Small-Molecule Screening Identifies the Selanazal Drug Ebselen as a Potent Inhibitor of DMT1-Mediated Iron Uptake

doi:10.1016/j.chembiol.2006.08.005

Chem Biol. Author manuscript; available in PMC 2008 September 18.

Published in final edited form as:

Chem Biol. 2006 September; 13(9): 965–972.

doi: 10.1016/j.chembiol.2006.08.005

PMCID: PMC2542486

NIHMSID: NIHMS53531

Small-Molecule Screening Identifies the Selanazal Drug Ebselen as a Potent Inhibitor of DMT1-Mediated Iron Uptake

Herbert A. Wetli,^1,² Peter D. Buckett,^1,² and Marianne Wessling-Resnick¹^*

¹ Department of Genetics and Complex Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115

^* Correspondence: Email: wessling/at/hsph.harvard.edu

²These authors contributed equally to this work.

The publisher's final edited version of this article is available at Chem Biol

See other articles in PMC that cite the published article.

Summary

HEK293T cells overexpressing divalent metal transporter-1 (DMT1) were established to screen for small-molecule inhibitors of iron uptake. Using a fluorescence-based assay, we tested 2000 known bioactive compounds to find 3 small molecules that potently block ferrous iron uptake. One of the inhibitors, ebselen, is a seleno compound used in clinical trials as a protective agent against ischemic stroke. Ebselen inhibited Fe(II) uptake (IC₅₀ of ~0.22 μM), but did not influence Fe(III) transport or DMT1-mediated manganese uptake. An unrelated antioxidant, pyrrolidine dithiobarbamate (PDTC), also inhibited DMT1 activity (IC₅₀ of ~1.54 μM). Both ebselen and PDTC increased cellular levels of reduced glutathione. These observations indicate that Fe(II) transport by DMT1 can be modulated by cellular redox status and suggest that ebselen may act therapeutically to limit iron-catalyzed damage due to transport inhibition.

Introduction

Small molecules can help to define biological pathways by inhibiting protein function to discover the factors involved in dynamic cellular processes. In particular, studies of membrane transport by carriers and channels have been significantly advanced by the use of pharmacological inhibitors to analyze transport mechanisms. Recent developments in the area of iron transport have led to the discovery of several novel membrane transporters and a new understanding of the regulation of iron absorption [1, 2]. Unfortunately, this area of research has been hampered by the lack of pharmacological reagents to probe the underlying molecular mechanisms involved in these processes. To identify small-molecule inhibitors of iron transport, we previously established a cell-based screening assay that takes advantage of iron-induced quenching of calcein fluorescence [3]. Using this approach, we discovered ten inhibitors of nontransferrin bound iron (NTBI) uptake [4]. Two other pathways of iron uptake are known to be mediated by divalent metal transporter-1 (DMT1). DMT1 is the transporter responsible for dietary iron absorption across the apical membrane of intestinal enterocytes [5] and is also involved in the delivery of iron to peripheral tissues by transferrin [6]. Defects in the DMT1 gene cause microcytic anemia in the mk mouse, an animal model that displays defective dietary iron absorption [7]. Defective transferrin-mediated iron uptake is also well characterized for a different animal model, the Belgrade rat, which harbors the same genetic defect in DMT1 [6]. Electrophysiological studies have shown that DMT1 not only mediates uptake of ferrous iron, but that it also interacts with other divalent metals, including Cd²⁺, Co²⁺, Cu²⁺, Mn²⁺, Zn²⁺, Ni²⁺, and Pb²⁺ [8]. In addition, the DMT1 mutation present in the b rat and mk mouse (G185R) confers Ca²⁺ transport activity to the transporter [9]. DMT1 activity has been characterized to be voltage and pH dependent [8], but despite intense effort to understand the transporter’s molecular properties [10], relatively little is known about cellular control of its function. To further our understanding of DMT1-mediated iron uptake, we established a HEK293T cell line that stably overexpresses this transporter, and we adapted the cell-based calcein assay to screen for small-molecule inhibitors of ferrous iron uptake in chemical libraries of known bioactive compounds. Among the inhibitors identified in this chemical genetic screen was ebselen, an antioxidant, anti-inflammatory selenium compound that has been found to be useful in treating patients with ischemic stroke [11, 12] and aneurismal subarachoid hemorrhage [13]. This report characterizes inhibition of DMT1 activity by ebselen and another unrelated antioxidant, pyrrolidine dithiocarbamate (PDTC). Based on these results, we propose that DMT1 activity is inversely regulated by cellular redox status. This study demonstrates the utility of cell-based assays using transporter overexpression as a means of identifying small-molecule inhibitors as well as the usefulness of chemical genetic screening as a tool for determining cellular factors involved in fundamental biological processes like membrane transport.

Results

A Screen for DMT1 Transport Inhibitors

HEK293T cells were transfected with DMT1 cDNA subcloned in the sense (coding) or antisense (noncoding) orientations [14] and selected for stable expression by using puromycin resistance. Figure 1A confirms robust expression of the transporter in cells transfected with sense DMT1 cDNA; DMT1 could not be detected either in nontransfected control cells (data not shown) or HEK293T cells transfected with antisense cDNA. Transport assays to determine the uptake of ⁵⁵Fe presented in the ferrous form at pH 6.75 indicated that DMT1 activity was ~25-fold greater in the HEK293T(DMT1) cells over-expressing the transporter (Figure 1B). Indirect immuno-fluorescence microscopy experiments with anti-DMT1 performed to cytolocalize exogenously expressed transporters revealed cell surface as well as punctate intracellular staining (Figure 1C).

Figure 1

Stable Expression of DMT1 Allows for a Chemical Genetic Screen for Transport Inhibitors

Using the HEK293T(DMT1) cell line, we adapted a cell-based screening assay for iron uptake based on calcein fluorescence to identify small-molecule inhibitors of DMT1. Briefly, the metal-sensitive fluorophore calcein is used to measure intracellular “labile” or free iron. In the presence of iron or other metals, the fluorescence of calcein is quenched [3]. Calcein does not bind to calcium or magnesium at physiological pH, and since the intracellular concentrations of other metal ions are low, a loss of cell-associated calcein fluorescence in the presence of extracellular iron is indicative of an increase in free intracellular iron levels due to transport. Using a 384-well format, 2000 compounds from the NINDS Custom Collection and SpecPlus Collection (Microsource Discovery Systems), available through the Institute of Chemistry and Cell Biology at Harvard Medical School, were robotically pin transferred and assayed at 50 μM for the ability to block iron uptake by HEK293T(DMT1) cells, as measured by fluorescence quenching upon addition of 1 μM ferrous iron (nominal cutoff for potency ≥ 50%).

From the survey of the 2000 bioactive compounds contained in these 2 libraries, 23 were “cherry picked” and rescreened by using the calcein assay in a 384-well format to validate results. Of these, eight compounds were obtained from outside suppliers and further studied by using a secondary screen for transport inhibition based on cellular uptake of radioactive ⁵⁵Fe (Figure 1D). From this final analysis, only three compounds were confirmed to be bona fide transport inhibitors: ebselen, Δ⁹-tetrahydrocannabinol (THC), and 4-methoxy-dalbergione. Acetyl-tryptophanamide, deoxodeoxydihydrogendurin, and dihydromunduletone were relatively weak inhibitors in the secondary screen, while cantharidin appeared to slightly enhance ⁵⁵Fe uptake. One other compound, methyl 7-deshydroxypryogallin-4-carboxylate, produced a more profound stimulatory effect (data not shown). ⁵⁵Fe in the assay mixture was collected onto filters with or without cells; therefore, the effects of this particular compound in the screening assay were most likely due to extracellular interactions with ferrous iron to indirectly block uptake. Such results emphasize the importance of secondary screens with a separate methodological approach in validation studies.

Using the ⁵⁵Fe tracer assay, ebselen and Δ⁹-THC were determined to inhibit DMT1 activity with IC₅₀ values of ~0.22 and 0.45 μM, respectively (Figure 2). While 4-methoxy-dalbergione alsoinhibited uptake, onlyabout 50% of the measured activity was blocked at concentrations above 0.1 μM, suggesting that limited cell permeability may reduce the efficacy of this compound. Because previous animal studies suggested that ebselen might affect tissue iron levels [15], our subsequent experiments focused on characterizing its capacity to inhibit DMT1-mediated iron uptake.

Figure 2

Dose-Response Studies of Potent DMT1 Inhibitors

Ebselen Does Not Influence DMT1 Activity by Indirect Effects on Fe(II)

To maintain iron in the ferrous state during transport assays, a 50 molar-fold excess of ascorbic acid is added. To determine if ebselen influenced DMT1-mediated transport by altering levels of ascorbate or Fe(II) present in the assay system, the following control experiments were performed. First, the stability of ascorbic acid over time was measured. The presence of 1 μM FeNTA (1:50 complex ratio) led to a loss of ascorbic acid over time, as measured by absorbance at 260 nm (Figure 3A). The half-life of ascorbic acid under the same conditions was nearly doubled by the addition of 50 μM ebselen. In the absence of FeNTA, ascorbic acid levels were relatively stable with or without the addition of ebselen. Likewise, ebselen itself was also stable under the assay conditions with ascorbic acid in the presence or absence of 1 μM FeNTA (Figure 3B). Finally, the amount of ferrous iron, measured spectrophotometrically by using the Fe(II)-specific iron chelator ferrozine (A_{563 nm}), was determined (Figure 3C). Fe(II) levels were stable in the presence of ascorbic acid for 1 hr, and were further stabilized by the presence of ebselen. Since our transport assays to measure ⁵⁵Fe(II) transport determined uptake within 20 min of the initial mixing of reaction components, these combined results confirmed that uptake of ferrous iron was measured and that ebselen did not affect the redox state of the transport substrate.

Figure 3

Stability of Assay Components in the Presence of Ebselen

Ebselen Does Not Influence Fe(III) or Mn(II) Uptake

To examine whether ebselen interfered with ferric iron transport, both transferrin bound and NTBI uptake were measured in the presence of this inhibitor. NTBI uptake by HeLa cells was determined as previously described [16]. Briefly, cells were incubated at 37°C with or without ebselen in the presence of 1 μM ⁵⁵FeNTA (1:4 complex) in serum-free DMEM for 1 hr. After quenching reaction components on ice, excess unlabeled FeNTA was added to remove any surface bound radiolabel and cell-associated ⁵⁵Fe was counted. As shown in Figure 4A, addition of up to 100 μM ebselen did not affect HeLa cell ⁵⁵Fe uptake compared to control cells incubated with vehicle alone (1% DMSO).

Figure 4

Ebselen Does Not Inhibit Nontransferrin Bound Iron, NTBI, or Transferrin Bound Iron, TBI, Uptake

To examine whether ebselen influenced the uptake of transferrin bound iron, apotransferrin was first loaded with ⁵⁵Fe and then incubated with cells in serum-free medium for 4 hr at 37°C (Figure 4B). Uptake of ⁵⁵Fe from transferrin was studied by using HeLa and HEK293T cells, including both the parental and HEK293T(DMT1) cell lines, as previously described [4]. The presence of up to 50 μM ebselen did not inhibit transferrin-mediated iron delivery to any of the cell lines tested. One interesting finding from these experiments was that transferrin-mediated iron uptake was not enhanced in HEK293T(DMT1) cells, suggesting that transferrin receptor number rather than endosomal iron transport is limiting for iron uptake by this pathway. Because DMT1 has a defined role in endosomal transport of iron, the lack of ebselen inhibition was also somewhat surprising. However, unlike the DMT1 transport assays to measure Fe(II) uptake, determination of NTBI and transferrin-mediated uptake was conducted at neutral pH. Previous studies have shown that ebselen interactions with the thioredoxin/thioredoxin reductase system to reduce dehydroascorbate are optimal at pH 6.4 [17], which is closer to the pH range used to detect the proton-coupled DMT1 activity (pH ~6.75). It is possible that a similar pH profile modifies the response of cellular iron uptake to ebselen, or that this inhibitor affects factors specifically involved in mediating ferrous iron import across the cell surface, but not endosomal membrane compartments.

DMT1 is known to transport other divalent cations in addition to Fe(II). Compared to parental control cells, HEK293T(DMT1) cells take up 25-fold greater levels of ⁵⁴Mn, consistent with DMT1 transport of this metal (Figure 5A). Inhibition studies, however, revealed that ebselen did not inhibit ⁵⁴Mn uptake at concentrations of up to 50 μM (Figure 5B). This result implies that inhibition of DMT1 by ebselen is specific to the transport substrate Fe(II). This observation is important because ebselen can potentially modify thiols to inactivate enzymatic activity by forming a selenylsulfide [18]. For example, the Na⁺, K⁺-ATPase is one target for ebselen inhibition, which occurs through the chemical modification of its cysteine residues [19]. Consistent with the idea that ebselen does not chemically modify DMT1 to inhibit its transport activity, the addition of dithiothreitol (DTT) as a reducing agent did not reverse ebselen’s effects on Fe(II) uptake (data not shown). Moreover, inhibition of Fe(II) uptake by ebselen was fully reversed after treatment of cells and subsequent wash out of the drug (Figure 5C).

Figure 5

Ebselen Inhibits Fe(II), but Not Mn(II), Uptake and Acts in a Reversible Manner

PDTC Inhibits DMT1-Mediated Fe(II) Uptake

Because ebselen appeared to target the pathway of Fe(II) uptake rather specifically, we reasoned that the antioxidant effects of this compound might influence transport of this redox-active metal. We therefore studied another antioxidant, PDTC, which is structurally unrelated to ebselen. This antioxidant is commonly used to probe redox-sensitive NF-κB activation in vivo [20–22]. Inhibition studies demonstrated that PDTC inhibited DMT1 ⁵⁵Fe transport activity (Figure 6). The IC₅₀ was determined to be 1.54 μM, although it should be noted that the 95% confidence interval for this value is large (0.68–3.45 μM). To directly compare the effects of both ebselen and PDTC on cellular glutathione levels, the ratio of reduced to oxidized glutathione was measured in HEK293T(DMT1) cells treated with 10 μM ebselen, 50 μM PDTC, or 0.5 % DMSO (vehicle control) for 20 min at 37°C under the transport assay conditions. The GSSG, GHS + GSSG, and GSH/GSSG values determined in these experiments are summarized in Table 1. Both compounds increased cellular levels of reduced GSH, as anticipated based on their antioxidant capacity. A simple interpretation of these combined results is that DMT1-mediated Fe(II) uptake activity is modulated by cellular redox status.

Figure 6

Antioxidant Activity Inhibits DMT1-Mediated Iron Uptake Inhibition of ⁵⁵Fe uptake by HEK293T(DMT1) cells was determined in the presence of 0.001–100 μM PDTC, an antioxidant that is structurally unrelated to ebselen. Shown are mean values (more ...)

Table 1

Antioxidant Effects on GSH/GSSG Ratio

Discussion

The use of small molecules to alter cellular function provides new opportunities to determine mechanistic elements involved in complex biological pathways. While molecular genetic approaches have provided new insights into iron transport, such as the identification of the iron transporter DMT1 [7, 8], there is a significant need to develop pharmacological tools to gain further insight into the molecular basis of metal uptake and regulation. To discover small-molecule inhibitors of DMT1-mediated transport activity, we established a phenotypic screen based on calcein fluorescence quenching in a cell-based assay for iron uptake. Use of the stable HEK293T(DMT1) cell line provided the necessary resource to detect DMT1 Fe(II) transport activity in an amplified and sensitive manner. This report demonstrates the utility of this cell-based approach in a screen of 2000 known bioactive compounds. Two potent transport inhibitors were identified: ebselen (IC₅₀ of ~0.22 μM) and Δ⁹-THC (IC₅₀ of ~0.47 μM). Δ⁹-THC, the major psychoactive component of the marijuana plant Cannabis sativa, is known to produce a number of behavioral and pharmacological effects mediated through interactions with the central nervous system cannabinoid receptor CB₁ and the peripheral receptor CB₂. Endogenous cannabinoids also activate these G protein-coupled receptors to negatively regulate adenylate cyclase activity and positively regulate inward rectifying K⁺ channels [23]. It is interesting to note that receptor-independent signal transduction pathways have also been recently reported to negatively regulate a number of ion channels, including T-type Ca²⁺ channels, TASK-1 channels, and Na⁺ channels [24]. There is significant interest in identifying signaling targets for cannabinoids since drugs that modify endocannabinoid activity are currently being developed to control obesity (Rimonabant), prevent osteoporosis (HU-308), and treat multiple sclerosis (Sativex). Marinol (pure THC) is still often used in treating AIDS and cancer patients. Thus, future studies must address both the mechanism of DMT1 inhibition by Δ⁹-THC as well as the significance of these effects.

Our immediate efforts focused on defining how ebselen affected DMT1-mediated iron uptake. Ebselen (also called PZ51), or 2-phenyl-1,2-benzisoselenazol-3[2H]-one, is thought to exert antioxidant effects as a glutathione peroxidase mimic (reviewed by Schewe [18]). More recent studies have shown that ebselen rapidly oxidizes reduced thioredoxin to interact with the thioredoxin reductase system [25, 26]. Ebselen is also known to directly inhibit several inflammatory enzymes by thiol modification to form a selenosulfide [18]. Interestingly, previous in vivo studies have demonstrated that ebselen treatment is associated with reduced tissue iron in a model of iron overload, suggesting its potential inhibition of iron uptake [15]. Although the antioxidant, anti-inflammatory actions of ebselen provide a mechanistic explanation for its efficacy in clinical trials, many animal studies have shown that iron chelation also successfully limits damage in models of ischemic stroke [27], consistent with the idea that ebselen could act therapeutically by inhibiting tissue iron uptake.

A previous study of DMT1 activity with Xenopus oocytes suggested that oxidative agents could inhibit transporter function by direct modification of protein thiols [28]. Both zinc and iron uptake were blocked by treatment with H₂O₂ and Hg²⁺, and DMT1-mediated transport activity was restored by the addition of DTT. We find that ebselen’s influence on DMT1 function is unlikely to be mediated by such direct effects on this transporter since the compound failed to block uptake of DMT1-mediated manganese and DTT failed to reverse inhibition of DMT1-mediated iron uptake. There are at least two possible explanations for how changes in cellular redox might promote inhibition of ferrous iron in a specific manner. First, the reduced cellular environment promoted by ebselen and PDTC could allow an expansion in the “labile” or free iron pool under our assay conditions. The total concentration of cytosolic free iron is balanced between Fe(II) and Fe(III), and the ratio of these forms is known to be determined by the cellular redox capacity [29, 30]. The antioxidants also may influence the activity of other factors known to modulate intracellular levels of free divalent iron, for example, ferric reductases [31]. Mathematical modeling of the mechanisms of DMT1-mediated transport [32] suggests that carrier-mediated uptake of Fe(II) into the cell could become limiting when intracellular concentrations of the transport substrate increased. Alternatively, the anti-oxidants could possibly influence the activity of specific factors involved in the intracellular targeting of Fe(II) to fulfill specific metabolic functions, for example, transfer to mitochondria for heme synthesis or iron-sulfur cluster formation [33]. This scenario is based on analogy to the family of copper chaperones that mediate movement of this metal throughout the cell to achieve metabolic targeting after import [34]. The discovery of ebselen’s inhibitory effects sheds new light on how the cellular redox environment can influence iron uptake. Use of anti-oxidants like ebselen and PDTC as pharmacological inhibitors of DMT1-mediated iron uptake should help to provide further molecular insights into the pathway’s cellular factors involved in this process.

Significance

There is a significant need to develop pharmacological tools to gain further insight into the molecular basis for iron uptake and its regulation. To develop a cell-based screen for inhibitors, a stable HEK293T(DMT1) cell line was established to detect DMT1 Fe(II) transport activity in an amplified and sensitive manner. Using calcein fluorescence quenching to assay ferrous iron transport inhibition, 2000 known bioactive compounds were screened and 3 potent transport inhibitors were identified. This report further characterizes the activity of one of these inhibitors, ebselen. This seleno compound is a protective agent against ischemic stroke. Ebselen inhibited Fe(II) uptake (IC₅₀ of ~0.22 μM), but did not influence Fe(III) or DMT1-mediated manganese uptake. Studies with an unrelated antioxidant, PDTC, confirmed that the cellular redox environment influences DMT1 iron uptake activity. Future use of antioxidants like ebselen and PDTC as pharmacological inhibitors of DMT1-mediated iron uptake should help to provide further molecular insights into the pathway’s cellular factors involved in this process.

Experimental Procedures

Generation of a Stable DMT1 Cell Line

HEK293T cells were grown to 60% confluency in Dulbecco’s modified Eagle’s medium (DMEM) containing 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum. The cells were cotransfected with pMT2-Nramp2 in a coding (sense) or noncoding (antisense) orientation (a kind gift from Dr. Nancy Andrews) and pBABEPuro (carrying a puromycin-resistance gene) at a 20:1 ratio by using Effectine (QIAGEN) according to the manufacturer’s instructions. After allowing 48 hr for gene expression, selection was performed by adding 2 μg/ml puromycin to the culture medium. After stable selection under these restrictive growth conditions, cells were subsequently grown in α minimal essential medium (αMEM) supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% fetal bovine serum.

Small-Molecule Library Screen

HEK293T(DMT1) cells were plated in 384-well poly-D-lysine-coated plates (Becton Dickinson) in columns 1–23 (15,000 cells/well) by using a Wellmate liquid dispensing apparatus (Matrix Technologies Corp.). Column 24 was left empty. After overnight incubation, the plates were washed three times with 65 μl serum- and phenol red-free αMEM by using a Bio-Tek ELx405 plate washer. Calcein-AM was added to a final concentration of 0.25 μM. After a 1 hr incubation at 37°C, the plates were washed ten times with 65 μl phosphate-buffered saline containing 1 mM MgCl₂ and 0.1 mM CaCl₂ (PBS++) with 5 mM glucose added. A final volume of 40 μl of this same buffer was added to each well, and a baseline fluorescence reading was measured by using an Analyst plate reader (485 nm excitation; 535 nm emission; Molecular Devices Corp.). Using a robotic pin-transfer apparatus, ~250 nl of each compound was transferred from master plates to duplicate HEK293T(DMT1) plates, which were then incubated at 37°C for 30 min, after which time a second fluorescence reading was taken. To each well in column 1, 10 μl aliquots of 200 mM HEPES, 200 mM Tris (pH 6.0), and 250 μM ascorbic acid were added. To each well in columns 2–24, 10 μl of the same assay mix with 5 μM FeNTA (1:50 metal:chelate ratio) was added. Columns 1 and 2 were used as controls, and no compounds were transferred to these wells. The plates were incubated at 37°C for an additional 20 min, and a final fluorescence reading was taken.

Inhibition Studies with Radioisotopic Tracer

HEK293T(DMT1) cells were incubated at 37°C for 20 min in assay buffer (25 mM Tris, 25 mM MES, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, 5 mM glucose [pH 6.75]) containing 1 μM ⁵⁵Fe or ⁵⁴Mn and 50 μM ascorbic acid (pH 6.75) (05–1.5 ± 10⁶ cells/transport reaction). Inhibitors were added at the concentrations noted in the figure legends immediately prior to the start of uptake. Cells were chilled on ice and either collected on nitrocellulose filters or by centrifugation at 2040 ± g for 5 min, then washed three times with PBS to remove any unbound ⁵⁵Fe or ⁵⁴Mn. Cell-associated radioactivity was determined by scintillation counting and was normalized to control (vehicle alone) or adjusted to cell protein measured in lysates by using the Bradford assay [35]. Values reported in figures and the text are means ± absolute deviation unless otherwise noted.

Acknowledgments

We wish to thank the Harvard Institute of Chemistry and Biology (ICCB), who assisted with this screen, and the National Cancer Institute’s Initiative for Chemical Genetics, who provided support for the ICCB. We also wish to acknowledge the help of Dr. Bryan MacKenzie (University of Cincinnati), who provided critical comments and helpful advice prior to submission of our manuscript. Support for this research was provided by National Institutes of Health grant DK064750. Herbert A. Wetli also thanks the Novartis Foundation for financial support.

References

1. Andrews NC. Iron homeostasis: insights from genetics and animal models. Nat Rev Genet. 2000;1:208–217. [PubMed]

2. Donovan A, Roy CN, Andrews NC. The ins and outs of iron homeostasis. Physiology (Bethesda) 2006;21:115–123. [PubMed]

3. Cabantchik ZI, Glickstein H, Milgram P, Breuer W. A fluorescence assay for assessing chelation of intracellular iron in a membrane model system and in mammalian cells. Anal Biochem. 1996;233:221–227. [PubMed]

4. Brown JX, Buckett PD, Wessling-Resnick M. Identification of small molecule inhibitors that distinguish between non-transferrin bound iron uptake and transferrin-mediated iron transport. Chem Biol. 2004;11:407–416. [PubMed]

5. Gunshin H, Fujiwara Y, Custodio AO, Direnzo C, Robine S, Andrews NC. Slc11a2 is required for intestinal iron absorption and erythropoiesis but dispensable in placenta and liver. J Clin Invest. 2005;115:1258–1266. [PMC free article] [PubMed]

6. Fleming MD, Romano MA, Su MA, Garrick LM, Garrick MD, Andrews NC. Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci USA. 1998;95:1148–1153. [PubMed]

7. Fleming MD, Trenor CC, III, Su MA, Foernzler D, Beier DR, Dietrich WF, Andrews NC. Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet. 1997;16:383–386. [PubMed]

8. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature. 1997;388:482–488. [PubMed]

9. Xu H, Jin J, DeFelice LJ, Andrews NC, Clapham DE. A spontaneous, recurrent mutation in divalent metal transporter-1 exposes a calcium entry pathway. PLoS Biol. 2004;2:E50. [PMC free article] [PubMed]

10. Mackenzie B, Hediger MA. SLC11 family of H+-coupled metal-ion transporters NRAMP1 and DMT1. Pflugers Arch. 2004;447:571–579. [PubMed]

11. Ogawa A, Yoshimoto T, Kikuchi H, Sano K, Saito I, Yamaguchi T, Yasuhara H. Ebselen in acute middle cerebral artery occlusion: a placebo-controlled, double-blind clinical trial. Cerebrovasc Dis. 1999;9:112–118. [PubMed]

12. Yamaguchi T, Sano K, Takakura K, Saito I, Shinohara Y, Asano T, Yasuhara H. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Stroke. 1998;29:12–17. [PubMed]

13. Saito I, Asano T, Sano K, Takakura K, Abe H, Yoshimoto T, Kikuchi H, Ohta T, Ishibashi S. Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage. Neurosurgery. 1998;42:269–277. [PubMed]

14. Su MA, Trenor CC, Fleming JC, Fleming MD, Andrews NC. The G185R mutation disrupts function of the iron transporter Nramp2. Blood. 1998;92:2157–2163. [PubMed]

15. Davis MT, Bartfay WJ. Ebselen decreases oxygen free radical production and iron concentrations in the hearts of chronically iron-overloaded mice. Biol Res Nurs. 2004;6:37–45. [PubMed]

16. Inman RS, Wessling-Resnick M. Characterization of transferrin-independent iron transport in K562 cells. Unique properties provide evidence for multiple pathways of iron uptake. J Biol Chem. 1993;268:8521–8528. [PubMed]

17. Zhao R, Holmgren A. Ebselen is a dehydroascor-bate reductase mimic, facilitating the recycling of ascorbate via mammalian thioredoxin systems. Antioxid Redox Signal. 2004;6:99–104. [PubMed]

18. Schewe T. Molecular actions of ebselen—an antiinflammatory antioxidant. Gen Pharmacol. 1995;26:1153–1169. [PubMed]

19. Borges VC, Rocha JB, Nogueira CW. Effect of diphenyl diselenide, diphenyl ditelluride and ebselen on cerebral Na(+), K(+)-ATPase activity in rats. Toxicology. 2005;215:191–197. [PubMed]

20. Weber C, Erl W, Pietsch A, Strobel M, Ziegler-Heitbrock HW, Weber PC. Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-κ B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals. Arterioscler Thromb. 1994;14:1665–1673. [PubMed]

21. Gupta S, Young D, Sen S. Inhibition of NF-κB induces regression of cardiac hypertrophy, independent of blood pressure control, in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol. 2005;289:H20–H29. [PubMed]

22. Flohe L, Brigelius-Flohe R, Saliou C, Traber MG, Packer L. Redox regulation of NF-κB activation. Free Radic Biol Med. 1997;28:1115–1126. [PubMed]

23. Massa F, Storr M, Lutz B. The endocannabinoid system in the physiology and pathophysiology of the gastrointestinal tract. J Mol Med. 2005;83:944–954. [PubMed]

24. Demuth DG, Molleman A. Cannabinoid signalling. Life Sci. 2006;78:549–563. [PubMed]

25. Zhao R, Masayasu H, Holmgren A. Ebselen: a substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidant. Proc Natl Acad Sci USA. 2002;99:8579–8584. [PubMed]

26. Zhao R, Holmgren A. A novel antioxidant mechanism of ebselen involving ebselen diselenide, a substrate of mammalian thioredoxin and thioredoxin reductase. J Biol Chem. 2002;277:39456–39462. [PubMed]

27. Selim MH, Ratan RR. The role of iron neurotoxicity in ischemic stroke. Ageing Res Rev. 2004;3:345–353. [PubMed]

28. Marciani P, Trotti D, Hediger MA, Monticelli G. Modulation of DMT1 activity by redox compounds. J Membr Biol. 2004;197:91–99. [PubMed]

29. Esposito BP, Epsztejn S, Breuer W, Cabantchik ZI. A review of fluorescence methods for assessing labile iron in cells and biological fluids. Anal Biochem. 2002;304:1–18. [PubMed]

30. Petrat F, de Groot H, Sustmann R, Rauen U. The chelatable iron pool in living cells: a methodically defined quantity. Biol Chem. 2002;383:489–502. [PubMed]

31. Petrat F, Paluch S, Dogruoz E, Dorfler P, Kirsch M, Korth HG, Sustmann R, de Groot H. Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool. J Biol Chem. 2003;278:46403–46413. [PubMed]

32. MacKenzie B, Ujwal ML, Chang MH, Romero MF, Hediger MA. Divalent metal-ion transporter DMT1 mediates both H+-coupled Fe2+ transport and uncoupled fluxes. Pflugers Arch-Eur J Physiol. 2006;451:544–558. [PubMed]

33. Napier I, Ponka P, Richardson DR. Iron trafficking in the mitochondrion: novel pathways revealed by disease. Blood. 2005;105:1867–1874. [PubMed]

34. O’Halloran TV, Culotta VC. Metallochaperones, an intracellular shuttle service for metal ions. J Biol Chem. 2000;275:25057–25060. [PubMed]

35. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. [PubMed]