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The metabolism of L-tryptophan by isolated rat liver cells. Effect of albumin binding and amino acid competition on oxidatin of tryptophan by tryptophan 2,3-dioxygenase.

ABSTRACT: 1. Novel methods, using L-[ring-2-14C]tryptophan, are described for the measurement of tryptophan 2,3-dioxygenase activity and tryptophan accumulation in isolated rat liver cells. 2. The effects of bovine serum albumin, non-esterified fatty acids and neutral amino acids on tryptophan oxidation by hepatocytes and on the partition of tryptophan between free and albumin-bound forms were investigated. 3. Oxidation of physiological concentrations (0.1 mM) of tryptophan was inhibited by approx. 50% in the presence of 2% (w/v) bovine serum albumin; no effects were found at tryptophan concentrations of 0.5 mM and above. 4. Increases in free tryptophan concentrations produced by displacement of 0.1 mM-tryptophan from albumin-binding sites by palmitate resulted in increased flux through tryptophan dioxygenase. 5. Addition of a mixture of neutral amino acids, at plasma concentrations, to hepatocyte incubations had no effect on the rate of tryptophan oxidation. 6. It is concluded that alterations in free tryptophan concentrations consequent to changes in albumin binding may be an important factor in regulating tryptophan uptake and catabolism by the liver. The results are briefly discussed with reference to possible consequences on brain tryptophan metabolism.

SUBMITTER: Smith SA

PROVIDER: S-EPMC1161737 | biostudies-other | 1980 Mar

REPOSITORIES: biostudies-other

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Project description:Kynurenine (Kyn), a metabolite of tryptophan (Trp), is a key regulator of mammal immune responses such as cancer immune tolerance. Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) are main enzymes regulating the first and rate-limiting step of the Kyn pathway. To identify new small molecule inhibitors of TDO, we selected A172 glioblastoma cell line constitutively expressed TDO. Characterization of this cell line using kinase inhibitor library resulted in identification of MEK/ERK pathway-dependent TDO expression. After knowing the properties for TDO expression, we further proceeded to screen chemical library for TDO inhibitors. We previously determined that S-benzylisothiourea derivatives are enzymatic inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and suggested that the isothiourea moiety could be an important pharmacophore for binding to heme. Based on this premise, we screened an in-house library composed of various isothiourea derivatives and identified a bisisothiourea derivative, PVZB3001, as an inhibitor of TDO. Interestingly, PVZB3001 also inhibited the enzymatic activity of IDO1 in both cell-based and cell-free assays but did not inhibit other heme enzymes. Molecular docking studies suggested the importance of isothiourea moieties at the ortho position of the phenyl ring for the inhibition of catalytic activity. PVZB3001 showed competitive inhibition against TDO, and this was supported by the docking simulation. PVZB3001 recovered natural killer (NK) cell viability and functions by inhibiting Kyn accumulation in conditioned medium of both IDO1- and TDO-expressing cells. Furthermore, oral administration of IDO1-overexpressing tumor-bearing mice with PVZB3001 significantly inhibited tumor growth. Thus, we identified a novel selective dual inhibitor of IDO1 and TDO using the Kyn production assay with a glioblastoma cell line. This inhibitor could be a useful pharmacological tool for modulating the Kyn pathway in a variety of experimental systems.

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The metabolism of L-tryptophan by isolated rat liver cells. Effect of albumin binding and amino acid competition on oxidatin of tryptophan by tryptophan 2,3-dioxygenase.

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