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Noncompetitive inhibition of human CYP2C9 in vitro by a commercial Rhodiola rosea product.

ABSTRACT: A commercial Rhodiola rosea (R. rosea) product has previously demonstrated CYP2C9 inhibition in humans. The purpose of this study was to provide in vitro inhibitory data for this particular interaction and to classify the mechanism of the interaction. Another aim was to examine the in vitro influence of ethanol on the CYP2C9 activity. Human CYP2C9 (wild type) isolated from a baculovirus-infected cell system was incubated with 0.8 ?mol/L losartan for 20 min. Sulfaphenazole was used as a positive control. The commercial R. rosea product "Arctic Root" was used as test inhibitor. Formation of the CYP2C9-produced losartan metabolite EXP-3174 was determined by validated LC-MS/MS methodology. Possible mechanism-based (irreversible) inhibition was evaluated using time- and NADPH-dependent inhibition assays. Kinetic constants (Km , Vmax , and Ki ) were calculated from a Lineweaver-Burk plot. Mode of inhibition was determined. CYP2C9 was inhibited by "Arctic Root" with an IC50 (extract concentration yielding 50% reduction in enzyme activity) of 19.2 ± 2.7 ?g/mL. Inhibitor concentrations of 20 ?g/mL and 40 ?g/mL yielded Ki values of 16.37 ?g/mL and 5.59 ?g/mL, respectively. The Lineweaver-Burk plot showed noncompetitive inhibition mode. No time- or NADPH-dependent inhibition was observed. The presence of ethanol inhibited CYP2C9 activity in a concentration-dependent manner. In conclusion, the commercial R. rosea product "Arctic Root" demonstrated noncompetitive inhibition of CYP2C9 in vitro. Further work identifying the constituents responsible for this inhibition is needed.

SUBMITTER: Thu OKF

PROVIDER: S-EPMC5684854 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Noncompetitive inhibition of human CYP2C9 in vitro by a commercial Rhodiola rosea product.

Thu Ole Kristian Forstrønen OKF Spigset Olav O Hellum Bent B

Pharmacology research & perspectives 20170801 4

A commercial Rhodiola rosea (R. rosea) product has previously demonstrated CYP2C9 inhibition in humans. The purpose of this study was to provide in vitro inhibitory data for this particular interaction and to classify the mechanism of the interaction. Another aim was to examine the in vitro influence of ethanol on the CYP2C9 activity. Human CYP2C9 (wild type) isolated from a baculovirus-infected cell system was incubated with 0.8 μmol/L losartan for 20 min. Sulfaphenazole was used as a positive ...[more]

PMID: 28805981

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Project description:Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 ?M. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

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Noncompetitive inhibition of human CYP2C9 in vitro by a commercial Rhodiola rosea product.

Publications

Noncompetitive inhibition of human CYP2C9 in vitro by a commercial Rhodiola rosea product.

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