Metabolism of 5-hydroxytryptamine by brain synaptosomes and microsomes in the presence of cysteine and glutathione

Singh, Satendra; Dryhurst, Glenn

doi:10.1021/jm00092a018

Cited by 10 publications

(10 citation statements)

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“…These results suggest that this combination of a free amine and a free thiol group is necessary for the reaction with 5-HIAL to occur. In contrast to our findings, Singh and Dryhurst (13) showed that 5-HITCA analogs formed with glutathione, and that little or no condensation product was observed unless the homogenate was incubated with both 5-HT and L-Cys or glutathione. Although the presence of L-Cys did increase the amount of 5-HITCA formed, upon 5-HT incubation without additional L-Cys-containing compounds, the metabolite formed at levels equivalent to 5-HIAA in central nervous system tissues, as well as in differentiated rat PC-12 cells.…”

Section: Discussioncontrasting

confidence: 99%

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Serotonin Catabolism and the Formation and Fate of 5-Hydroxyindole Thiazolidine Carboxylic Acid

Squires

Jakubowski

Stuart

et al. 2006

Journal of Biological Chemistry

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Significant research efforts have focused on advancing our understanding of serotonin (5-HT) 2 function and its mechanism of release, uptake, and metabolism. The majority of this research has involved the central nervous system, where imbalances in 5-HT levels have been linked to various diseases, including Parkinson, Huntington, Alzheimer, Alzheimer-like dementia, anxiety, and depression (1), and its regulation depends on a multitude of 5-HT receptors and neurochemical pathways (2). Other 5-HT functions within the brain involve learning and memory (3, 4) and regulation of various stages of development (5). However, neither 5-HT nor its effects are limited to the central nervous system; 5-HT is found in most smooth muscles in the body and is responsible for the induction of the contractile responses of the gastrointestinal, pulmonary, and genito-urinary systems (6). Specifically, researchers estimate that 95% of the approximate 10 mg of 5-HT in the human body is produced in the enteric nervous system, which includes both the peripheral nervous system of the gastrointestinal tract, as well as the 5-HT-secreting enterochromaffin cells of the gut lining (7). In the enteric nervous system, 5-HT fulfills all criteria necessary for classification as a neurotransmitter (8). Imbalances in 5-HT levels within the enteric nervous system have been observed in association with various disorders, including irritable bowel syndrome, functional dyspepsia, non-cardiac chest pain, and gastric ulcer formation (6, 9). Our focus is to gain insight into the pathways by which 5-HT is catabolized and the compounds into which it is converted. Because of its high biological potency, tight regulation of 5-HT levels in specific nervous system regions is necessary, and 5-HT catabolism plays an important role in this regulation. Because 5-HT conversion into these other compounds affects the overall levels of 5-HT, formation of these conversion products can be a fundamental factor in 5-HT regulation.Scheme 1 represents the main 5-HT metabolic pathways and enzymes; however, these represent only a subset of the pathways of 5-HT metabolism, as other lower abundance serotonin metabolites, such as 5-HT sulfate, are known. In immune response pathways, compounds such as formyl 5-HT and 5-hydroxykynuremine (10) are additional serotonin metabolic products. Research performed in the 1950s to track the metabolism of radiolabeled tryptophan demonstrated a number of unknown 5-HT metabolites, proposed to result from additional branches of the monoamine oxidase (MAO) pathway (11). MAO exists in two forms, MAOa and MAOb (12); the former is the primary form of the enzyme responsible for the conversion of 5-HT, although, in the absence of MAOa, MAOb takes over the 5-HT conversion process. With a more in-depth understanding of this, and other potential tissue-specific 5-HT catabolic pathways, it may be possible to develop methods for controlling serotonergic levels in a tissue-specific manner.In this study, we identify unique 5-HT metabolites by analyzing 5-HTp...

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Section: Discussioncontrasting

confidence: 99%

“…This data further confirmed the structure of 5-HITCA, which was reported to form from 5-HIAL and L-Cys in aqueous media and in tissue incubated with 5-HT and L-Cys (13)(14)(15).…”

Section: Discussionsupporting

confidence: 87%

Serotonin Catabolism and the Formation and Fate of 5-Hydroxyindole Thiazolidine Carboxylic Acid

Squires

Jakubowski

Stuart

et al. 2006

Journal of Biological Chemistry

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“…16 Route B 5 describes the nucleophilic addition of the Cys-thiol group to the aldehydic carbon generating the substituted mercaptomethanol intermediate and subsequently, following elimination of water, a carbosulfonium intermediate. 5 Here, ring contraction occurs via nucleophilic attack of the Cys-amino group on the carbosulfonium intermediate to form the thiazolidine−glycine-adduct. 5,15−17 While recent literature data highlighted examples of desired reversible binding, such as saxagliptin (Onglyza) where prolonged binding via the cyanide functional group 19 to the target peptidase inhibitor balanced maximizing pharmacodynamic effects with minimizing drug levels, 20,21 the formation of the reactive aldehyde intermediate as described in this article was an entirely unexpected and undesired effect.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Reactive metabolite trapping screens with liver microsomes (usually human) fortified with nucleophiles such as GSH, cysteine, potassium cyanide, and methoxylamine are routinely employed to trap reactive electrophilic species at sufficient concentration to form stable, recognizable products/adducts which can be identified by their expected MS changes. However, unexpected or structurally unusual products from nonstandard biotransformations such as structural rearrangements − could escape detection during the MSMS investigations due to changes in molecular structure, molecular weight, and the lack of expected fragmentation patterns. Of particular concern are cyclized GSH adducts − which can produce a false negative result in conventional LC-MSMS GSH trapping screens, as GSH adducts are generally identified by their expected target mass, a neutral loss of 129 Da in positive ion mode, or/and a m / z of 273 in a precursor ion scan in negative ion mode.…”

Section: Introductionmentioning

confidence: 99%

Reactive Metabolite Trapping Screens and Potential Pitfalls: Bioactivation of a Homomorpholine and Formation of an Unstable Thiazolidine Adduct

Lenz

Martin

Schmidt

et al. 2014

Chem. Res. Toxicol.

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Successful early attrition of potential problematic compounds is of great importance in the pharmaceutical industry. The lead compound in a recent project targeting neuropathic pain was susceptible to metabolic bioactivation, which produced reactive metabolites and showed covalent binding to protein. Therefore, as a part of the backup series for this compound several structural modifications were explored to mediate the reactive metabolite and covalent binding risk. A homomorpholine containing series of compounds was identified without compromising potency. However, when these compounds were incubated with human liver microsomes in the presence of GSH, Cys-Gly adducts were identified, instead of intact GSH conjugates. This article examines the formation of the Cys-Gly adduct with AZX ([M+H]+ 486) as a representative compound for this series. The AZX-Cys-Gly-adduct ([M+H]+ 662) showed evidence of ring contraction by formation of a thiazolidine-glycine and was additionally shown to be unstable. During its isolation for structural characterization by 1H NMR spectroscopy, it was found to have decomposed to a product with [M+H]+ 446. The characterization and identification of this labile GSH-derived adduct using LC-MS/MS and 1H NMR are described, along with observations around stability. In addition, various structurally related trapping reagents were employed in an attempt to further investigate the reaction mechanism along with a methoxylamine trapping experiment to confirm the structure of the postulated reactive intermediate.

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“…A minor pathway involves the reduction of 25 to the primary alcohol 5-hydroxytryptophol ( 27 ) by aldehyde reductase. Singh and Dryhurst demonstrated that incubation of 24 with NADPH- and l -cysteine (or GSH)-supplemented pig or bovine brain microsomes or with synaptosomes resulted in the formation of the (2 R ,4 R )- and (2 S ,4 R )-epimers of 2-[(5-hydroxy-1 H -indol-3-yl)methyl]thiazolidine (compounds 28 and 29 , respectively) or α-amino-4-[[(carboxymethyl)amino]carbonyl]-2-[(5-hydroxy-1 H -indol-3-yl)methyl-δ-oxo-3-thiazolidinepentanoic acid (compounds 30 and 31 , respectively) from a reaction of cysteine or GSH with the aldehyde 25 . While the rates of formation of thiazolidine adducts in brain microsomes or synaptosomes were relatively faster with l -cysteine than with GSH, it is interesting to note that the γ-glutamate residue was retained in the GSH portion of the adducts 30 and 31 .…”

Section: Literature Examples Pertaining To Cyclized Cysteinyl Adductsmentioning

confidence: 99%

Liabilities Associated with the Formation of “Hard” Electrophiles in Reactive Metabolite Trapping Screens

Kalgutkar

2016

Chem. Res. Toxicol.

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Soft electrophiles (e.g., epoxides, quinones, quinone-imines, quinone-methides, etc.) generated via the oxidative bioactivation of phenyl, phenolic, amino-, and alkylphenolic substituents can be trapped with nucleophiles of comparable softness (e.g., glutathione or cysteine) in reactive metabolite screens. In contrast, hard nucleophiles such as cyanide and amines are frequently utilized to trap hard electrophiles (e.g., iminiums and aldehydes) that result from the oxidative bioactivation of cyclic (or acylic) amines and primary alcohols. In some instances, soft sulfydryl nucleophiles have also been utilized to trap aldehydes to yield cyclized thiazolidine adducts. Case studies where hard electrophiles are thought to be responsible for cytochrome P450 inactivation, genotoxicity, and/or target organ toxicity in animals have been presented. The association of hard electrophiles with immune-mediated idiosyncratic adverse drug reactions is less clear given the paucity of available examples and the fact that several marketed drugs containing cyclic amine motifs can generate hard electrophiles via α-carbon ring oxidation. This perspective examines available data associating toxicity with the formation of hard electrophilic intermediates from small molecule drugs/drug candidates. Pragmatic risk mitigation strategies around unwarranted idiosyncratic toxicity risks with drug candidates that generate hard electrophiles are also discussed against the backdrop of marketed agents that possess analogous cyclic amine framework.

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Metabolism of 5-hydroxytryptamine by brain synaptosomes and microsomes in the presence of cysteine and glutathione

Cited by 10 publications

References 1 publication

Serotonin Catabolism and the Formation and Fate of 5-Hydroxyindole Thiazolidine Carboxylic Acid

Serotonin Catabolism and the Formation and Fate of 5-Hydroxyindole Thiazolidine Carboxylic Acid

Reactive Metabolite Trapping Screens and Potential Pitfalls: Bioactivation of a Homomorpholine and Formation of an Unstable Thiazolidine Adduct

Liabilities Associated with the Formation of “Hard” Electrophiles in Reactive Metabolite Trapping Screens

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