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 μM) 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.
Highlights
Ro 31–8220 is a potent pan-protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs), that contains 11 chemicals, numbered from BIM-I to BIM-XI, initially characterized for their putative interaction with PKCs [1]
OCT1 activity in HEK-OCT1 cells was inhibited by pre-treatment with the calcium/calmodulin-dependent protein kinase (CaMK) inhibitor KN62 and by the Src kinase inhibitor PP2, used here as positive controls of OCT1 modulation because the calcium/CaMK pathway as well as the Src kinase Lck have been already shown to be involved in OCT1 activity regulation [40]
Various off-targets have already been described for the potent PKC inhibitor Ro 31–8220, including blockage of the ATP-binding cassette (ABC) transporter BCRP [25]
Summary
Ro 31–8220 is a potent pan-protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs), that contains 11 chemicals, numbered from BIM-I to BIM-XI, initially characterized for their putative interaction with PKCs [1]. Ro 31–8220 ( known as BIM-IX) inhibits PKC activity in various types of cells, including platelets and T lymphocytes [2], which is consistent with the fact that this lipophilic chemical is a cell-permeable compound, that most likely enters cells through passive diffusion as well-established for hydrophobic chemicals [3]. Ro 31–8220 notably inhibits mitogen-activated protein kinase (MAPK) phosphatase-1 [13], RSK1, RSK2 and RSK3 isoforms of the p90 ribosomal S6 kinase [14], p70 ribosomal S6 kinase [15, 16], CDC2 histone H1 kinase [17] and glycogen synthase kinase-3 [18] It activates phosphoinositide phospholipase C and c-Jun N-terminal kinase, induces apoptosis in tumoral cells and blocks voltagedependent sodium channels in a PKC-independent manner [19,20,21,22]
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