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M300358

Cross-inhibition of SR-BI- and ABCA1-mediatedcholesterol transport by the small molecules BLT-4and glyburide Thomas J. F. Nieland,*,†,§ Angeliki Chroni,** Michael L. Fitzgerald,†† Zoltan Maliga,†,§
Vassilis I. Zannis,** Tomas Kirchhausen,† and Monty Krieger1,*
Department of Biology,* Massachusetts Institute of Technology, Cambridge, MA 02139; Department of Cell Biology,† Harvard Medical School, and The CBR Institute for Biomedical Research, Inc., Boston, MA02115-5701; Molecular Genetics, Whitaker Cardiovascular Institute, Department of Medicine and Biochemistry,** Boston University School of Medicine, Boston, MA 02118; Lipid Metabolism Unit,†† Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; and Harvard Institute for Chemistry and Cell Biology,§ Seeley G. Mudd 604, Boston, MA 02115 Scavenger receptor class B type I (SR-BI) and
teins such as scavenger receptor class B type I (SR-BI) and ABCA1 are structurally dissimilar cell surface proteins that
ABCA1 plays an important role in cellular and whole body play key roles in HDL metabolism. SR-BI is a receptor that
lipid metabolism and can profoundly influence the risk binds HDL with high affinity and mediates both the selec-
of atherosclerosis and coronary heart disease (1–8). The tive lipid uptake of cholesteryl esters from lipid-rich HDL
pathologic consequences of naturally arising or experi- to cells and the efflux of unesterified cholesterol from cells
to HDL. ABCA1 mediates the efflux of unesterified choles-
mentally induced disruptions in the SR-BI and ABCA1 terol and phospholipids from cells to lipid-poor apolipo-
genes clearly demonstrate that these lipid transport pro- protein A-I (apoA-I). The activities of ABCA1 and other
teins serve physiologically important, yet dramatically dis- ATP binding cassette superfamily members are inhibited by
tinct, purposes in HDL metabolism (1, 2).
the drug glyburide, and SR-BI-mediated lipid transport is
SR-BI, a member of the CD36 superfamily of proteins, blocked by small molecule inhibitors called BLTs . Here, we
is a 509 residue glycoprotein (apparent mass of 82 kDa) show that one BLT, [1-(2-methoxy-phenyl)-3-naphthalen-2-yl-
containing two predicted transmembrane helices adjacent urea] (BLT-4), blocked ABCA1-mediated cholesterol efflux
to very short N- and C-terminal cytoplasmic domains, with to lipid-poor apoA-I at a potency similar to that for its inhi-
the bulk of the protein consisting of a large extracellular bition of SR-BI (IC
55–60 M). Reciprocally, glyburide
blocked SR-BI-mediated selective lipid uptake and efflux
loop (3, 4). Alterations in SR-BI expression can influence at a potency similar to that for its inhibition of ABCA1 (IC
profoundly several physiologic processes, including biliary 275–300 M). As is the case with BLTs, glyburide in-
cholesterol secretion, female fertility, red blood cell matu- creased the apparent affinity of HDL binding to SR-BI.
ration, and the development of atherosclerosis and coro- The reciprocal inhibition of SR-BI and ABCA1 by BLT-4
nary heart disease (5–7, 9–14). SR-BI tightly binds large, and glyburide raises the possibility that these proteins may
spherical, cholesteryl ester-rich HDL particles, primarily share similar or common steps in their mechanisms of lipid
via its major apolipoprotein, apolipoprotein A-I (apoA-I), transport.—Nieland, T. J. F., A. Chroni, M. L. Fitzgerald, Z.
but lipid-free apoA-I is a poor ligand (15). SR-BI mediates Maliga, V. I. Zannis, T. Kirchhausen, and M. Krieger. Cross-
inhibition of SR-BI- and ABCA1-mediated cholesterol trans-
the selective uptake of HDL cholesteryl esters (16–19) port by the small molecules BLT-4 and glyburide J. Lipid
and other lipids (3, 20, 21) into cells, after which the lipid- Res. 2004. 45: 1256–1265.
depleted particles dissociate from the cells. The mecha-nism of selective lipid uptake differs markedly from that Supplementary key words
scavenger receptor class B type I • ATP of the classic coated pit-mediated endocytic uptake of LDL binding cassette transporter A1 • blockers of lipid transport receptors (22). The binding of native spherical HDL par- Cholesterol and cholesteryl ester movement into and out of cells mediated by lipoproteins and cell surface pro- Abbreviations: ABC, ATP binding cassette; apoA-I, apolipoprotein A-I; BLT, small molecules that block lipid transport; COE, cholesteryloleyl ether; DSP, dithiobis (succinimidyl propionate); Kd, dissociationconstant; SR-BI, scavenger receptor class B type I; SUR, sulfonylurea re- Manuscript received 21 August 2003 and in revised form 13 February 2004. Published, JLR Papers in Press, April 21, 2004. To whom correspondence should be addressed.
e-mail: [email protected] Copyright 2004 by the American Society for Biochemistry and Molecular Biology, Inc.
Journal of Lipid Research
This article is available online at http://www.jlr.org
ticles or reconstituted apoA-I/phospholipid/cholesteroldisks to SR-BI also results in the efflux of unesterified cho-lesterol from cells to the particles (23–25), but the physio-logical significance of this activity has not yet been deter-mined.
ABCA1, a member of the ATP binding cassette (ABC) superfamily of proteins, has a structure strikingly differentfrom that of SR-BI. ABCA1 is a much larger protein (200kDa) that contains 12 membrane-spanning helices, two cy-toplasmic ATP binding domains, and two large extracellu-lar loops (2, 26, 27). It mediates the transfer of unesteri-fied cholesterol and phospholipids from cells to lipid-poor apoA-I (28–31), which can subsequently mature intospherical, cholesteryl ester-rich HDL particles (32–35).
Chemical structures of glyburide (A) and 1-(2-methoxy- These mature HDLs are poor acceptors for ABCA1-medi- phenyl)-3-naphthalen-2-yl-urea (BLT-4) (B).
ated cholesterol efflux (36). Mutations in ABCA1 are theunderlying cause of Tangier disease, which is character-ized by the virtual absence of spherical, cholesteryl ester-rich HDL particles in the plasma and an accumulation of that in the low nanomolar to micromolar range block cholesterol in peripheral macrophages (28, 37–39). Mice lipid transport (selective uptake from and efflux to HDL) with targeted homozygous inactivating mutations in the mediated by SR-BI (45). Unexpectedly, they enhance ABCA1 gene exhibit phenotypes similar to those of hu- rather than decrease the apparent affinity of HDL bind- man Tangier patients (40–42). In contrast, SR-BI-deficient ing to SR-BI.
mice (homozygous null mutations) have increased levels Here, we report that 1-(2-methoxy-phenyl)-3-naphtha- of abnormally large lipid-rich HDL particles in their len-2-yl-urea (BLT-4) (Fig. 1B) blocks ABCA1-mediated plasma (43). The differences in the structures of SR-BI cholesterol efflux to lipid-free apoA-I. The other BLTs did and ABCA1 suggest that they use different mechanisms to not inhibit ABCA1 activity. Conversely, glyburide pre- transfer lipids between cells and HDL. This is supported vented SR-BI-mediated selective lipid uptake from and by experiments showing that the structural or conforma- cholesterol efflux to HDL. Similar to the BLTs, glyburide tional determinants on apoA-I that are crucial for its inter- enhanced the affinity of HDL binding to SR-BI. The action with ABCA1 (e.g., apoA-I's C terminus) appear to concentrations at which glyburide inhibited SR-BI- and differ from those necessary for productive interactions ABCA1-mediated lipid efflux were similar (IC with SR-BI (15, 24, 44). However, their common abilities 300 M), as were those for BLT-4, but BLT-4 was the more to mediate cholesterol efflux from cells led us to probe potent inhibitor (IC 55–60 M). The reciprocal or their activities using small molecules that had previously cross-inhibition of these two lipid transport proteins by been shown to inhibit their lipid transport activities.
BLT-4 and glyburide raises the possibility that there may These included small molecules that block lipid transport be similarities in at least one step of the mechanisms of (BLTs) mediated by SR-BI (45) and the drug glyburide SR-BI- and ABCA1-mediated lipid transport (29, 31, 46–51), which blocks ABCA1-mediated choles-terol efflux.
Glyburide (also called glybenclamide; Fig. 1A) is a sulfo-
EXPERIMENTAL PROCEDURES nylurea that has received wide attention in clinical settingsas a potent drug for the treatment of non-insulin-depen- Stock solutions of BLTs (Chembridge Corp.) and glyburide dent diabetes mellitus (52). Glyburide binds tightly to and (Sigma) were prepared in 100% DMSO and diluted into the as- inhibits sulfonylurea receptors 1 and 2 (SUR1 or SUR2), say medium immediately before use. Dithiobis (succinimidyl pro- members of the ABC superfamily of proteins that form pionate) (DSP; from Pierce Biotechnology, Inc.), which contains the SUR subunit of the ATP-sensitive potassium channel.
thiol-sensitive bonds, was dissolved in dimethyl sulfoxide andthen diluted to 250 M with PBS immediately before use.
SUR1 and SUR2 are responsible for the binding and hy-drolysis of ATP and effectively control the closing of the Lipoproteins and cells
channel and the regulation of insulin release. The dissoci- Human HDL (density of 1.09–1.16 g/ml) and recombinant ation constant (Kd) for glyburide binding to SUR1, which lipid-free human apoA-I were isolated and labeled with either apparently involves association with its transmembrane 125I (125I-HDL or 125I-apoA-I) or [3H]cholesteryl oleyl ether domains, is in the low nanomolar range [reviewed in ref.
([ H]COE, [ H]COE-HDL) (3, 19, 25, 44, 57, 58). LDL receptor- (53)]. Glyburide also inhibits the activities of other ABC deficient Chinese hamster ovary cells that express low levels of en-dogenous SR-BI (ldlA-7) (59), ldlA-7 cells stably transfected to ex- proteins (54–56), including ABCA1. Inhibition of ABCA1 press high levels of murine SR-BI (ldlA[mSR-BI]) (19), and HEK293- by glyburide has been reported to occur in the concentra- EBNA-T human embryonic kidney (HEK) cells (27) were main- tion range of 100–1,000 M (29, 31, 46–51).
tained in culture as previously described. All assay media contained By using a high-throughput chemical library screen, we 0.5% or 0.29% (v/v) DMSO and 0.5% (w/v) BSA (fatty acid-free recently identified five small molecules (BLT-1 to BLT-5) form in efflux assays), and all experiments were conducted at 37C.
Nieland et al. Small molecule inhibitors of cholesterol efflux
Lipid transport and binding assays
is defined as the difference between the efflux values deter- All data presented are representative of results from two or mined for incubations in medium that did or did not contain more independent experiments.
lipid-free apoA-I. The ABCA1- and apoA-I-dependent efflux is SR-BI. Assays for the uptake of [3H]COE from [3H]COE- defined as the difference between the apoA-I-dependent efflux HDL, efflux of [3H]cholesterol from labeled cells, and 125I-HDL values determined using cells that express the ABCA1 transgene binding were performed using ldlA[mSR-BI] cells and control (HEK[ABCA1]) and the empty vector controls (HEK[control]).
untransfected ldlA-7 cells as previously described (19, 23, 25).
Efflux values are presented as the percentage of total cellular We also determined the SR-BI-mediated efflux of [3H]choles- 1,2[ H]cholesterol present in the cells at the beginning of the 4 h terol from HEK293-EBNA-T cells transiently transfected with an SR-BI expression plasmid (19), as described for ABCA1 below.
The IC50 values are those concentrations of the compounds On the day of the assay, cells were preincubated for 1 h at 37C in that caused a half-maximal inhibition of lipid transport mediated assay medium (Ham's F12, 0.5% BSA, 0.5% DMSO, and 25 mM by either ABCA1 or SR-BI.
HEPES, pH 7.4) containing compounds at the indicated concen-trations. Subsequently, the cells were incubated for an additional Cross-linking assay of apoA-I binding to ABCA1
2 h with the same concentration of small molecules and with the The binding assays were conducted as described by Wang et indicated concentrations of 125I-HDL (binding), [3H]COE-HDL al. (62) with minor modifications. HEK293-EBNA-T cells were (uptake), or unlabeled HDL (efflux). 125I-HDL saturation bind- plated as described above on 12-well poly-d-lysine-coated plates ing assays were performed as previously described, in which a 40- and 3 days later were transfected with plasmids when the cells fold excess of unlabeled HDL was included in some of the incu- were 95% confluent, as described above. On day 4, the cells bations to permit the correction for nonspecific binding (19, were preincubated in DMEM (high glucose) and 0.2% (w/v) 45). Analysis of saturation binding curves was performed using fatty acid-free BSA with the indicated amounts of compounds at GraphPad Prism3 software from GraphPad Software, Inc. (San 37C for 1 h before incubation at 37C for 1 h with 2 g/ml 125I-apoA-I and the indicated compounds in the presence or For some experiments, the values presented were normalized absence of a 30-fold excess of unlabeled apoA-I. Cells were so that 100% of control represents activity in the absence of com- then washed, reversibly cross-linked with DSP (2 ml/well), and pounds. In Figs. 2 and 3B, 0% activity was defined as the activity washed again as previously described (62). For immunoprecipi- determined in the presence of a 1:800 dilution of the anti-SR-BI tation, cell lysates prepared in buffer RI [50 mM Tris, pH 7.6, 150 blocking antibody KKB-1 (25), a generous gift from Karen Kozar- mM NaCl, 0.25% sodium deoxycholate, 1% Nonidet P-40] con- sky. In other experiments, the 0% values were defined as the taining a protease inhibitors mixture (Roche Molecular Bio- amount of activity either in ldlA-7 control cells in the presence of chemicals GmbH), and 1 mM phenylmethylsulfonyl fluoride drugs (Fig. 4C) or in ldlA[mSR-BI] cells in the presence of a were subjected to centrifugation at 1,000 g in a microcentrifuge 40-fold excess of unlabeled HDL (Fig. 5). The amount of cell- for 10 min. The supernatant was collected and precleared with associated [3H]COE is expressed as the equivalent amount of 10 l of GammaBind G Sepharose beads for 1 h at room temper- [3H]COE-HDL protein (nanograms) to permit direct compari- ature with rotation. Polyclonal anti-ABCA1 antibody (50 g) di- son of the relative amounts of 125I-HDL binding and [3H]COE rected against the C terminus of ABCA1 (61) was added to the uptake (60).
preabsorbed cell lysates and incubated with rotation at room ABCA1. ABCA1-dependent efflux of [3H]cholesterol was temperature for 2 h. Twenty microliters of GammaBind G Sepha- measured using HEK293-EBNA-T cells transiently transfected rose beads was then added, and the incubation with rotation was with either an ABCA1 pcDNA1 expression vector or the empty continued at room temperature for 1 h. The samples were sub- pcDNA1 vector control, as previously described (44, 61). On day jected to a brief centrifugation, and the pellets were washed 0, HEK293-EBNA-T cells were plated at 200,000 cells/well on 24- three times with cold buffer RI at room temperature. The bound well poly-d-lysine-coated plates (Becton Dickinson) in medium A proteins were eluted from the beads by incubation and boiling in [DMEM containing high glucose and 10% (v/v) fetal calf serum Laemmli sample buffer in the presence of 5% -mercaptoetha- without antibiotics]. On day 1, cells were transfected with either nol, which also serves to break the DSP-mediated cross-links be- the ABCA1 vector or an "empty" vector control using Lipo- tween apoA-I and ABCA1. The eluted proteins were fractionated fectamine 2000 (Invitrogen) according to the manufacturer's by 12% SDS-polyacrylamide gel electrophoresis, and the radioac- suggestions. The media were removed 16 h later, and cells were tivity in the dried gels was analyzed using a PhosphorImaging incubated for 24 h in medium A supplemented with 0.5 Ci/ml STORM860 system (Molecular Dynamics, Inc., Sunnyvale, CA) 1,2[3H]cholesterol (Perkin-Elmer). The cells were then washed and ImageQuant software.
twice with DMEM (high glucose) and incubated for 1 h in assaymedium [DMEM (high glucose) supplemented with 0.2% fatty Determination of ABCA1 expression levels
acid-free BSA, 0.5% DMSO, and 25 mM HEPES, pH 7.4]. The To determine the effect of the compounds on the cell surface cells were then pretreated with the indicated concentrations of expression of ABCA1, we used previously described quantitative small molecules in the assay medium for 1 h at 37C and then in- assays for cell surface ABCA1 that detects a FLAG tag inserted cubated with the same concentrations of compounds in the pres- in the first large extracellular loop of ABCA1 (27). In brief, ence or absence of 10 g/ml lipid-free recombinant human HEK293-EBNA-T cells on 24-well plates were transfected with a apoA-I for an additional 4 h to permit the efflux of cellular cho- FLAG-ABCA1 cDNA or empty vectors as described above. The lesterol. The supernatant was harvested and clarified by centrifu- media containing DNA and lipofectamine were removed 1 day gation (5 min, 6,000 g), the cells were lysed in 0.1 M NaOH, and after transfection, and 1 day later the cells were exposed for 5 h radioactivity in both fractions was measured by liquid scintilla- at 37C to assay medium [DMEM (high glucose) supplemented tion counting.
with 0.2% fatty acid-free BSA, 0.5% DMSO, and 25 mM HEPES, Cholesterol efflux was defined as the amount of radioactivity pH 7.4] with or without 150 M BLT-4 or 500 M glyburide. The in the extracellular media at the end of the 4 h incubation di- cells were then chilled on ice for 10 min, and cell surface vided by the total amount of cellular radioactivity in the media ABCA1-FLAG expression was measured as described by Fitzger- plus cells (the percentage efflux). The apoA-I-dependent efflux ald et al. (27).
Journal of Lipid Research
Total cellular ABCA1 expression was measured by immunoblot fects of BLTs on ABCA1-mediated cholesterol efflux from analysis using a polyclonal antibody as previously described (61).
HEK293 cells transiently transfected with either an ABCA1- Transfected cells were treated for 5 h at 37C with 500 M glyburide expression vector (HEK[ABCA1]) or an empty vector or 150 M BLT-4 in assay medium [DMEM (high glucose) supple- mented with 0.2% fatty acid-free BSA, 0.5% DMSO, and 25 mM Cholesterol efflux was measured from cells labeled with HEPES, pH 7.4] and subsequently lysed in a hypotonic buffer (250 mM sucrose, 10 mM HEPES, pH 7.4, and 1 mM EDTA) supple- unesterified [ H]cholesterol for 24 h, followed by a 1 h mented with a protease inhibitor mini-cocktail containing EDTA preincubation with the BLT. This was followed by a 4 h in- (Roche). Nuclei and cell debris were removed by centrifugation cubation with the BLT in the presence or absence of 10 (800 g for 10 min). The amount of protein was measured in the g of protein per milliliter of lipid-free apoA-I, which postnuclear supernatants by the method of Bradford (63) and 15 served as an acceptor for cholesterol efflux. The amounts g of each sample were separated by 6% SDS-PAGE and transferred of [3H]cholesterol in the incubation media and the to nitrocellulose membranes. The membranes were processed for amounts remaining associated with the cells were mea- antibody staining, and ABCA1 was detected using an enhanced sured, and efflux was expressed as the percentage of cellu- chemiluminescence system (Pierce) as previously described (61).
lar [3H]cholesterol released into the medium during the Measurement of total cellular cholesterol and cholesteryl
4 h incubation. Figure 2 shows that the ABCA1-mediated
ester levels by TLC
[3H]cholesterol efflux to apoA-I (closed bars) was not in- HEK, ldlA7, and ldlA[mSR-BI] cells were labeled with [3H]cho- lesterol as described above for cholesterol efflux assays. After incu-bation for 5 h with or without 500 M glyburide or 150 M BLT-4,the cells were lysed (lysis buffer: 50 mM Tris, pH 7.6, 150 mMNaCl, 0.25% sodium deoxycholate, and 1% Nonidet P-40) for 30min at room temperature. A mixture of 2:1 chloroform-methanol(v/v) containing unlabeled lipids (200 g/l cholesterol and 200 g/l cholesteryl oleate) was added to the lysate as an internal standard. The organic phase of the resulting mixture was evapo-rated under nitrogen, dissolved in 75 l of chloroform, and frac-tionated by TLC using ITLC SA plates (Pal Corporation) usingpetroleum ether-ether-acetic acid (85:15:1). Spots containing cho-lesteryl ester, cholesterol, and origin spots were cut out, and radio-activity was measured by liquid scintillation counting.
Effects of BLTs on ABCA1- and scavenger receptor class B Synthesis of BLT-4
type I (SR-BI)-dependent efflux of [3H]cholesterol to extracellularacceptors. HEK293-EBNA-T cells were transiently transfected with Naphthyl isocyanate (Sigma-Aldrich) was dissolved in ethyl either an ABCA1 expression vector or the control pcDNA1 vector.
acetate and N-methyl-pyrrolidinone (Sigma-Aldrich) and then One day after transfection, the cells were labeled for 24 h with cooled to 0C. 2-Anisidine was added drop-wise by syringe over [3H]cholesterol and then preincubated for 1 h at 37C in assay me- 10 min. Solid naphthyl isocyanate (1.00 g, 5.92 mmol) was dis- dium containing 0.5% (v/v) DMSO with or without the indicated solved in 45 ml of anhydrous 1:2 N-methyl pyrrolidinone-ethyl concentrations of BLTs. After the preincubation, the cells were in- acetate (Acros Organics) under a nitrogen atmosphere and cubated for an additional 4 h at 37C with the same concentrations cooled to 0C. 2-Anisidine (900 l, 7.32 mmol) was added drop- of BLTs in the presence or absence of 10 g/ml lipid-free recombi- wise by syringe over 10 min and the reaction was stirred, first at nant human apolipoprotein A-I (apoA-I) to measure cholesterol ef- 0C for 2 h and then for an additional 2 h at room temperature.
flux. The amounts of [3H]cholesterol transferred from the cells to The reaction was quenched by the addition of 100 ml of 0.1 N the medium and the values for ABCA1- and apoA-I-dependent ef- HCl and extracted three times with 50 ml of dichloromethane.
flux were determined as described in Experimental Procedures The pooled organic fractions were dried over anhydrous potas- (closed bars). The control efflux values (percentage of cellular cho- sium carbonate, concentrated by rotary evaporation, and puri- lesterol released into the medium) measured in the absence of fied by flash chromatography using silica gel 60 (EMD Pharma- BLTs were as follows: HEK[ABCA1] with apoA-I, 7.4%; HEK ceuticals) equilibrated in 2:1 (v/v) hexane-ethyl acetate. BLT-4 [ABCA1] without apoA-I, 4%; HEK[control] with apoA-I, 1.7%; was eluted with 1:1 (v/v) hexane-ethyl acetate and lyophilized HEK[control] without apoA-I, 1%. The 100% control value deter- with a final yield of 22%. The product was more than 95% pure mined in the absence of BLTs for ABCA1- and apoA-I-dependent ef- as determined by liquid chromatography-mass spectrometry and flux was 2.7% [calculated as (7.4-4)(1.7-1)%]. Error bars repre- NMR spectroscopy. The 1H-NMR resonance frequencies for BLT-4 sent the range of duplicate determinations. In independent are as follows: (d experiments, SR-BI-mediated and HDL-dependent [3H]cholesterol 6-Me2SO): 3.89 (3H, s), 6.90 (1H, td, J efflux was determined in [3H]cholesterol-labeled ldlA[mSR-BI] Hz, 1.5 Hz), 6.96 (1H, td, J  1.5, 5.5 Hz), 7.02 (1H, dd, J  7, cells (open bars). Cells were preincubated with the indicated 1.5 Hz), 7.34 (1H, T, J  8 Hz), 7.44 (3H, m), 7.77 (1H, s), 7.79 amounts of BLTs for 1 h and then incubated for 2 h at 37C with (1H, s), 7.81 (1H, d, J  4 Hz), 7.84 (1H, s), 8.12 (1H, d, J  2 500 g of protein per milliliter of HDL and the same concentra- Hz), 8.17 (1H, dd, J  1.5, 6.5 Hz), 8.30 (1H, s), 9.52 (1H, s).
tions of BLTs in the presence or absence of a 1:800 dilution of theanti-SR-BI blocking antibody KKB-1 (25). The 100% of control RESULTS AND DISCUSSION value represents efflux in the absence of compound (50% of initialcellular radiolabeled cholesterol for efflux to HDL), and the 0% of Effects of BLTs on ABCA1-mediated [3H]cholesterol
control value represents efflux in the presence of HDL and the efflux to apoA-I
KKB-1 antibody (1:800 dilution) (12% of initial cellular radiola-beled cholesterol). There was no effect of the compounds on efflux To determine if SR-BI- and ABCA1-mediated choles- to HDL in the presence of KKB-1. Error bars represent standard de- terol efflux share common features, we examined the ef- viations from triplicate measurements.
Nieland et al. Small molecule inhibitors of cholesterol efflux
hibited by BLT-1 (10 M), BLT-3 (150 M), or BLT-5 (150 the presence of apoA-I as an acceptor in the incubation M), at concentrations that inhibit SR-BI-mediated [3H] medium. As the concentration of BLT-4 increased, there cholesterol efflux to HDL from ldlA[mSR-BI] cells, a cell was a substantial inhibition of efflux from HEK[ABCA1] line stably transfected with murine SR-BI (open bars) cells incubated with apoA-I, but there was little or no in- (45). At these concentrations, all three BLTs inhibited hibition of the relatively low background efflux from cholesterol efflux to HDL from HEK cells transfected with HEK[ABCA1] cells in the absence of apoA-I or from a cDNA construct encoding murine SR-BI, indicating that HEK[control] cells either with or without apoA-I. Figure HEK cells are not intrinsically resistant to BLT-1, BLT-3, 3B shows that the ABCA1- and apoA-I-dependent [3H] and BLT-5 (data not shown). Because the structures of cholesterol efflux, calculated as described in Experimen- BLT-1 and BLT-2 are very similar (45), we did not test BLT-2 tal Procedures, was inhibited by BLT-4 with an IC50 of 60 in these studies. In contrast to the other BLTs, BLT-4 (Fig.
M. This value is similar to that for its inhibition of SR-BI- 1B) at a concentration of 150 M inhibited ABCA1- and dependent [3H]cholesterol efflux to HDL (IC50  55 M; apoA-I-dependent efflux by 90%, approximately the Fig. 3B) (45).
same extent of its inhibition of cholesterol efflux observed The shared sensitivities to BLT-4 suggest that there may for SR-BI-mediated efflux to HDL (Fig. 2) from trans- be similarities in the mechanisms by which BLT-4 inhib- fected ldlA-7 (45) or HEK293 (data not shown) cells.
ited ABCA1- and SR-BI-mediated cellular cholesterol ef- Figure 3A shows the concentration dependence of
flux. The inability of BLT-1, BLT-3, and BLT-5 to inhibit BLT-4-mediated inhibition of [3H]cholesterol efflux. HEK ABCA1 raises the possibility that the mechanisms by which [ABCA1] and HEK[control] cells were labeled with [3H] they block SR-BI activity may differ from that of BLT-4, al- cholesterol and incubated with or without lipid-free apoA-I though additional studies will be required to directly ad- and the indicated concentrations of BLT-4. As described dress this question.
previously (28, 29, 44, 61, 64), we found that [3H]choles- terol efflux depended on the expression of ABCA1 and Effects of the ABCA1 inhibitor glyburide
on SR-BI-mediated [3H]cholesterol efflux to HDL
The inhibition of ABCA1-mediated cholesterol efflux by BLT-4 prompted us to determine if the ABCA1 inhibi-
tor glyburide (29, 31, 46–51) (Fig. 1A) could block SR-
BI-mediated [3H]cholesterol efflux to HDL. Figure 4
compares the concentration dependence of glyburide'sinhibition of ABCA1-mediated [3H]cholesterol efflux toapoA-I (A, C) with its effects on SR-BI-mediated [3H]cho- lesterol efflux to HDL (B, C).
As expected from previous studies (29, 31, 48–50), gly- buride inhibited ABCA1-mediated [3H]cholesterol effluxfrom HEK[ABCA1] cells to apoA-I (10 g protein/ml)with an IC50 of 300 M (Fig. 4A), and it had little effecton the low background efflux in the absence of ABCA1transgene expression, in the absence of apoA-I, or both.
This IC50 value is substantially greater than that for BLT-4(60 M). Thus, BLT-4 was a more potent inhibitor ofABCA1 than was glyburide.
Strikingly, glyburide's effects on SR-BI-mediated efflux were virtually identical to those on ABCA1-mediated ef-flux. Figure 4B shows that glyburide inhibited SR-BI-medi- Concentration dependence of the inhibition of ABCA1- ated [3H]cholesterol efflux from ldlA[mSR-BI] cells to and SR-BI-mediated [3H]cholesterol efflux by BLT-4. A: [3H]cho- lesterol efflux from HEK[ABCA1] (closed symbols) or HEK[con- g protein/ml) with a IC50 of 275 M, and it trol] (open symbols) cells was determined in the presence had little effect on the low background efflux in the ab- (squares) or absence (triangles) of 10 g/ml lipid-free recombi- sence of SR-BI transgene expression (ldlA-7 control cells), nant human apoA-I and the indicated concentrations of BLT-4 as in the absence of HDL, or both. Figure 4C, which com- described in the legend to Fig. 2 and Experimental Procedures. Val- pares directly glyburide's inhibition of ABCA1- and apoA- ues are expressed as the percentage of cellular [3H]cholesterol re- I-dependent cholesterol efflux and its inhibition of SR-BI- leased into the medium after a 4 h incubation at 37C. B: The effect and HDL-dependent cholesterol efflux, shows that the of BLT-4 on ABCA1-mediated and apoA-I-dependent [3H]choles-terol efflux (closed squares) was calculated from the data in A as drug is equally potent in inhibiting these cell surface described in the legend to Fig. 2 and Experimental Procedures. In transport proteins.
an independent experiment, SR-BI-mediated and HDL (500 g We also found that lipid-free apoA-I (10 g protein/ml) protein/ml)-dependent [3H]cholesterol efflux (open circles) was was a poor acceptor of SR-BI-mediated [3H]cholesterol ef- determined as described in the legend to Fig. 2 and Experimental flux compared with HDL (500 g protein/ml) [2% vs.
Procedures. The maximum efflux in the absence of BLT-4 (60% oftotal cellular cholesterol) was set to 100% (the 0% of control was 61%, respectively; also see ref. (36)]. The low level of lipid-free apoA-I-dependent [3H]cholesterol efflux from Journal of Lipid Research
of glyburide interfering with other cellular processes, in-cluding features of cellular cholesterol metabolism. Wewere unable to detect glyburide inhibition of cholesterolesterification (thin layer chromatographic assay) or of thetotal cellular cholesterol levels in either HEK cells orldlA[mSR-BI] cells loaded with [3H]cholesterol (data notshown). Thus, at least some aspects of cellular cholesterolmetabolism appear to be unaffected by high-dose gly-buride treatment under the conditions used in these ex-periments. We previously have shown that BLT-4 is a rela-tively specific inhibitor in that it does not affect multiplemembrane transport processes, such as clathrin-depen-dent endocytosis, lipid-raft-dependent endocytosis, andthe protein secretory pathway, nor does it have a detect-able impact on the integrity of the actin and microtubularnetworks (45). As was the case with glyburide, BLT-4 didnot appear to inhibit cholesterol esterification or to altertotal cellular cholesterol levels in either HEK cells orldlA[mSR-BI] cells loaded with [3H]cholesterol (data notshown).
Effects of glyburide on SR-BI-mediated [3H]COE uptake
from [3H]COE-HDL and 125I-HDL binding
Previous studies have shown a close relationship be- Concentration dependence of the inhibition of ABCA1- tween SR-BI-mediated binding to HDL with both SR-BI- and SR-BI-mediated [3H]cholesterol efflux by glyburide. A: mediated cholesterol efflux to HDL and selective lipid up- [3H]cholesterol efflux from HEK[ABCA1] (closed symbols) or take from HDL (24, 57, 65–68). We tested the effects of HEK[control] (open symbols) cells was determined in the pres- glyburide on these two additional activities of SR-BI. Fig-
ence (squares) or absence (triangles) of 10 g/ml lipid-free re- ure 5 shows that glyburide suppressed SR-BI-mediated
combinant human apoA-I and the indicated concentrations ofglyburide as described in Experimental Procedures. B: [3H]choles- [3H]COE uptake from [3H]COE-HDL by ldlA[mSR-BI] terol efflux from SR-BI-expressing ldlA[mSR-BI] (closed symbols) cells (IC50  150 M). Flow cytometric analysis of the sur- or control ldlA-7 (open symbols) cells was determined in the pres- face expression of SR-BI established that the effects of gly- ence (circles) or absence (triangles) of 500 g of protein per milli- buride (at concentrations of 500 M) on SR-BI's activi- liter of HDL and the indicated concentrations of glyburide as ties were not attributable to a reduction in the steady-state described in the legend to Fig. 3B. C: ABCA1-mediated and apoA-I-dependent [3H]cholesterol efflux (closed squares) was calculated level of SR-BI on the cell surface (data not shown).2 Thus, from the data in A as described in Experimental Procedures. SR-BI- glyburide inhibited both lipid transport activities of SR-BI, mediated and HDL-dependent [3H]cholesterol efflux (open circles) selective uptake and cholesterol efflux, as is the case for was calculated from the data in B. Efflux to HDL in ldlA[mSR-BI] cells in the absence of compound, and in ldlA-7 cells in the pres- BLTs 1–5 increase the affinity of 125I-HDL binding to ence of compound, was used to set the 100% and 0% of control SR-BI at 37C (lower apparent Kd) (45). The decreased values, respectively. There was a small, KKB-1-inhibitable effect ofglyburide on cholesterol efflux to HDL in ldlA-7 cells, which repre- apparent Kd values are attributable, at least in part, to sents the efflux mediated by the low levels of endogenous SR-BI decreased dissociation rates and are not accompanied that is expressed in these cells.
by substantially altered maximal binding values. Figure5 shows that increasing concentrations of glyburide in- ldlA[mSR-BI] and untransfected ldlA-7 control cells (2%)was insensitive to glyburide (data not shown). It is thus un- 2 When ldlA[mSR-BI] or ldlA7 cells were treated with a very high likely that glyburide's ability to affect SR-BI's activity concentration of glyburide (1 mM) for a total of 3 h, we observed mor- has complicated the interpretation of previous studies phological changes (e.g., changes in the appearance of the nucleus) of ABCA1 activity, because those studies used lipid-free and occasionally signs of toxicity as measured by trypan blue exclusion.
3 Glyburide at a concentration of 1 mM has been reported to pre- apoA-I as an acceptor of glyburide-sensitive cholesterol ef- vent specific ABCA1- and apoA-I-dependent phospholipid (phosphati- dylcholine) efflux from cells, determined as the difference in phospho- We and others have found that the concentration of gly- lipid efflux from ABCA1-expressing cells measured in the presenceand absence of apoA-I in the extracellular medium (29). We also ob- buride required to inhibit ABCA1 is substantially higher served this inhibition at very high doses of glyburide (500 M and than its reported in vitro Ki for other ABC proteins, such higher). However, we found in our experiments that the glyburide- as SUR1/2 [reviewed in ref. (53)]. It is possible that the induced decrease in specific apoA-I-dependent phospholipid efflux waslargely attributable to an increase in ABCA1-mediated efflux to the inhibition of ABCA1-mediated cholesterol efflux by gly- apoA-I-free medium rather than to a substantial decrease in efflux to buride is a secondary consequence of high concentrations the apoA-I containing medium (data not shown).
Nieland et al. Small molecule inhibitors of cholesterol efflux
Effects of glyburide on the concentration dependence of 125I-HDL binding to SR-BI-expressing cells. SR-BI-expressing Effects of glyburide on SR-BI-mediated [3H]cholesteryl ldlA[mSR-BI] cells were preincubated in the absence (closed oleyl ether ([3H]COE) uptake from [3H]COE-HDL and 125I-HDL squares) or presence (open squares) of 250 M glyburide and then binding. SR-BI-expressing ldlA[mSR-BI] or control ldlA-7 cells were incubated for 2 h at 37C with the same amount of glyburide and preincubated with the indicated concentrations of glyburide, and the indicated concentrations of 125I-HDL in the presence (single in- then the uptake of [3H]COE from [3H]COE-HDL (10 g protein/ cubations) or absence (duplicate incubations) of a 40-fold excess of ml) (closed circles) or the binding of 125I-HDL (10 g protein/ml) unlabeled HDL. The specific binding values are shown.
(open circles) was determined in the presence or absence of a 40-fold excess of unlabeled HDL (duplicate incubations). The specificvalues (difference between the values in the absence and presence 2–4). We did not detect a decrease in either the cell sur- of excess HDL) were normalized such that the value in the absenceof glyburide was set to 100%. The 100% of control values (nano- face levels or the total protein levels of ABCA1 in cells grams of HDL protein per milligram of cell protein) for ldlA[mSR- treated under similar conditions with either compound BI] cells were as follows: [3H]COE uptake, 1,962; 125I-HDL binding, (data not shown). Thus, glyburide and BLT-4 inhibition of 198. The 100% of control values for ldlA-7 cells were as follows: 125I-apoA-I binding to ABCA1 cannot be explained by de- [3H]COE uptake, 289; 125I-HDL binding, 21.
creases in either cell surface or total levels of ABCA1. Theinhibition of apoA-I binding to ABCA1 by glyburide andBLT-4 contrasts their abilities to enhance the affinity of creased the binding of 125I-HDL (10 g protein/ml) to HDL binding to SR-BI.
ldlA[mSR-BI] cells in a manner that was correlated in- It is possible that the reciprocal inhibition of SR-BI and versely with its inhibition of lipid transport (Figs. 4 and 5).
ABCA1 by the structurally distinct small molecules BLT-4 The increased binding of 125I-HDL to SR-BI observed at a and glyburide was simply a coincidence of multiple inde- subsaturating concentration of 125I-HDL appeared to be pendent activities of these compounds. An alternative, attributable, at least in part, to a glyburide-induced in- more appealing explanation is that, despite the very dif- crease in the affinity of SR-BI for 125I-HDL without a sub- ferent structures and physiologic functions of SR-BI and stantial change in the maximal binding values (Fig. 6).
For the 125I-HDL concentrations used in these experi-
ments [a relatively broad density range (23)], the appar-
ent Kd values were 26.2 3.7 g protein/ml in the ab-
sence of glyburide and 4.7 0.7 g protein/ml in the
presence of 250 M glyburide. Thus, the effects of the
ABCA1 inhibitor glyburide on SR-BI's activities were simi-
lar to the activities of the BLTs: increased HDL binding
and decreased lipid transport.
Effects of BLT-4 and glyburide on apoA-I binding
to ABCA1
The increase in affinity of SR-BI for HDL induced by Effects of BLT-4 and glyburide on 125I-apoA-I binding to BLT-4 [see also ref. (45)] and glyburide prompted us to ABCA1. HEK293-EBNA-T cells were transiently transfected with ei- examine the binding of apoA-I to ABCA1. Figure 7 shows
ther an ABCA1 expression vector (ABCA1) or a control pcDNA1 a representative assay that involved incubating ABCA1- vector (No ABCA1). One day after transfection, the cells were pre- expressing cells with 125I-apoA-I, treating the cells with a incubated for 1 h at 37C in assay medium with the indicated con- cleavable cross-linking agent (DSP), immunoprecipitating centrations of BLT-4 or glyburide. The binding of 125I-apoA-I (2 g/ml, 1 h, 37C) in the presence or absence of a 30-fold excess of cross-linked complexes with anti-ABCA1 antiserum, and unlabeled apoA-I and the indicated concentrations of inhibitors then detecting the coprecipitated 125I-apoA-I after break- was measured using a dithiobis (succinimidyl propionate) cross- ing the cross-links and fractionating the samples by SDS- linking assay as described in Experimental Methods. Bound 125I- PAGE (62). As expected, there was no detectable binding apoA-I was fractionated by SDS-PAGE and detected in the gels us- of 125I-apoA-I to control cells transfected with empty vec- ing a PhosphorImaging STORM860 system (top), and the relative tor (no ABCA1). Both glyburide [250–1,000 M; see also amounts of 125I-apoA-I in the lanes were determined using Im-ageQuant software (bottom). Values shown in the lower portion of ref. (62)] and BLT-4 (150 M) effectively blocked the the figure are representative of two independent experiments per- binding of 125I-apoA-I to ABCA1 at concentrations that in- formed in duplicate. Error bars represent the ranges of duplicate hibit ABCA1-mediated cholesterol efflux to apoA-I (Figs.
Journal of Lipid Research
ABCA1, some features of their mechanisms of lipid trans- ously providing the KKB-1 antibody, and Mason Freeman, Tim port may have similarities or even share common steps Mitchison, and the members of the Harvard Institute of Chem- that are sensitive to BLT-4 and glyburide. This explanation istry and Cell Biology (ICCB) for helpful discussions and is supported by the observation that the potencies (IC support. The experiments shown in Figs. 2–6 and 7 were per- for glyburide inhibition of SR-BI and ABCA1 are virtually formed by T.J.F.N. and A.C., respectively. Z.M. synthesized BLT-4, identical, as they are for BLT-4.
M.L.F. determined the expression levels of ABCA1, and A.C.
determined the total cellular cholesterol and cholesteryl ester The molecular targets of BLT-4 and glyburide responsi- levels. This work was supported by National Institutes of Health ble for their inhibition of SR-BI- and ABCA1-mediated Grants HL-48739, HL-66105, and HL-52212 (M.K.), HL-48739 lipid transport activities have not yet been identified, and and HL-68216 (V.I.Z.), GM-62566 (T.K.), and HL-68988 to Ma- they need not be the same. However, these lipid transport son Freeman, and Grants CA-78048 and GM-62566 supported proteins themselves are obvious candidates. Glyburide Z.M. through the ICCB.
and BLT-4 might bind to either the same or distinct siteson each protein. It is also possible that the direct targetsof these inhibitors are not the transporters themselves but rather some other protein(s) or lipids (e.g., a specificmembrane domain). It is noteworthy that, even though 1. Rigotti, A., H. E. Miettinen, and M. Krieger. 2003. The role of the the structures of glyburide and BLT-4 (with the exception high-density lipoprotein receptor SR-BI in the lipid metabolism of of the methoxyphenyl and the urea functionalities) differ endocrine and other tissues. Endocr. Rev. 24: 357–387.
2. Oram, J. F. 2003. HDL apolipoproteins and ABCA1: partners in (Fig. 1), they both can inhibit the activities of several ABC the removal of excess cellular cholesterol. Arterioscler. Thromb. Vasc. proteins. BLT-4 inhibits ethidium bromide efflux from Biol. 23: 720–727.
Staphylococcus aureus mediated by NorA, a member of the 3. Acton, S. L., P. E. Scherer, H. F. Lodish, and M. Krieger. 1994. Ex- pression cloning of SR-BI, a CD36-related class B scavenger recep- ABC superfamily (69), as well as ABCA1 activity (this tor. J. Biol. Chem. 269: 21003–21009.
study). Glyburide, which was originally identified as a 4. Krieger, M. 1999. Charting the fate of the "good cholesterol": iden- drug that binds to and inhibits the ABC subunit (SUR) of tification and characterization of the high-density lipoprotein re-
ceptor SR-BI. Annu. Rev. Biochem. 68: 523–558.
the ATP-sensitive K ATP channels, can interfere with path- 5. Trigatti, B., H. Rayburn, M. Vinals, A. Braun, H. Miettinen, M.
ways involving the ABC proteins cystic fibrosis trans- Penman, M. Hertz, M. Schrenzel, L. Amigo, A. Rigotti, and M.
membrane regulator (54), bile salt export pump (56), Krieger. 1999. Influence of the high density lipoprotein receptor and multidrug resistance P-glycoprotein (55), as well as SR-BI on reproductive and cardiovascular pathophysiology. Proc. Natl. Acad. Sci. USA. 96: 9322–9327.
ABCA1 (29, 31, 46–51). It is possible that the target of the 6. Kozarsky, K. F., M. H. Donahee, J. M. Glick, M. Krieger, and D. J.
inhibitory activities of both drugs could be a cell surface Rader. 2000. Gene transfer and hepatic overexpression of the or intracellular ABC transporter(s) other than ABCA1 HDL receptor SR-BI reduces atherosclerosis in the cholesterol-fed
LDL receptor-deficient mouse. Arterioscler. Thromb. Vasc. Biol. 20:
that may directly or indirectly influence lipid transport mechanisms mediated by both SR-BI and ABCA1.
7. Arai, T., N. Wang, M. Bezouevski, C. Welch, and A. R. Tall. 1999.
Despite the similar abilities of either glyburide or BLT-4 Decreased atherosclerosis in heterozygous low density lipoproteinreceptor-deficient mice expressing the scavenger receptor BI to inhibit lipid transport mediated by ABCA1 and SR-BI, transgene. J. Biol. Chem. 274: 2366–2371.
there are several striking differences in the mechanisms 8. Covey, S. D., M. Krieger, W. Wang, M. Penman, and B. L. Trigatti.
by which these transporters function. Lipid-free apoA-I 2003. Scavenger receptor class B type I-mediated protectionagainst atherosclerosis in LDL receptor-negative mice involves its binds preferentially to ABCA1, and along with pre-apoA-I expression in bone marrow-derived cells. Arterioscler. Thromb. Vasc. it is the preferred cholesterol acceptor from ABCA1 rela- Biol. 23: 1589–1594. [Erratum. 2003. 23: 1950.]
tive to spherical, lipid-rich HDL particles (32–36). In con- 9. Holm, T. M., A. Braun, B. L. Trigatti, C. Brugnara, M. Sakamoto, trast, spherical, lipid-rich HDL particles are the preferred M. Krieger, and N. C. Andrews. 2002. Failure of red blood cell mat-uration in mice with defects in the high-density lipoprotein recep- ligands for SR-BI (15). The effects of glyburide and BLT-4 tor SR-BI. Blood. 99: 1817–1824.
on ligand binding also highlight the differences between 10. Miettinen, H. E., H. Rayburn, and M. Krieger. 2001. Abnormal these transporters. Both inhibitors block ligand binding lipoprotein metabolism and reversible female infertility in HDL
receptor (SR-BI)-deficient mice. J. Clin. Invest. 108: 1717–1722.
to ABCA1, whereas they enhance the ligand binding affin- 11. Ueda, Y., E. Gong, L. Royer, P. N. Cooper, O. L. Francone, and ity of SR-BI. Additional studies will be required to eluci- E. M. Rubin. 2000. Relationship between expression levels and date the mechanisms underlying the complex effects of atherogenesis in scavenger receptor class B, type I transgenics. J.
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12. Kozarsky, K. F., M. H. Donahee, A. Rigotti, S. N. Iqbal, E. R. Edel- In summary, we have identified BLT-4 as a new and rela- man, and M. Krieger. 1997. Overexpression of the HDL receptor tively potent inhibitor of ABCA1. We also show that gly- SR-BI alters plasma HDL and bile cholesterol levels. Nature. 387:
414–417.
buride is a heretofore unrecognized inhibitor of SR-BI.
13. Braun, A., B. L. Trigatti, M. J. Post, K. Sato, M. Simons, J. M. Edel- The reciprocal inhibition of these transport proteins by berg, R. D. Rosenberg, M. Schrenzel, and M. Krieger. 2002. Loss of BLT-4 and glyburide raises the possibility that the mecha- SR-BI expression leads to the early onset of occlusive atheroscle- nisms of lipid transport by SR-BI and ABCA1 might possi- rotic coronary artery disease, spontaneous myocardial infarctions,severe cardiac dysfunction, and premature death in apolipopro- bly involve another ABC transporter.
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Nieland et al. Small molecule inhibitors of cholesterol efflux

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