Dataset Information

Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220.

ABSTRACT: Ro 31-8220 is a potent protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs). Various PKC-independent effects of Ro 31-8220 have however been demonstrated, including inhibition of the ATP-binding cassette drug transporter breast cancer resistance protein. In the present study, we reported that the BIM also blocks activity of the solute carrier organic cation transporter (OCT) 1, involved in uptake of marketed drugs in the liver, in a PKC-independent manner. Ro 31-8220, in contrast to other pan-PKC inhibitors such as staurosporine and chelerythrine, was thus shown to cis-inhibit uptake of the reference OCT1 substrate tetraethylammonium in OCT1-transfected HEK293 cells in a concentration-dependent manner (IC50 = 0.18 ?M) and without altering membrane expression of OCT1. This blockage of OCT1 was also observed in human hepatic HepaRG cells that constitutionally express OCT1. It likely occurred through a mixed mechanism of inhibition. Ro 31-8220 additionally trans-inhibited TEA uptake in OCT1-transfected HEK293 cells, which likely discards a transport of Ro 31-8220 by OCT1. Besides Ro 31-8220, 7 additional BIMs, including the PKC inhibitor LY 333531, inhibited OCT1 activity, whereas 4 other BIMs were without effect. In silico analysis of structure-activity relationships next revealed that various molecular descriptors, especially 3D-WHIM descriptors related to total size, correspond to key physico-chemical parameters for inhibition of OCT1 activity by BIMs. In addition to activity of OCT1, Ro 31-8220 inhibited those of other organic cation transporters such as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K, whereas, by contrast, it stimulated that of OCT2. Taken together, these data extend the nature of cellular off-targets of the BIM Ro 31-8220 to OCT1 and other organic cation transporters, which has likely to be kept in mind when using Ro 31-8220 and other BIMs as PKC inhibitors in experimental or clinical studies.

SUBMITTER: Mayati A

PROVIDER: S-EPMC4675551 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220.

Mayati Abdullah A Bruyere Arnaud A Moreau Amélie A Jouan Elodie E Denizot Claire C Parmentier Yannick Y Fardel Olivier O

PloS one 20151210 12

Ro 31-8220 is a potent protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs). Various PKC-independent effects of Ro 31-8220 have however been demonstrated, including inhibition of the ATP-binding cassette drug transporter breast cancer resistance protein. In the present study, we reported that the BIM also blocks activity of the solute carrier organic cation transporter (OCT) 1, involved in uptake of marketed drugs in the liver, in a PKC-independent mann ...[more]

PMID: 26657401

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Project description:1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.

Project description:The organic cation transporter (OCT) 3 is widely expressed in various organs in humans, and involved in the disposition of many exogenous and endogenous compounds. Several lines of evidence have suggested that OCT3 expressed in the brain plays an important role in the regulation of neurotransmission. Relative to wild-type (WT) animals, Oct3 knockout (KO) mice have displayed altered behavioral and neurochemical responses to psychostimulants such as amphetamine (AMPH) and methamphetamine. In the present study, both in vitro and in vivo approaches were utilized to explore potential mechanisms underlying the disparate neuropharmacological effects observed following AMPH exposure in Oct3 KO mice. In vitro uptake studies conducted in OCT3 transfected cells indicated that dextroamphetamine (d-AMPH) is not a substrate of OCT3. However, OCT3 was determined to be a high-capacity and low-affinity transporter for the neurotransmitters dopamine (DA), norepinephrine (NE), and serotonin (5-HT). Inhibition studies demonstrated that d-AMPH exerts relatively weak inhibitory effects on the OCT3-mediated uptake of DA, NE, 5-HT, and the model OCT3 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide. The IC(50) values were determined to be 41.5 +/- 7.5 and 24.1 +/- 7.0 microM for inhibiting DA and 5-HT uptake, respectively, while 50% inhibition of NE and 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide uptake was not achieved by even the highest concentration of d-AMPH applied (100 microM). Furthermore, the disposition of d-AMPH in various tissues including the brain, liver, heart, kidney, muscle, intestine, spleen, testis, uterus, and plasma were determined in both male and female Oct3 KO and WT mice. No significant difference was observed between either genotypes or sex in all tested organs and tissues. Our findings suggest that OCT3 is not a prominent factor influencing the disposition of d-AMPH. Additionally, based upon the inhibitory potency observed in vitro, d-AMPH is unlikely to inhibit the uptake of monoamines mediated by OCT3 in the brain. Differentiated neuropharmacological effects of AMPHs noted between Oct3 KO and WT mice appear to be due to the absence of Oct3 mediated uptake of neurotransmitters in the KO mice.

Project description:The importance of the organic cation transporter OCT2 in the renal excretion of cationic drugs raises the possibility of drug-drug interactions (DDIs) in which an inhibitor (perpetrator) drug decreases OCT2-dependent renal clearance of a victim (substrate) drug. In fact, there are clinically significant interactions for drugs that are known substrates of OCT2 such as metformin. To identify drugs as inhibitors for OCT2, individual drugs or entire drug libraries have been investigated in vitro by using experimental probe substrates such as 1-methyl-4-phenylpyridinium (MPP+) or 4-4-dimethylaminostyryl-N-methylpyridinium (ASP+). It has been questioned whether the inhibition data obtained with an experimental probe substrate such as MPP+ or ASP+ might be used to predict the inhibition against other, clinical relevant substrates such as metformin. Here we compared the OCT2 inhibition profile data for the substrates metformin, MPP+ and ASP+. We used human embryonic kidney (HEK 293) cells stably overexpressing human OCT2 as the test system to screen 125 frequently prescribed drugs as inhibitors of OCT2-mediated metformin and MPP+ uptake. Data on inhibition of OCT2-mediated ASP+ uptake were obtained from previous literature. A moderate correlation between the inhibition of OCT2-mediated MPP+, ASP+, and metformin uptake was observed (pairwise rs between 0.27 and 0.48, all P < 0.05). Of note, the correlation in the inhibition profile between structurally similar substrates such as MPP+ and ASP+ (Tanimoto similarity T = 0.28) was even lower (rs = 0.27) than the correlation between structurally distinct substrates, such as ASP+ and metformin (T = 0.01; rs = 0.48) or MPP+ and metformin (T = 0.01; rs = 0.40). We identified selective as well as universal OCT2 inhibitors, which inhibited transport by more than 50% of one substrate only or of all substrates, respectively. Our data suggest that the predictive value for drug-drug interactions using experimental substrates rather than the specific victim drug is limited.

Dataset Information

Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220.

Publications

Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220.

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