Kinetic basis of metformin-MPP interactions with organic cation transporter OCT2

Organic cation transporter 2 (OCT2) clears the blood of cationic drugs. Efforts to understand OCT2 selectivity as a means to predict the potential of new molecular entities (NMEs) to produce unwanted drug-drug interactions typically assess the influence of the NMEs on inhibition of transport. However, the identity of the substrate used to assess transport activity can influence the quantitative profile of inhibition. Metformin and 1-methyl-4-phenylpyridinium (MPP), in particular, display markedly different inhibitory profiles, with IC50 values for inhibition of MPP transport often being more than fivefold greater than IC50 values for the inhibition of metformin transport by the same compound, suggesting that interaction of metformin and MPP with OCT2 cannot be restricted to competition for a single binding site. Here, we determined the kinetic basis for the mutual inhibitory interaction of metformin and MPP with OCT2 expressed in Chinese hamster ovary cells. Although metformin did produce simple competitive inhibition of MPP transport, MPP was a mixed-type inhibitor of metformin transport, decreasing the maximum rate of mediated substrate transport and increasing the apparent Michaelis constant ( Ktapp) for OCT2-mediated metformin transport. Furthermore, whereas the IC50 value for metformin’s inhibition of MPP transport did not differ from the Ktapp value for metformin transport, the IC50 value for MPP’s inhibition of metformin transport was less than its Ktapp value for transport. The simplest model to account for these observations required the influence of a distinct inhibitory site for MPP that, when occupied, decreases the translocation of substrate. These observations underscore the complexity of ligand interaction with OCT2 and argue for use of multiple substrates to obtain the needed kinetic assessment of NME interactions with OCT2.

Download Full-text

Multiple mechanisms of ligand interaction with the human organic cation transporter, OCT2

AJP Renal Physiology ◽

10.1152/ajprenal.00486.2012 ◽

2013 ◽

Vol 304 (1) ◽

pp. F56-F67 ◽

Cited By ~ 34

Author(s):

Jaclyn N. Harper ◽

Stephen H. Wright

Keyword(s):

Organic Cation ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Organic Cation Transporter ◽

Ligand Interaction ◽

Ovary Cells ◽

Tandem Mass Spectroscopy ◽

Mechanistic Basis ◽

Independent Assessment ◽

Structural Classes

OCT2 is the entry step for organic cation (OC) secretion by renal proximal tubules. Although many drugs inhibit OCT2 activity, neither the mechanistic basis of their inhibition nor their transport status is generally known. Using representatives of several structural classes of OCT2-inhibitory ligands described recently (Kido Y, Matsson P, Giacomini KM. J Med Chem 54: 4548–4558, 2011), we determined the kinetic basis of their inhibition of 1-methyl-4-phenylpyridinium (MPP) transport into Chinese hamster ovary cells that stably expressed hOCT2. The “cluster II” inhibitors (which contain known OCT2 substrates) metformin and cimetidine interacted competitively with MPP. However, other cluster II compounds, including tetraethylammonium (TEA), diphenidol and phenyltoloxamine, were mixed-type inhibitors of MPP transport (i.e., decreasing Jmax and increasing Kt). A cluster III (neutral steroid) representative, adrenosterone, and a cluster I (large, flexible cation) representative, carvedilol, displayed noncompetitive inhibitory profiles. Competitive counterflow (CCF) was used to determine whether the inhibitory ligands served as substrates of hOCT2. Carvedilol (cluster I) and adrenosterone (cluster III) did not support CCF, consistent with the prediction that members of these structural classes are likely to be nontransported inhibitors of OCT2. The cluster II representatives MPP, metformin, cimetidine, and TEA all supported CCF, consistent with independent assessments of their OCT2-mediated transport. However, the other cluster II representatives, diphenidol and phenyltoloxamine, failed to support CCF, suggesting that neither compound is transported by OCT2. An independent assessment of diphenidol transport (using liquid chromatography with tandem mass spectroscopy) confirmed this observation. The results underscore the caution required for development of predictive models of ligand interaction with multidrug transporters.

Download Full-text

Functional significance of conserved cysteines in the human organic cation transporter 2

AJP Renal Physiology ◽

10.1152/ajprenal.00038.2012 ◽

2012 ◽

Vol 303 (2) ◽

pp. F313-F320 ◽

Cited By ~ 14

Author(s):

Ryan M. Pelis ◽

Yodying Dangprapai ◽

Yaofeng Cheng ◽

Xiaohong Zhang ◽

Jennifer Terpstra ◽

...

Keyword(s):

Organic Cation ◽

Chinese Hamster Ovary Cells ◽

Transmembrane Helix ◽

Chinese Hamster ◽

Organic Cation Transporter ◽

Disulfide Bridge ◽

Cell Surface Expression ◽

Extracellular Loop ◽

Intact Cells ◽

Organic Cation Transporter 2

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants ( Kt) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO2-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the Kt value for MPP. In contrast, the Kt value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.

Download Full-text

The effect of levocetirizine and dextrocetirizine on the transport of tetraethylammonium in chinese hamster ovary cells transfected with the human organic cation transporter OCT2

World Allergy Organization Journal ◽

10.1097/01.wox.0000301359.87170.3d ◽

2007 ◽

Vol &NA; ◽

pp. S82

Author(s):

Rhys Whomsley ◽

Margherita Strolin Benedetti ◽

Theresa Wunz ◽

Stephen Wright

Keyword(s):

Chinese Hamster Ovary ◽

Organic Cation ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Organic Cation Transporter ◽

Ovary Cells

Download Full-text

Role of MAPK and PKA in regulation of rbOCT2-mediated renal organic cation transport

AJP Renal Physiology ◽

10.1152/ajprenal.00043.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. F21-F27 ◽

Cited By ~ 13

Author(s):

Sunhapas Soodvilai ◽

Atip Chatsudthipong ◽

Varanuj Chatsudthipong

Keyword(s):

Organic Cation ◽

Chinese Hamster ◽

Organic Cation Transporter ◽

Inhibitory Effect ◽

Cell System ◽

Proximal Tubules ◽

Common Pathway ◽

Renal Proximal Tubules ◽

Organic Cation Transporter 2 ◽

Heterologous Cell

The effects of protein kinases MAPK and PKA on the regulation of organic cation transporter 2 (OCT2) were investigated both in a heterologous cell system [Chinese hamster ovary (CHO-K1) cells stably transfected with rabbit (rb)OCT2] and in native intact rabbit renal proximal S2 segments. Inhibition of MEK (by U-0126) or PKA (by H-89) reduced transport activity of rbOCT2 in CHO-K1 cells. The inhibitory effect of U-0126 combined with H-89 produced no additive effect, indicating that the action of PKA and MAPK in the regulation of rbOCT2 is in a common pathway. Activation of PKA by forskolin stimulated rbOCT2 activity, and this stimulatory effect was eliminated by H-89, indicating that the stimulation required PKA activation. In S2 segments of rabbit renal proximal tubules, activation of MAPK (by EGF) and PKA (by forskolin) stimulated activity of rbOCT2, and this activation was abolished by U-0126 and H-89, respectively. This is the first study to show that MAPK and PKA are involved, apparently in a common pathway, in the regulation of OCT2 activity in both a heterologous cell system and intact renal proximal tubules.

Download Full-text

Interaction of H+ with the extracellular and intracellular aspects of hMATE1

AJP Renal Physiology ◽

10.1152/ajprenal.00075.2011 ◽

2011 ◽

Vol 301 (3) ◽

pp. F520-F528 ◽

Cited By ~ 18

Author(s):

Yodying Dangprapai ◽

Stephen H. Wright

Keyword(s):

Time Course ◽

Organic Cation ◽

Physiological Function ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Ovary Cells ◽

Molecular Identity ◽

Luminal Membrane ◽

Ph Conditions ◽

Kinetics Of

Human multidrug and toxin extrusion 1 (hMATE1, SLC47A1) is a major candidate for being the molecular identity of organic cation/proton (OC/H+) exchange activity in the luminal membrane of renal proximal tubules. Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake, i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction, i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, we measured the time course of hMATE1-mediated efflux of the prototypic MATE1 substrate, [3H]1-methyl-4-phenylpyridinium, under a variety of intra- and extracellular pH conditions, from Chinese hamster ovary cells that stably expressed the transporter. In this study, we showed that an IC50/ Ki for interaction between extracellular H+ and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] was not significantly different ( P = 0.6). Furthermore, kinetics of interaction between intracellular H+ and inward-facing hMATE1 determined using the efflux protocol revealed an IC50 for H+ of 11.5 nM (pH 7.91), consistent with symmetrical interactions of H+ with the inward-facing and outward-facing aspects of hMATE1.

Download Full-text

Fluorescent organic cations for human OCT2 transporters screening: uptake in CHO cells stably expressing hOCT2

ADMET & DMPK ◽

10.5599/admet.5.2.389 ◽

2017 ◽

Vol 5 (2) ◽

pp. 135 ◽

Cited By ~ 3

Author(s):

Malachy C. Ugwu ◽

Ryan Pelis ◽

Charles O. Esimone ◽

Remigius U. Agu

Keyword(s):

Cho Cells ◽

Organic Cation ◽

Rhodamine 6G ◽

Chinese Hamster ◽

Organic Cation Transporter ◽

Rhodamine 123 ◽

Intracellular Uptake ◽

Wild Type ◽

Transfected Cells ◽

Organic Cation Transporter 2

The aim of this study was to assess the suitability of amiloride, rhodamine 6G and rhodamine 123 as non-radioactive substrates for characterizing hOCT2 using CHO cells. The uptake characteristics of these compounds were compared in wild-type (WT) and human organic cation transporter 2 (hOCT2)-stably transfected Chinese Hamster Ovary (CHO) cells. All the compounds were accumulated by the CHO-hOCT2 cells. Intracellular uptake of the compounds was higher in CHO cells stably-expressing hOCT2 compared to the WT. The uptake was concentration–dependent and saturable (except for rhodamine 123). The affinities of the compounds for the hOCT2 (in descending order) were: amiloride (Km = 72.63 ± 12.02 µM) > rhodamine 6 G (Km = 82.47 ± 29.15 µM). Uptake of amiloride in transfected cells was pH -dependent and significantly inhibited by hOCT2 inhibitors (quinine, verapamil and quinidine). Based on our kinetic data and other considerations, we recommend the use of amiloride for characterizing hOCT2 transporters.

Download Full-text

Competitive Inhibition of the Luminal Efflux by Multidrug and Toxin Extrusions, but Not Basolateral Uptake by Organic Cation Transporter 2, Is the Likely Mechanism Underlying the Pharmacokinetic Drug-Drug Interactions Caused by Cimetidine in the Kidney

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.111.184986 ◽

2011 ◽

Vol 340 (2) ◽

pp. 393-403 ◽

Cited By ~ 127

Author(s):

Sumito Ito ◽

Hiroyuki Kusuhara ◽

Miyu Yokochi ◽

Junko Toyoshima ◽

Katsuhisa Inoue ◽

...

Keyword(s):

Drug Interactions ◽

Organic Cation ◽

Competitive Inhibition ◽

Organic Cation Transporter ◽

Organic Cation Transporter 2 ◽

Pharmacokinetic Drug

Download Full-text

Molecular and cellular physiology of organic cation transporter 2

AJP Renal Physiology ◽

10.1152/ajprenal.00422.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. F1669-F1679 ◽

Cited By ~ 1

Author(s):

Stephen H. Wright

Keyword(s):

Organic Cation ◽

Critical Role ◽

Organic Cation Transporter ◽

Initial Step ◽

Organic Cations ◽

Molecular Characteristics ◽

Ligand Interaction ◽

Organic Cation Transporter 2 ◽

Influence Of Substrate ◽

Inhibitory Potential

Organic cation transporters play a critical role in mediating the distribution of cationic pharmaceuticals. Indeed, organic cation transporter (OCT)2 is the initial step in the renal secretion of organic cations and consequently plays a defining role in establishing the pharmacokinetics of many cationic drugs. Although a hallmark of OCTs is their broad selectivity, this characteristic also makes them targets for unwanted, adverse drug-drug interactions (DDIs), making them a focus for efforts to develop models of ligand interaction that could predict and preempt these adverse interactions. This review discusses the molecular characteristics of these transporters as well as the evidence that established the OCTs as key players in the distribution of organic cations. However, the primary focus is the present understanding of the complexity of ligand interaction with OCTs, particularly OCT2, including evidence for the presence of multiple ligand-binding sites and the influence of substrate structure on the affinity of the transporter for inhibitory ligands. This leads to a discussion of the complexities associated with the development of protocols for assessing the inhibitory potential of new molecular entities to perpetrate unwanted DDIs, the criteria that should be considered in the interpretation of the results of such protocols, and the challenges associated with development of models capable of predicting unwanted DDIs.

Download Full-text

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649597 ◽

1993 ◽

Vol 70 (03) ◽

pp. 418-422 ◽

Cited By ~ 23

Author(s):

Masaharu Aritomi ◽

Naoko Watanabe ◽

Rika Ohishi ◽

Komakazu Gomi ◽

Takao Kiyota ◽

...

Keyword(s):

Protein C ◽

Antithrombin Iii ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Time Lag ◽

Chinese Hamster ◽

Ovary Cells ◽

Soluble Thrombomodulin ◽

Simulation Based ◽

Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Download Full-text