Combinatorial Peptide Libraries: Robotic Synthesis and Analysis by Nuclear Magnetic Resonance, Mass Spectrometry, Tandem Mass Spectrometry, and High-Performance Capillary Electrophoresis Techniques

Boutin, Jean A.; Hennig, Philippe; Lambert, Pierre‐Herve; Bertin, Sophie; Petit, Laurence; Mahieu, Jean-Pierre; Serkiz, Bernard; Volland, Jean‐Paul; Fauchère, Jean‐Luc

doi:10.1006/abio.1996.0064

Cited by 67 publications

(40 citation statements)

References 7 publications

(9 reference statements)

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“…Liver microsomes from rats pretreated with PB (an inducer of CYP2B1 and CYP2B2), ISF (a CYP1A inducer) or BNF (a CYP1A inducer) [16], yielded the same four glucuronides. The relative proportions of these glucuronides was not a¡ected by the di¡erent CYP inducers which is in agreement with previous studies and which shows that the microsomal UGTs responsible for the conjugation of £avonoids are not inducible by CYP1A and CYP2B inducers [12]. Based on integrated peak areas, G1 and G2 represented approximately 89% and 10% of total glucuronides formed in incubations, respectively.…”

Section: Resultssupporting

confidence: 91%

“…A typical 0.5-ml incubation mixture consisted of 0.5 mg protein of microsomes from untreated or PB-, ISF-, or BNF-treated rats or human liver microsomes, 52 mM HEPES/NaOH bu¡er pH 7.4 containing 10 mM MgCl 2 , 0.25 mM Triton X-100, 52 mM UDPGA, and 100 WM XN (dissolved in 3 Wl ethanol) as a substrate [12]. The reaction was initiated by adding UDPGA.…”

Section: Xn Glucuronidation By Rat or Human Liver Microsomesmentioning

confidence: 99%

“…In a recent study, we showed that XN was metabolized to four di¡erent metabolites by isosafrole/L-naphtho£avone (ISF/ BNF) -induced rat liver microsomes [11]. Numerous studies have been published demonstrating that many types of £avo-noids are excreted as glucuronides by humans or other mammals [12,4]. Glucuronidation is the main pathway of the Phase II detoxi¢cation processes for most xenobiotics, including £a-vonoids, and is catalyzed by UDP-glucuronosyl transferases which are membrane-bound and located mainly in the liver endoplasmic reticulum [13].…”

Section: Introductionmentioning

confidence: 99%

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In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes

2001

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Xanthohumol (XN) is the major prenylated flavonoid of hop plants and has been detected in beer. Previous studies suggest a variety of potential cancer chemopreventive effects for XN, but there is no information on its metabolism. The aim of this study was to investigate in vitro glucuronidation of XN by rat and human liver microsomes. Using high-performance liquid chromatography, two major glucuronides of XN were found with either rat or human liver microsomes. Release of the aglycone by enzymatic hydrolysis with L L-glucuronidase followed by liquid chromatography/mass spectrometry and nuclear magnetic resonance analysis revealed that these were C-4P P and C-4 monoglucuronides of XN. ß

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Section: Resultssupporting

confidence: 91%

Section: Xn Glucuronidation By Rat or Human Liver Microsomesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes

2001

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“…The libraries were synthesized using a robotic instrument built around a Zymark arm (40). The robot was able to handle 1 g of resin with 2 ϫ 10 6 beads per reactor, therefore ensuring a high ratio of the number of beads to the number of tetrapeptides (41).…”

Section: Peptide Library Synthesismentioning

confidence: 99%

“…The position 3 was composed of ⑀-aminocaproic acid, Ala, Arg, Asn, Asp, ␤-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). The tetrapeptide library was synthesized as a Cterminal amide library.…”

Section: Peptide Library Synthesismentioning

confidence: 99%

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Ferry¹,

Loynel²,

Kucharczyk³

et al. 2000

Journal of Biological Chemistry

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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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Evaluation of high performance liquid chromatography/electrospray mass spectrometry with selected ion monitoring for the analysis of large synthetic combinatorial peptide libraries

Lambert

Bertin

Fauchère

et al. 1997

Rapid Commun. Mass Spectrom.

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Combinatorial Peptide Libraries: Robotic Synthesis and Analysis by Nuclear Magnetic Resonance, Mass Spectrometry, Tandem Mass Spectrometry, and High-Performance Capillary Electrophoresis Techniques

Cited by 67 publications

References 7 publications

In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes

In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Evaluation of high performance liquid chromatography/electrospray mass spectrometry with selected ion monitoring for the analysis of large synthetic combinatorial peptide libraries

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