Why we should be vigilant: Drug cytotoxicity observed with in vitro transporter inhibition studies

doi:10.1016/j.bcp.2010.06.012

Biochemical Pharmacology

Volume 80, Issue 7, 1 October 2010, Pages 1087-1092

https://doi.org/10.1016/j.bcp.2010.06.012 Get rights and content

Abstract

From routine in vitro drug-transporter inhibition assays, observed inhibition is typically assumed from direct interaction with the transporter. Other mechanisms that possibly reduce substrate uptake are not frequently fully examined. The objective of this study was to investigate the association of transporter inhibition with drug cytotoxicity. From a pool of drugs that were identified as known ASBT or OCTN2 inhibitors, 21 drugs were selected to screen inhibitory potency of their prototypical substrate and cytotoxicity against three human sodium-dependent solute carrier (SLC) transporters: apical sodium-dependent bile acid transporter (ASBT), organic cation/carnitine transporter (OCTN2), and the excitatory amino acid transporter 4 (EAAT4) in stable cell lines. Twenty drugs showed apparent inhibition in OCTN2–MDCK and ASBT–MDCK. Four dihydropyridine calcium channel blockers were cytotoxic to MDCK cells, and the observed cytotoxicity of three of them accounted for their apparent OCTN2 inhibition, and consequently were classified as non-OCTN2 inhibitors. Meanwhile, since their cytotoxicity only moderately contributed to ASBT inhibition, these three were still considered ASBT inhibitors. Four other drugs showed apparent inhibition in EAAT4–HEK cells, and cytotoxicity of three drugs corresponded with their inhibition of this transporter. Therefore, cytotoxicity significantly affected EAAT4 observations. Results showed the potential of cytotoxicity as a mechanism that can account for apparent in vitro transporter inhibition. Drug cytotoxicity varied in different cell lines, which could increase false positives for pharmacophore development.

Graphical abstract

Introduction

Computational screening is a popular approach to rapidly identify potential inhibitors of transporters [1], where “hits” are then experimentally validated via cell-based inhibition assays. In addition to specific drug binding to the protein, other possible modes of drug inhibition of a transporter can be regarded as false positives but are not frequently considered. Such modes of inhibition include cell toxicity, modulation of the energy source for active transport, and non-specific interaction of a drug with the cell membrane. Chemical modification of protein thiol groups [2], [3] and interference in binding interactions by compound aggregates have recently been identified as non-specific mechanisms for compound candidates to inhibit enzymes [4], [5].

The objective of this study was to evaluate cytotoxicity as a mechanism for apparent transporter inhibition. Twenty-one drugs, along with the Na⁺/K⁺ pump inhibitor ouabain, were evaluated against three solute carrier proteins: human apical sodium-dependent bile acid transporter (ASBT; SLC10A2), human organic cation/carnitine transporter (OCTN2; SLC22A5), and the excitatory amino acid transporter 4 (EAAT4; SLC1A6). Previously, using a combined in vitro and pharmacophore-based approach, several drugs were found to be inhibitors of ASBT or the OCTN2 [6], [7]. Eleven ASBT inhibitors and ten OCTN2 inhibitors were selected to screen against these three transporters. Ouabain was used to evaluate the effect of sodium gradient modulation [8]. Drug cytotoxicity was also evaluated.

ASBT, OCTN2, and EAAT4 were selected since they are all sodium-dependent, active influx transporters, and recognize dissimilar substrates with little known overlap for inhibitors. The transporters are energized by co-transporting sodium ion down the membrane sodium gradient. ASBT is responsible for the intestinal recovery of bile acids recovery [9]. Substrate translocation is coupled with sodium in a 2:1 sodium: bile acid stoichiometry [10]. OCTN2 mediates the reabsorption of organic cations in the kidney, particularly carnitine [11]. OCTN2-mediated translocation can be either sodium-dependent or sodium-independent, although translocation of l-carnitine is sodium-dependent [12]. EAAT4 is enriched in the Purkinje cells of the cerebellum and is a subtype of the five known human glutamate transporters in neurons [13]. It precisely regulates extracellular glutamate concentrations to maintain critical signaling yet avoiding excitotoxicity by uptake of glutamate from the synaptic cleft [14]. Both OCTN2 and ASBT have hydrophobes as features in common, while ASBT prefers a negative charge and OCTN2 requires a positive ionisable feature [6], [7], [15]. A general EAAT pharmacophore includes two acidic functional groups and a protonatable nitrogen [16].

Results from drug screening of three sodium-depended transporters in this study indicate that for some drugs, their cytotoxicity contributed to apparent transporter inhibition, and we identified cytotoxicity differences between MDCK and HEK cells.

Section snippets

Materials

[³H]-Taurocholic acid and [³H]-glutamic acid were purchased from Perkin Elmer (Waltham, MA). [³H]-l-carnitine was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Taurocholate and glutamic acid were obtained from Sigma–Aldrich (St. Louis, MO). l-Carnitine was purchased from Spectrum Pharmacy Products (Tucson, AZ). Fetal bovine serum (FBS), trypsin, and Dulbecco's modified Eagle's medium (DMEM) were procured from Invitrogen Corporation (Carlsbad, CA). WST reagent was

Transporters inhibition and cytotoxicity

Twenty-one FDA approved drugs were subjected to ASBT, OCTN2, and EAAT4 inhibition in vitro assays, as well as cytotoxicity assay. Fig. 1 plots inhibition vs. cytotoxicity results for each transporter. Inhibition results are presented in terms of percent of substrate (i.e. taurocholate, l-carnitine, or glutamate, respectively). Drugs were considered cytotoxic if they reduced the cell viability more than 20%, and their names are labeled in Fig. 1.

In ASBT–MDCK assay (Fig. 1A), 16 out of 21 drugs

Discussion

We have evaluated drug inhibition of three SLC sodium-dependent transporters, which possess different substrate requirements. From a pool of drugs that were known to inhibit either ASBT or OCTN2, the majority inhibited both ASBT and OCTN2. Only one drug, fluvastatin, inhibited EAAT4, suggesting the cellular membrane sodium gradient was not altered by the remaining drugs. Both ASBT and OCTN2 were stably transfected in MDCK cells, while EAAT4 was not transfected in MDCK cells but in HEK cells.

Acknowledgements

This work was supported in part by National Institutes of Health grant DK67530. The authors kindly acknowledge Dr. Xin Ming and Dhiren R. Thakker (University of North Carolina-Chapel Hill) for providing the hOCTN2-MDCK cell line. The authors kindly acknowledge Svetlana Vidensky and Dr. Jeffrey D. Rothstein (Johns Hopkins University) for providing the stably transfected EAAT4–HEK cell line.

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Why we should be vigilant: Drug cytotoxicity observed with in vitro transporter inhibition studies

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Transporters inhibition and cytotoxicity

Discussion

Acknowledgements

Adv Drug Deliv Rev

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

J Lipid Res

Eur J Pharmacol

Biochem Pharmacol

J Biol Chem

Chem Biol Interact

J Pharm Sci

Eur J Pharmacol

Eur J Pharmacol

Int J Pharm

Toxicology

J Pharmacol Toxicol Methods

Eur J Pharm Sci

Enhancement of chemical rules for predicting compound reactivity towards protein thiol groups

J Comput Aided Mol Des

Toxicological evaluation of thiol-reactive compounds identified using a la assay to detect reactive molecules by nuclear magnetic resonance

Chem Res Toxicol

Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors

J Am Chem Soc

Identification and prediction of promiscuous aggregating inhibitors among known drugs

J Med Chem