Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/07/14/mol.116.105056.DC2 1521-0111/90/3/254–264$25.00 http://dx.doi.org/10.1124/mol.116.105056 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:254–264, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Lack of Influence of Substrate on Ligand Interaction with the Human Multidrug and Toxin Extruder, MATE1 s Lucy J. Martínez-Guerrero, Mark Morales, Sean Ekins, and Stephen H. Wright Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona (L.J.M.-G., M.M., S.H.W.); and Collaborations in Chemistry, Fuquay-Varina, North Carolina (S.E.) Received May 13, 2016; accepted June 30, 2016 ABSTRACT Downloaded from Multidrug and toxin extruder (MATE) 1 plays a central role in Transport was measured in Chinese hamster ovary cells that mediating renal secretion of organic cations, a structurally stably expressed the human ortholog of MATE1. Comparison of diverse collection of compounds that includes ∼40% of pre- the resulting inhibition profiles revealed no systematic influence of scribed drugs. Because inhibition of transport activity of other substrate structure on inhibitory efficacy. Similarly, IC50 values for multidrug transporters, including the organic cation transporter 26 structurally diverse compounds revealed no significant influence (OCT) 2, is influenced by the structure of the transported of substrate structure on the kinetic interaction of inhibitor with molpharm.aspetjournals.org substrate, the present study screened over 400 drugs as MATE1. The IC50 data were used to generate three-dimensional inhibitors of the MATE1-mediated transport of four structur- quantitative pharmacophores that identified hydrophobic regions, ally distinct organic cation substrates: the commonly used H-bond acceptor sites, and an ionizable (cationic) feature as key drugs: 1) metformin and 2) cimetidine; and two prototypic determinants for ligand binding to MATE1. In summary, in contrast cationic substrates, 3) 1-methyl-4-phenylpyridinium (MPP), to the behavior observed with some other multidrug transporters, and 4) the novel fluorescent probe, N,N,N-trimethyl-2-[methyl(7- including OCT2, the results suggest that substrate identity exerts nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium iodide. comparatively little influence on ligand interaction with MATE1. at ASPET Journals on October 1, 2021 Introduction by organic cation transporter (OCT) 2 (Motohashi et al., 2002, 2013). The second step in this process involves exit of the OC The kidney, particularly the proximal tubule, plays the into the tubular filtrate across the apical or luminal mem- principal role in clearing organic cations (OCs) from the body, brane of renal proximal tubule cells by a process that uses i.e., molecules that carry a net positive charge at physiologic pH electroneutral OC/H1 exchange. In humans the luminal step (Hagenbuch, 2010). These OCs include approximately 40% of is dominated by the multidrug and toxin extruders (MATEs), all prescribed and over-the-counter drugs (including cimeti- MATE1 and MATE2/2-K (Motohashi et al., 2013). The pres- dine, procainamide, pindolol, and metformin) (Neuhoff et al., ence within the kidney of this common pathway for the 2003; Ahlin et al., 2008). Thus, renal OC secretion is a critical secretion of OCs sets the stage for unwanted drug-drug in- element in the chain of processes defining the pharmacoki- teractions (DDIs) (Lepist and Ray, 2012). The clinical cost netics of almost half of the drugs to which people are exposed. of DDIs is substantial and responsible for approximately 1% The secretion of OCs by the kidney is the consequence of two of hospital admissions (almost 5% in elderly populations) sequential transport processes in the renal proximal tubule. [Becker et al., 2007; U.S. Food and Drug Administration, The first of these is entry of the OC from the blood into a renal Preventable adverse drug reactions: A focus on drug interac- proximal tubule cell across the basolateral membrane by a tions (http://www.fda.gov/Drugs/DevelopmentApprovalProcess/ process that involves electrogenic-facilitated diffusion. In DevelopmentResources/DrugInteractionsLabeling/ucm110632. humans the basolateral element of OC secretion is dominated htm)]; therefore, the ability to predict potential DDIs could lead to decreased morbidity and increased monetary savings. This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant 1R01DK080801]; MATE-mediated OC efflux is both the active and rate- the National Institutes of Health National Institute of Environmental Health limiting element of the secretory process (Schäli et al., 1983; Sciences [Grant 5P30ES006694]; and the National Institutes of Health National Pelis and Wright, 2011) and has been implicated in several Heart, Lung, and Blood Institute [Grant 5T32HL07249]. Portions of this work were a part of a dissertation that was submitted by clinically relevant DDIs (Ito et al., 2012; Lepist and Ray, L.J.M.-G. to the University of Arizona in accordance with academic requirements. 2012). To date, a primary focus of studies of MATE function dx.doi.org/10.1124/mol.116.105056. has been establishing the interaction of MATE transporters s This article has supplemental material available at molpharm. aspetjournals.org. (typically, MATE1) with specific structural classes of drugs ABBREVIATIONS: ASP, 4-(4-dimethylamino)styryl)-N-methylpyridinium; CHO, Chinese hamster ovary; DDI, drug-drug interaction; FCFP6, function class fingerprints of maximum diameter 6; hMATE, human multidrug and toxin extruder; MATE, multidrug and toxin extruder; MPP, 1- methyl-4-phenylpyridinium; NBD-MTMA, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium iodide; NCC, Na- tional Institutes of Health Clinical Collection; OC, organic cation; OCT, organic cation transporter; S.A., specific activity; 3D, three-dimensional. 254 Substrate Independence of MATE1 Inhibition 255 (e.g., Yonezawa et al., 2006; Nies et al., 2012; Lee et al., 2014). were used for transport studies. Subculture of the cells was performed The increasing attention given to the clinical impact of every 3 to 4 days. unwanted DDIs, and the growing acceptance of the critical Uptake Experiments with Cultured Cells. CHO cells express- role played by MATE1 in renal OC secretion, has led to the ing hMATE1 or wild-type control cells were plated in 96-well cell development of several predictive models of ligand interaction culture plates (Greiner/VWR International; Arlington Heights, IL) at densities sufficient for the cells to reach confluence within 24 hours with human MATE1 (hMATE1) (Astorga et al., 2012; Wittwer (50,000 cells per well). For experiments of MATE1 transport activity et al., 2013; Xu et al., 2015), each based on assessing profiles of the cells (MATE1-expressing and control cells) were typically pre- ligand inhibition of MATE1 transport activity. However, little incubated for 20 minutes (room temperature) in buffer containing 1 attention has been given to a critical issue relevant to un- 20 mM NH4Cl (the first step in establishing an outwardly directed H derstanding the influence of MATE1 on unwanted DDI: the gradient) (Roos and Boron, 1981). Plates were then placed in an potential impact of substrate identity on the profile of drug automatic fluid aspirator/dispenser (Model 406, BioTek, Winooski, interaction with MATE1. Increasing evidence suggests that VT) and automatically rinsed/aspirated three times with room tem- the effectiveness of cationic drugs as inhibitors of multi- perature Waymouth’s Buffer (pH 7.4), and transport was initiated by aspirating this medium and replacing it with 60 mlofaNH4Cl-free drug transporters can be significantly influenced by the 1 substrate used to monitor transport activity (Belzer et al., medium (thereby rapidly establishing an outwardly directed H gradient) containing labeled substrate. Following the experimental 2013; Thévenod et al., 2013; Hacker et al., 2015), which may ∼ – incubation, the transport reaction was stopped by the rapid ( 2 complicate the interpretation of decision tree based assays for seconds) addition (and simultaneous aspiration) of 0.75 ml cold (4°C) Downloaded from assessing potential DDIs (Giacomini et al., 2010; Hillgren Waymouth’s Buffer. Following aspiration of the cold stop, 200 mlof et al., 2013). However, the extent to which MATE transporters scintillation cocktail (Microscint 20; Perkin-Elmer, Waltham, MA) display such behavior is not clear. was added to each well and the plates were sealed (Topseal-A, Perkin- In the current study, we screened over 400 drugs as inhibitors Elmer) and allowed to sit for at least 2 hours before radioactivity was of the MATE1-mediated transport of four structurally distinct assessed in a 12-channel, multiwell scintillation counter (Wallac OC substrates: the commonly used drugs 1) metformin and 2) Trilux 1450 Microbeta, Perkin-Elmer). Substrate uptake was typically molpharm.aspetjournals.org cimetidine; and two prototypic cationic substrates, 3) 1-methyl- normalized to nominal surface area of confluent cells. For the purpose 4-phenylpyridinium (MPP), and 4) the novel fluorescent probe, of comparison with rates reported in studies that normalize transport to cell protein, we find the factor of 0.035 mg cell protein×cm22 to be N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-