reverse-phase HPLC using a Platinum EPS C8, (53 × 7 mm, Alltech, France) column on a Hewlett-Packard 1100 system. The column was eluted with a linear gradient of 5–35% acetonitrile in H2O, 0.1% trifluoroacetic acid at a flow rate of 2 ml/min for 5 min. While the separation took about 7.5 min, efficient mass spectrometry detection required longer separation times (10 min) as did also more hydrophobic substrates with higher gradient curves (30 min). This type of separation was obtained in the same buffer using a different polymeric analytical column (C4 ASTEC, 4.6 × 150 mm, CIL Cluzeau, Ste-Foy-la-Grande, France). The column was eluted with a linear gradient 5–35 or 0–100% acetonitrile in H2O, 0.1% trifluoroacetic acid at a flow rate of 1 ml/min for 10 or 30 min. The C4 Astec polymeric column and tritiated serotonin (NEN Life Science Products, 1 TBq/mmol, 10 μl in incubation with cold serotonin 4 mm final) were used to study other cosubstrates than acetyl-CoA. HPLC gradient and buffer were the same as described above for the 15-min run. The radioactivity was followed on-line after addition of the scintillation mixture (2 ml/min) using a Berthold detector (EGG, Bad Wildbad, Germany). Deguchi's extraction assay was used as the reference assay (27.Deguchi T. J. Neurochem. 1975; 24: 1083-1085Crossref PubMed Scopus (35) Google Scholar) for serotonin N-acetyltransferase. Reaction products from the incubation (substrate was 1 mmtryptamine in this assay) were extracted into chloroform, and the organic phase was rinsed once with sodium phosphate buffer (0.1m, pH 6.8) and twice with 1 m NaOH. The chloroform phase containing [3H]acetyl-CoA was evaporated and its radioactivity estimated in a β counter (Packard Instrument Co.). Except when otherwise noted, all the measurements were done using the HPLC assay. Basically, the affinity data have been collected using the classical process described by Segel (38.Segel I.C. Enzyme Kinetics. Wiley Interscience, New York1975Google Scholar) for bisubstrate enzymes as applied on this transferase (30.DeAngelis J. Gastel J. Klein D.C. Cole P.A. J. Biol. Chem. 1998; 273: 3045-3050Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 31.Khalil E.M. DeAngelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) or on other ones (39.Boutin J.A. Antoine B. Siest G. J. Pharmacol. Sci. 1994; 83: 591-596Abstract Full Text PDF PubMed Scopus (4) Google Scholar). For the apparent K m value, the substrate concentrations were varied from 0.06 to 4 mm, whereas the cosubstrate (acetyl-CoA) concentration was 1 mm. For apparent K m values of acetyl-CoA, a saturating concentration of 4 mm was used for the substrate (usually, serotonin). Although the rule is that the saturating concentration should be 10 times the K m value (38.Segel I.C. Enzyme Kinetics. Wiley Interscience, New York1975Google Scholar), for most of the natural bioamine substrates of AANAT, this concentration could not be reached (∼10 mm). For real K mdeterminations, experiments were also conducted according to the guidelines given by Segel (38.Segel I.C. Enzyme Kinetics. Wiley Interscience, New York1975Google Scholar). At least five concentration points were routinely used, and the experiments were repeated three times. Finally, for treatment of the data, double-reciprocal plots were used as the most accurate method when less than 10 concentration points are available. Mass spectra were recorded on a TSQ 7000 triple quadrupole instrument (Finnigan, San Jose, CA) operating in positive atmospheric pressure chemical ionization mode (APCI+). The API vaporizer was operated at 400 °C with a capillary temperature of 250 °C. Nitrogen was used as auxiliary gas (35 mL/min) and as sheath gas (70 pounds/square inch). The corona voltage was 5 kV. All spectra were acquired in centroid mode. Collision-induced dissociation (CID) experiments were carried out by setting Q1 to pass only the ion of interest ([M + H]+ ± 0.3 Da in 0.5 s), inducing collisions in Q2 and scanning Q3 from 10 to 600 Da with a scan time of 1 s. MS/MS analyses were performed with argon as collision gas at a pressure of 1 millitorr and a dissociation offset of −20 eV. Single ion-monitored (SIM) parameters were 0.9 Da/s scan time and ±0.3-Da scan window. LC separations for the LC/MS experiments were performed using a Hewlett-Packard 1090 binary pump equipped with a UV detector. The detection wavelength was set at 210 nm. The samples passed through a 20-μl injection loop, into the ASTEC polymeric C4 column (see above) at 1 ml/min flow rate and directly into the mass spectrometer. The eluents were as follows: eluent A, 0.01% trifluoroacetic acid in deionized water, and eluent B, 0.01% trifluoroacetic acid in acetonitrile, and the gradient sequence was from 95 to 50% of eluent A in 30 min. The libraries were synthesized using a robotic instrument built around a Zymark arm (40.Boutin J.A. Hennig P. Lambert P.H. Bertin S. Petit L. Mahieu J.P. Serkiz B. Volland J.P. Fauchère J.L. Anal. Biochem. 1996; 234: 126-141Crossref PubMed Scopus (56) Google Scholar). The robot was able to handle 1 g of resin with 2 × 106 beads per reactor, therefore ensuring a high ratio of the number of beads to the number of tetrapeptides (41.Boutin J.A. Fauchère A.L. Trends Pharmacol. Sci. 1996; 17: 8-13Abstract Full Text PDF PubMed Scopus (24) Google Scholar). The robot automatically handled the main steps of the mix and split procedure (42.Houghten R.A. Pinilla C. Blondelle S.E. Appel J.R. Dooley C.T. Cuervo J.H. Nature. 1991; 354: 84-86Crossref PubMed Scopus (1147) Google Scholar) using the Fmoc strategy for peptide synthesis on solid phase (43.Wang S.S. J. Am. Chem. Soc. 1973; 95: 1328-1333Crossref PubMed Google Scholar). The libraries obtained were prepared from sets of 24 different amino acids depending on the library types. The exact amino acid composition of the tripeptide library was at position 1: γ-aminobutyric acid, 4-aminophenylalanine, Arg, β-alanine, benzothienylalanine (Bta), cyclohexylalanine (Cha), 4-chlorophenylalanine (Clp), 4-fluorophenylalanine (Fpa), Gln, Glu, Gly, His, Lys, methionine sulfoxide, 4-nitrophenylalanine (Nip), norleucine (Nle), Phe, phenylglycine, 3-pyridylalanine,tert-butylglycine, thienylalanine, Thr, Tyr, andO-methyltyrosine. This “optidiverse” set of amino acids displays a high fingerprint diversity, compared with the natural series (44.Fauchère J.L. Henlin J.M. Boutin J.A. Can. J. Physiol. Pharmacol. 1997; 75: 683-689Crossref PubMed Scopus (10) Google Scholar). The position 2 was occupied by Trp, Bta, 1-naphthylalanine (Nal1), 2-naphthylalanine (Nal2), or Tyr. The position 3 was composed of ε-aminocaproic acid, Ala, Arg, Asn, Asp, β-alanine, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Nip, Nle, Orn, Phe, Sar, Ser, Thr, Trp, Tyr, and Val. This particular set is called the standard set of amino acids (40.Boutin J.A. Hennig P. Lambert P.H. Bertin S. Petit L. Mahieu J.P. Serkiz B. Volland J.P. Fauchère J.L. Anal. Biochem. 1996; 234: 126-141Crossref PubMed Scopus (56) Google Scholar). The tetrapeptide library was synthesized as a C-terminal amide library. It comprised at all four variable positions the optidiverse set of amino acids. The pentapeptide library was composed of Trp at position 1 and the standard set at positions 2–4. At position 5, the Pro residue was replaced by Nle. These mixtures were white to yellowish powders that could be handled in the assay, the same way as if they were discrete chemicals. They were analyzed using MS and MS/MS techniques (40.Boutin J.A. Hennig P. Lambert P.H. Bertin S. Petit L. Mahieu J.P. Serkiz B. Volland J.P. Fauchère J.L. Anal. Biochem. 1996; 234: 126-141Crossref PubMed Scopus (56) Google Scholar, 45.Boutin J.A. Gesson I. Henlin J.M. Bertin S. Lambert P.H. Volland J.P. Fauchère J.L. Mol. Diversity. 1997; 3: 43-60Crossref PubMed Scopus (22) Google Scholar, 46.Lambert P.H. Boutin J.A. Bertin S. Fauchere J.L. Volland J.P. Rapid Commun. Mass Spectrom. 1997; 11: 1971-1976Crossref Scopus (24) Google Scholar) providing enough information to ensure (i) the lack of major by-products in the mixture and (ii) the presence of the accounted structures in the sublibraries. Furthermore, in each sublibrary, individual amino acids were indeed found to be present in roughly equal amounts by two-dimensional NMR (40.Boutin J.A. Hennig P. Lambert P.H. Bertin S. Petit L. Mahieu J.P. Serkiz B. Volland J.P. Fauchère J.L. Anal. Biochem. 1996; 234: 126-141Crossref PubMed Scopus (56) Google Scholar). The mix and split deconvolution method used consists of the synthesis of a set of sublibraries in which the N-terminal position is fixed and known, followed by a sequential unrandomization of randomized fragments (47.Freier S.M. Konings D.A.M. Wyatt J.R. Ecker D.J. J. Med. Chem. 1995; 38: 344-352Crossref PubMed Scopus (72) Google Scholar) based on the biological activity of the sublibraries. The most active single peptides and several analogs were either resynthesized by solid phase synthesis using an Fmoc strategy on the Zymark robot in the parallel mode (no mixing step). The final compounds were obtained after cleavage and deprotection in 95% trifluoroacetic acid in the presence of scavengers. They were purified by preparative HPLC and lyophilized. Peptides were at least 95% (HPLC) pure. Furthermore, a series of single analogs were synthesized, including the five peptides of an Ala scan, the five peptides of a Gly scan of the pentapeptide inhibitor S 34461, and the five tetrapeptides corresponding to the successive deletion of each amino acid residue in S 34461. A series of amines was synthesized in order to find out which of the ethylamine derivatives was recognized by hAANAT as substrates. SD 219 was synthesized as described by Fournier and Boyer (48.Fournier C. Boyer F. C. R. Acad. Sci. Ser. C (Paris). 1970; 270: 1179-1182Google Scholar), SD 236 by Chan et al. (49.Chan J.H.T. Elix J.A. Ferguson B.A. Aust. J. Chem. 1975; 28: 1097-1111Crossref Scopus (20) Google Scholar), SD 552 by Sam et al. (50.Sam J. Aparajithan K. Shafik R. J. Pharmacol. Sci. 1968; 57: 564-568Abstract Full Text PDF PubMed Scopus (5) Google Scholar), SD 715 by Khalil and Cole (51.Khalil E. Cole P.A. J. Am. Chem. 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