Proc. Natl. Acad. Sci. USA
`Vol. 93. p . ll99l—ll995, October I996
`Pharmaco ogy
`
`An inhibitor of the microsomal triglyceride transfer
`inhibits apoB secretion from HepG2 cells
`(Iipoprotelns/ lipid transfer protein/protein disulflde isomerase/atherosclerosis)
`
`protein -
`
`Hams JAMn.*, DAVID A. GoRnoN*, DAVID C. Eus11cET¢, CIN-DY M. Bkooxsl, JOHN K. Dickson, R.§,
`YING CHEN§, BEVERLY RlCCl", CHING-HSUEN CHu", THOMAS W. HARRITY’
`, CARL P. CIOSEK, 112.’,
`Scorr A. Bn.LER§, RICHARD E. GREGG‘, AND JOHN R. WE'rrERAu‘ll
`
`.-
`
`‘Department of Metabolic Diseases and 5Division of Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000; and ‘Division
`of Biomolecular Screening, Bristol-Myers Squibb Company, Wallingford, Cl‘ 06492-5100
`:
`
`Communicated by Leon E. Rosenberg, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ,
`January 22, I 996)
`
`July 16,
`
`l9_967(received for review
`
`The microsomal triglyceride (TG) transfer
`ABSTRACT
`protein (MTP) is 5. heterodiineric lipid transfer protein that
`catalyzes the transport of triglyceride, cholesteryl ester, and
`phosphatidylcholine between membranes. Previous studies
`showing that the proximal cause ofabetalipoproteinemia is an
`absence of MTP indicate that MTP function is required for the
`assembly of the apolipoprotein B (apoB) containing plasma
`lipoproteins, i.e., very low density lipoproteins and chylomi-
`crons. However, the precise role of MTP in lipoprotein as-
`sembly is not known.
`ln this study,
`the role of MTP in
`lipoprotein assembly is investigated using an inhibitor of
`MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-
`piperidinyl]-2,3-dihydro-1H-isoindoI-l-one (BMS-200150).
`The similarity of the lC5o for inhibition of bovine MTP-
`medlated TG transfer (0.6 uM) to the K,. for binding of
`BMS-200150 to bovine MTP (1.3 uM) strongly supports that
`the inhibition of TG transfer is the result of a direct effect of
`the compound on MTP. BMS-200150 also inhibits the transfer
`of phosphatidylcholine, however to a lesser extent (30% at a
`concentration that almost completely inhibits TG and cho-
`lesteryl ester transfer). When BMS-200150 is added to cul-
`tured HepG2 cells, a human liver-derived cell line that secretes
`apoB containing lipoproteins, it inhibits apoB secretion in a
`concentration dependent manner. These results support the
`hypothesis that transport of lipid, and in particular,
`the
`transport of neutral lipid by MTP, plays a critical role in the
`assembly of apoB containing lipoproteins.
`
`Genetic studies (1-4) have demonstrated that an absence ‘of
`microsomal triglyceride (TG) transfer protein (MTP) causes
`abetalipoproteinemia, a disease characterized by a defect in
`the assembly and secretion of apolipoprotein B (apoB) con-
`taining plasma lipoproteins. These studies indicate that MTP
`is required for the production of the apoB containing lipopro-
`teins, very low density lipoproteins and chylomicrons by the
`liver and intestine. The requirement for MTP for lipoprotein
`production is further supported by studies using cell lines that
`are not of hepatic or intestinal origin. When a truncated form
`of apoB representing 53% of the full-length apoB-100 is
`expressed in HeLa cells, virtually no apoB is secreted (5).
`However, when MTP is coexpressed with apoB, apoB is
`secreted as a lipoprotein particle. Similar findings have been
`observed in COS cells (6).
`MTP is found in the lumen ofmicrosomes isolated from liver
`and intestine (7). The protein purified from bovine liver is a
`heterodimer consisting of the multifunctional enzyme, protein
`disulfide isomerase, and a unique, large 97-kDa subunit (8-
`10). In vitro, MTP catalyzes the transport of TG, cholesteryl
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked “advertisement” in
`accordance with 18 U.S.C. §l734 solely to indicate this fact.
`
`ester (CE), and phosphatidylchol.ine’(PC) between membranes
`(3),
`g
`.
`,
`3
`‘
`.:
`‘ Although MTP is required for the assembly of plasma
`lipoproteins, the precise role of MTP in the ‘assembly process
`is not known. We have proposed that MTP transports lipid
`molecules from the endoplasmic reticulum (ER) membrane
`where they are synthesized, to developing lipoprotein particles
`in the ER lumen. However, there has not been any direct
`experimental evidence to support this hypothesis to date. Here
`we report the identification and characterization of a small
`molecule inhibitor of MTP-mediated lipid ‘transport. This
`molecule, BMS-200150, selectively inhibits TG and CE trans-
`port. When the inhibitor is added to HepG2 cells, a human
`liver-derived cell line that assembles and secretes apoB con-
`taining lipoproteins, apoB secretion is inhibited. This supports
`our hypothesis that MTP-mediated lipid transport is required
`for lipoprotein assembly.
`
`MATERIALSAND METHODS
`Radiolabelcd 1,2-dipalmitoyl ~L-3-phosphatidyl[N-methyl-
`3H]choline and glycerol tri[1-"‘C]oleate were obtained from
`Amersham. All unlabeled lipids were obtained from Sigma.
`Lipids were stored under N2 gas incliloroform at —20"C.
`HepG2 human hepatoblastoma cells were obtained from the
`American Type Culture Collection, and were maintained as
`recommended by the supplier under standard cell culture
`conditions (37°C with a 5% CO2/95% air atmosphere). Tissue
`culture media and serum were obtained from GlBCO. Mono-
`clonal and polyclonal antibodies against apoB and apoAl were
`obtained from Biodesign International (Kennebunkport, ME).
`Synthesis of BMS-200150 and [3H]BMS-200150. A mixture
`of 4-amino-1-benzylpiperidine (101 mmol) and phthalic anhy-
`dride (101 mmol) was heated at 125°C for 30 min, cooled to
`room temperature, and purified bylflash chromatography
`(silica, 30% EtOAc/hexane) to yield?2-[1-(phenylmethyl)-4-
`piperidinyl]-1H-isoindol-l,3(2H)-dionc (compound A) (77%
`yield) as a white solid [melting point (mp) 151—154°C].
`A mixture of compound A (62.5 mmol) and Zn dust‘(438
`mmol) in ACOH (250 ml) was heated at reflux for 18 h, cooled
`to room temperature, filtered through Celite (Aldrich), and
`concentrated. The residue was dissolved in CH;Cl2 (500 ml),
`washed with saturated NaHCO3 (2 X .100 ml) and brine (100
`ml), dried over MgSO4, and concentrated. The crude product
`was recrystallized from i-PrOH to yield 2,3-dihydro-2-[l-
`
`Abbreviations: TG, triglyceride; MTP, microsomal TG transfer pro-
`tein; CE, cholesteryl ester; PC, phosphatidylcholine, ER, endoplasmic
`reticulum; SUV. small unilamellar vesicles; apo, apolipoprotein;
`DMSO, dimethyl sulfoxide.
`‘Present address: Bayer Corporation, West Heaven, CI 06516;‘
`‘To whom reprint requests should be addressed at: Bristol—Myers
`Squibb, P.O. Box 4000, Princeton, NJ 08543.
`'
`5
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`11992
`
`Pharmacology: Jamil el al.
`
`Proc. Natl. Acad. Sci. USA 93 (I996)
`
`(phenylmethyl)-4-piperidinyl]-1H-isoindol-1-one (compound
`B) (80% yield) as a white solid (mp 130—133°C).
`A mixture of compound B (26.4 mmol), AcOH (53 mmol),
`and 10% Pd/C (0.66 mmol Pd) in EtOH (65 ml) was agitated
`on a Parr hydrogenator at 45 psi of Hz for 48 h, filtered through
`Celite, and concentrated. The residue was dissolved in CHCI3
`(100 ml), washed with 1 M KOH saturated with NaCl (2 X 30
`ml), dried over MgSO4, and concentrated to yield 2-(4-
`piperidinyl)-2,3-dihydro-1H-isoindol-1-one (compound C)
`(77% yield) as a white solid (mp 137—140°C).
`'y-phenylbenze-
`A mixture of «compound C (9.26 mmol),
`nepropanol, 4-methylbenzenesulfonate ester (1 1) (9.26 mmol),
`and K;CO3 (14.8 mmol) in i-PrOH (25 ml) was heated at reflux
`for 18 h, cooled to room temperature, filtered, and concen-
`trated. The crude product was purified by flash chromatogra-
`phy (silica, 2.5% MeOH/CH2Cl¢) to yield the free amine (74%
`yield) as a colorless oil. A portion of the free amine (2.4 mmol)
`in MeOH (7.0 ml) was treated with 1 M HCI/Et2O (4.9 mmol)
`and concentrated. The residue was recrystallized from EtOH
`to yield 2-[1-(3, 3-dipheriylpropyl)-4-piperidinyl]-2,3-dihydro-
`1H-isoindol-1-one, monohydrochloride (BMS-200150) (68%
`yield) as a .white solid (mp 237—241°C): ‘H NMR (400 MHz,
`CD3OD) 6 7.76 (d, 1H, J = 7.3 Hz), 7.55 (m, 2H), 7.49 (t, 1H,
`J = 7.3 Hz), 4.50 (s, 2H), 4.39 (m, 1H), 4.05 (t, 1H, J = 7.7 Hz),
`3.68 (br d, 2H, J = 12 Hz), 3.12 (m, 4H), 2.59 (m, 2H), 2.20 (m,
`2H), 2.08 (br d, 2H, J = 12 Hz).
`Radiolabeled BMS-200150 was synthesized as follows. Di-
`methyl sulfide (14.5 mmol) was added to a solution of N-
`chlorosuccinimide (11.4 mmol) in CH2Cl2 (40 ml) at —40°C.
`The reaction was warmed to room temperature for 30 min and
`recooled to -40°C, and a solution of 3,3-diphenyI-2-propene-
`1-01 (12) (10.3 mmol) in CHZCI2 (3 ml) was added dropwise.
`The reaction was stirred at -40°C for 2 h, diluted with hexane
`(100 ml), washed with H20 (50 ml) and brine (2 X 50 ml), dried
`over Na2SO4, and concentrated to yield 1-chloro-3,3-diphenyl-
`2-propene (compound D) (81% yield) as a colorless oil.
`A mixture of compound C (7.56 mmol), compound D (8.32
`mmol), and KzCO3 (7.94 mmol) in dimethylformamide (35 ml)
`was heated at 50°C overnight, cooled to room temperature,
`and concentrated. The residue was dissolved in CHZCI2 (150
`ml), washed with H20 (2 X 50 ml) and brine (2 X 50 ml), dried
`over MgSO4, and concentrated. The crude solid was purified
`by flash chromatography (silica, 3% MeOH/CH;C|2) to yield
`2-[1-(3, 3-diphenyl-2-propenyl)-4-piperidinyl]-2,3-dihydro-
`1H-isoindol-1-one (compound E) (63% yield) as a white solid
`(mp 164—I67°C).
`Olefin E was reduced by catalytic tritiation performed by
`NEN to yield [3H]BMS-200150 with a specific activity of 44.0
`Ci/mmol (1 Ci = 37 GBq) and a radiochemical purity of 99%.
`Isolation and Purification of MTP. Bovine MTP was puri-
`fied from bovine liver as described previously (13). The
`purified protein had an activity of 3.0% TG transfer/min/pg
`of protein in our assay (see below), and showed only two bands
`with apparent molecular masses of 88 and 58 kDa on SDS/8%
`polyacrylamide gels.
`Human MTP was partially purified from 10/150 cm’ flasks
`of HepG2 cells (3 X 103 cells per flask) grown to confluency
`in RPMI 1640 medium containing 10% fetal bovine serum.
`Cells were homogenized in 50 ml of 50 mM Tris-HCI, pH
`' 7.4/5.0 mM EDTA/50 mM KCl containing 1.0 mM phenyl-
`methylsulfonylfluoride and 0.5 p.g/ml leupeptin (homogeni-
`zation buffer) using a Polytron PT3000 (Brinkmann) for 1 min
`at the half maximal setting. Cell homogenate was diluted with
`homogenization buffer to a protein concentration of 1.75
`mg/ ml. To release protein from the microsomal lumen, one
`part of sodium deoxycholate (0.54%, pH 7.5) was added to 10
`parts of diluted homogenate while vortexing, and then it was
`left to stand on ice for 30 min. Cell membranes were subse-
`quently removed by centrifugation at 100,000 X g for 1 h. The
`supernatant was dialyzed against 10 mM NaH2PO4, pH 7.1/1.0
`
`mM EDTA/0.02% sodium azide and protease inhibitors (1.0
`mM benzamidine/ 1.0 mM phenylmethylsulfonyl fluoride/0.01
`mM L-tosylamido-2-phenylethyl chloromethyl ketone/0.4
`pg/ml each leupeptin,.antipain, and pepstatin), loaded onto a
`DEAE~Sephacel (Sigma) column (20 ml) and eluted with a
`linear gradient of 0 to 500 mM_ NaCl. M_;’I‘P eluted between 200
`and 300 mM NaCl.
`‘
`.
`Inhibition of Lipid Transport. The inhibition of lipid trans-
`fer activity in the presence of BMS-200150 was measured in an
`assay similar to one that has been previously described (1, 14).
`Donor and acceptor small unilamellar vesicles (SUVs) were
`prepared by bath sonication in 15 mM Tris-HCI, pH 7.5 / 1 mM
`EDTA acid/40 mM NaCl/0.02% sodium azide (assay buffer).
`The lipid transfer assay mixture contained‘ donor membranes
`(40 nmol egg PC/7.5 mol % _cardiolipi'n/0.25 mol % radiola-
`beled substrate), acceptor membranes (240 nmol egg PC), 5.0
`mg BSA, and various concentrationsofj BMS-200150 in a total
`volume of 0.68 ml assay buffer. BMS-200150 was dissolved in
`dimethyl sulfoxide (DMSO) and added to the reaction mix-
`ture. The final concentration of DMSO was 0.5%. The reaction
`was started by the addition of MTP in 20 [JJ assay buffer. After
`a 60-min incubation at 37°C, the reaction was terminated by
`the addition of 0.5 ml of DE-52 cellulose (Whatman)~pre-
`equilibrated in 15 mM Tris-HCl, pH 7.4/1.0 mM EDTA/
`0.02% sodium azide (1:1, vol/vol). The mixture was agitated
`for 5 min and centrifuged at maximum speed in a Biofuge B
`centrifuge (Baxter Scientific Products, McGaw Park, IL) for 3
`min to pellet the DE-52 bound donor vesicles. First order
`kinetics were used to calculate the lipid transfer rate using the
`equation [S] = [S]oe-"' (8), where [S]o and [S] are the fraction
`of the available labeled lipid in the donor membrane at times
`0 and t, respectively, and k is the fraction of the available
`labeled lipid transfered per unit time. This calculation corrects
`for the depletion of labeled lipid in donor vesicles that occurs
`with time.
`"
`-
`.
`‘
`‘
`
`Equilibrium Binding Assay. Fifty or one hundred micro-
`grams of bovine MTP in 200 pl of assay buffer was pipetted
`into a 12-14 kDa cut-off dialysis bag (Spectra/For) and
`dialyzed against various concentrations of [3H]BMS-200150
`(0.1 to 100 p.M) in 1.0 ml of assay buffer in 1.5 ml Eppendorf
`tubes for 24 h. The concentration of bound and free inhibitor
`was quantitated by measuring the protein inside the dialysis
`bag and radioactivity both inside and outside the dialysis bag.
`Inhibition of Apolipoprotein Secretion from HepG2 Cells.
`HepG2 cells were seeded at 'a densityiof 50% confluency in
`48-well plates and allowed to grow for 48 h before treatment.
`At this time, the medium was replaced with fresh medium
`containing 0.5% DMSO and the indicated concentrations of
`BMS-200150. After a 16 h incubation under standard cell
`culture conditions, the medium was diluted with fresh tissue
`culture medium 30-fold for an apoB ELISA, and 60-fold for an
`apoAl ELISA. A sandwich ELISA wasiused to, measure apoB
`in the media as has been reported (15). A similar assay was
`used to quantitate apoAl. For the apoAl assay, the primary
`and secondary antibodies were a monoclonal anti-human
`apoAl (l:500 dilution) and a goat anti-human apoAl poly-
`clonal antibody (11500). The concentration of the'respective
`proteins was measured against a 2-fold dilution standard curve
`from l.25—40 ng/ml of the purified proteins. In this range of
`concentrations, both assays show ‘a linear response. ‘Each drug
`concentration was tested in duplicate cultures, and apoB and
`apoAl were measured by ELISA’ in each culture in triplicate.
`Analyses of ApoB Secretion from HepG2 Cells by [35S]Me-
`thionine Labeling and Immunoprecipitation. Duplicate
`100-mm culture dishes containing confluent monolayers of
`HepG2 cells were incubated for 30 min in 10 ml of methionine-
`free RPM] 1640 medium containing 16.5 mg/ml BSA and 0.8
`mM sodium oleate. This was followed by a 30-min incubation
`in 5 ml of the same medium containing 100 p.Cl/ml [35S]me-
`thionine and 50 p.M unlabeled L-methionine. At this point
`2 of 5
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`
`Pharmacology: Jamil et al.
`
`Proc. Natl. Acad.:Sci.
`
`if/sA.93 (1993)
`
`11993
`
`(time = 0 min) a 1-ml aliquot of medium was removed and
`saved for immunoprecipitation analysis. One milliliter of fresh
`medium containing 100 pCi/ml of [3-"S]methionine and 50 ptM
`unlabeled L-methionine was added. Immediately thereafter,
`one culture was brought
`to 10 p.M BMS-200150 + 0.5%
`DMSO, whereas the other was brought to 0.5% DMSO only.
`At
`the indicated times, 1-ml aliquots were removed and
`replaced with fresh labeling medium containing the appropri-
`ate concentrations of radiolabeled methionine, drug, and/or
`DMSO. The concentration of radiolabeled apoB in each media
`sample was measured by immunoprecipitation of 250-ul ali-
`quots with 5 pl of goat anti-human apoB polyclonal antiserum
`using a protocol previously described (15). Preimmune serum
`was used to determine background levels of radioactivity
`immunoprecipitated for each media sample. The data are
`expressed as the counts per minute immunoprecipitated, fol-
`lowing correction for background and dilution of the media
`over the course of the experiment.
`[~"H]Thymidine Incorporation into DNA. HepG2 cells were
`grown to 90% confluency in 24-well plates. To investigate the
`effect of BMS-200150 on HepG2 cell growth, the cells were
`incubated for an additional 16 h in the absence or presence of
`various concentrations of BMS-200150. Each concentration of
`compound was tested in triplicate. The cells were then pulsed
`with [3H]thymidine (0.5 uCi/ml) for 1 h at 37°C. After washing
`the monolayers two times with PBS, 0.5 ml
`ice cold 10%
`trichloroacetic acid was added, and the plates were incubated
`at 4°C for 5 min. This step was repeated, and then the plates
`were washed briefly with cold PBS. The precipitate was
`solubilized in 0.5 ml of 0.1 M NaOH for 15 min at room
`temperature, and the radioactivity was quantitated by liquid
`scintillation counting.
`
`RESULTS
`
`Identification and Characterization of BMS-200150. High-
`throughput screening of the Bristol-Myers Squibb compound
`collection identified BMS-200150 (Fig. 1) as a potent inhibitor
`of bovine MTP-mediated transport of TG between SUVs (Fig.
`2 Left). The IC5o for inhibition of TG transfer was 0.6 p.M.
`BMS-200150 also inhibits human MTP-mediated TG transfer
`(Fig. 3) with a similar lC5o of 2.2 uM. In a control experiment,
`BMS-200150 had no effect on human plasma cholesterol ester
`transfer protein-mediated CE transfer (data not shown).
`MTP binds and shuttles lipid molecules between SUVs (16).
`There are two possible mechanisms by which a small molecule
`could inhibit MTP-mediated lipid transport. First,
`it could
`partition into the SUVs in the assay and disrupt the ability of
`MTP to bind to the vesicle surfaces or disrupt the ability of
`MTP to extract lipid molecules from or deposit lipid molecule
`into the membrane. Alternatively,
`it could bind directly to
`MTP and render it unable to transport lipid. To investigate the
`means by which BMS-200150 inhibits MTP-mediated lipid
`transport, [3H]BMS-200150 was synthesized to study the in-
`teraction of BMS-200150 with bovine MTP in an equilibrium
`dialysis experiment. Scatc_hard analysis of the binding revealed
`a single binding site on MTP with a Kd value of 1.3 p.M (Fig.
`2 Right). The Kd is very similar to the lC5o for inhibition of lipid
`transfer (0.6 uM) (Fig. 2 Left), suggesting that
`the direct
`
`100
`
`_ so
`8
`
`E so
`v
`g 40
`2
`er
`n. 20
`
`to
`
`3? 0.3
`2
`
`5 0.6
`3
`1; 0.4
`'0
`5
`0-0.2
`an
`
`0
`
`.1
`
`_
`
`to
`I.
`BMS-200150 (pM)
`
`100
`
`.
`.
`;
`‘o.o
`o_.o.-o.2.o.4.o.s 20.5 1.0 12
`'
`. Bound
`
`FIG. 2. BMS-200150 binds to_ and inhibits bovine’ MTP. (Left) The
`lipid transfer activity of bovine liver MTP was measured in the
`presence of 0-15 p.M BMS-200150. The assay.‘ measures the rate of
`[“C]’l'G transfer from donor SUVs to acceptor SUVs as‘ described in
`Materials and Methods. TG transfer in the absence of inhibitor was 15%
`of the labeled TG in‘the donor vesicles. (Right) The binding of
`[3H]BMS-200150 to bovine MTP was'de'_termined by, equilibrium
`dialysis at various concentrations (0.1 to 100 uM) of the compound
`using 50 p.g (A) and 100 pg (0) of MTP. A Scatchard plot derived from
`the equation [L].,/[L]g[P]. = (-1 /K.) [L];,/[P]. + n/K, is shown, where
`[L].,/[P]. is moles BMS-200150 bound per mole of MTP (Bound), [L];
`is free BMS-200150 (Free), [P]. is the total MTP concentration, n is
`the number of BMS-200150 binding sites per molecule of MTP, and
`K, is the BMS-200150 dissociation constant of a site.
`
`binding of BMS-200150 to MTP is responsible for inhibition of
`MTP-mediated lipid transport.
`"
`MTP transports a wide variety of both neutral lipids and
`phospholipids (8, 14). The ability of BMS-200150 to inhibit the
`human MTP-mediated transport of different lipid substrates
`was investigated using donor vesicles containing 0.25 mol % of
`different "C-labeled substrates. The [C50 for the inhibition of
`CE transfer was 1.7 p.M, a value similar to the lC5o for TG
`transfer (Fig. 3). BMS-200150 also inhibited PC transport over
`a similar concentration range, but
`to a lower extent. The
`inhibition of PC transport was only 30%.
`I
`Effect of BMS-200150 On ApoB Secretion by HepG2 Cells.
`HepG2 cells are a human liver-derived cell line that assemble
`and secrete apoB containing lipoproteins. To test the role of
`MTP-mediated lipid transport in lipoprotein production, the
`effect of BMS-200150 on apoB secretion from HepG2 cells was
`investigated. HepG2 cells were incubated for 16 h with various
`concentrations of BMS-200150. The amount of apoB secreted
`120
`
`
`
`Percentcontrol
`
`0)O
`
`.1
`
`1
`
`‘ 10
`
`BMS-200150 (pM)
`
`100
`
`"
`
`- HCI O
`
`FIG. 1. Structure of BMS-200150.
`
`Inhibition of human MTP by BMS-200150. The lipid
`FIG. 3.
`transfer activity of human MTP was measured in the presence of 0-30
`p.M BMS-200150 using donor vesicles containing 0.25 mol % ["C]TG,
`CE, or PC. The assay measures the rate of lipid transfer from donor
`SUVs to acceptor SUVs as described in Materials and Methods. The
`100% control values for TG, CE, and PC transfer were 9%, 6%. and
`10%, respectively, of the total labeled lipid in the donor vesicles.
`PENN EX. 2152
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`IPR2015—01835
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`3 of 5
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`
`11994
`
`Pharmacology: Jamil er al.
`
`Proc. Natl. Acad. Sci. USA 93 (I996)
`
`6 1500
`8
`5 1200
`
`into the medium was quantitated by an ELISA assay. ApoB
`secretion was inhibited in a concentration-dependent manner
`with an lC5o of 1.8 uM (Fig. 4). The selectivity of the effect was
`confirmed by measuring apoAl secretion. At 30 pM BMS-
`200150, a concentration that is more than 15 times the lC5o for
`inhibition of apoB secretion and that inhibited apoB secretion
`90%, there was no effect on apoAl secretion (Fig. 4).
`The nonspecific toxicity of BMS-200150 to HepG2 cells was
`tested by measuring [3H]thymidine incorporation into DNA
`after incubation with varying concentrations of the compound
`for 16 h. At 2 p.M BMS-200150, the lC5o for inhibition of apoB
`secretion,
`the thymidine incorporation was 100% that of
`control cells. At 20 pm BMS-200150, a concentration that
`inhibits apoB secretion over 90%, thymidine incorporation was
`80% that of control (data not shown). This indicates that
`BMS-200150 is not toxic to HepG2 cells at concentrations that
`selectively inhibit apoB secretion.
`The time of onset of the effect of BMS-200150 on the
`secretion of apoB by HepG2 cells was investigated by mea-
`suring the inhibition of [ 5S]methionine—labeled apoB secre-
`tion into the medium (Fig. 5). After a lag period of ~30 min,
`the rate of apoB secretion in the media of drug treated cells
`was dramatically inhibited. No effect of BMS-200150 was seen
`on incorporation of [35S]methionine into total cellular protein.
`
`DISCUSSION
`
`Although genetic studies have clearly demonstrated a require-
`ment for MTP in lipoprotein assembly, they have not revealed
`its precise role in the process. Here we report that BMS-200150
`inhibits MTP-mediated transfer of TG and CE between mem-
`branes. Binding studies further demonstrated that BMS-
`200150 binds to MTP with a Kd similar to the lC5o for inhibition
`of lipid transfer. This strongly supports that the inhibition of
`lipid transport results from a direct effect of the inhibitor on
`MTP, rather than an indirect effect due to its partitioning into
`the substrate membranes. ln addition, BMS-200150 selectively
`inhibits apoB secretion from HepG2 cells with an 1C5.) similar
`to the lC5o for inhibition of human MTP TG transport (1.8 p.M
`compared with 2.2 p.M).
`MTP appears to possess two to three lipid molecule binding
`sites that can be divided into two classes (17). The primary fast
`site or sites appear to be involved in the transport of both
`neutral lipid and phospholipid. A second slow site appears to
`120
`
`100
`
`80
`
`so
`
`40
`
`20
`
`0
`0.1
`
` "OmO
`
`ApoB —c)—
`A Al —O—
`9°
`
`1
`
`10
`
`100
`
`BMS-200150 (pM)
`
`T)
`«E
`
`o 0
`
`-5
`0)
`E.’
`3
`
`FIG. 4. Effect of BMS-200150 on apoB and apoAl secretion from
`HepG2 cells. HepG2 cells were treated with various concentrations of
`BMS-200150 for 16 h. The amount of apoB and apoAl accumulated
`in the media over 16 h was determined by ELISA. The data are
`representative of four independent experiments. Control values for
`apoB and apoAl secretion were 372 t 60 and 446 : 91 ng/ml,
`respectively.
`
`4ofS
`
`--0- Control
`—o— BMS-200150
`
`
`
`.
`
`9oo
`
`600
`
`aoo
`
`0
`
`B2-
`
`5
`'5
`
`2 8
`
`'
`
`C3E
`
`.E
`EO.
`
`o
`
`o
`
`30
`
`so:
`
`'
`
`90
`
`120
`
`150
`
`Time (miri.) A
`FIG. 5. Two-hour time course of apoB secretion from HepG2‘cells
`following the administration of BMS-200150. HepG2 cells were la-
`beled with [35S]methionine in the presence or absence of 10 p.M
`BMS-200150. At the indicated time. aliquots of medium were removed
`and radiolabeled apoB was quantitated by immunoprecipitation.
`
`specifically bind phospholipid. BMS-200150 binds to MTP in
`a fashion that completely disrupts the transport of neutral
`lipid, but only partially disrupts phospholipid transfer. The
`simplest explanation of these results: would be that BMS-
`200150 binds to MTP at a location that disrupts a binding site
`on MTP that is responsible for T_G, CE, and PC transport. To
`determine if this is the result of BMS-200150 binding directly
`in this site or to an allosteric site will require further investi-
`gation.
`Newly synthesized apoB follows a sequence of steps that
`leads either to its degradation within the cell or the formation
`and secretion of a mature lipoprotein particle. The lipoprotein
`assembly process is initiated as apoB is translocated into the
`lumen of the ER (18). Smal|_apoB particles with a density of
`low density lipoproteins can be secreted from liver derived cell
`lines including HepG2 cells (reviewed in ref. 19). Similar
`particles are not detected in abetalipoproteinemia subjects,
`suggesting that the assembly defect resulting from an absence
`of MTP lies early in the assembly process prior to the forma-
`tion of a small, secretion competent, lipoprotein particle. The
`results of this study indicate that a crucial step in this early
`process is MTP-mediated transport of Elipid. TG or CE trans-
`port appears particularly impoitant early in the assembly
`process. These studies, however, do not address the extent that
`MTP mediates the transport of all the lipid incorporated into
`the mature lipoprotein particlesfi,
`Elevated plasma lipid levels cause premature atherosclerosis
`(20). Restriction of dietary fat'- and drug therapy are currently
`used to lower elevated plasma lipoprotein levels. However, in
`many cases these treatments are not capable of attaining the
`required decrease in plasma lipid levels. This has led to a
`search for better ways to control plasma lipid levels. The
`studies of the role of MTP in abetalipoproteinemia demon-
`strated that MTP is required for both‘ hepatic and intestinal
`apoB containing lipoprotein production (very low density
`lipoprotein and chylomicrons), and suggested that inhibition of
`MTP function may be an effective strategy ‘to prevent very low
`density lipoprotein and chylomicron assembly and to lower
`plasma lipid levels. The results of this work support
`this
`proposal, and demonstrate that inhibition of its lipid transfer
`activity is a viable approach to inhibit lipoprotein production.
`If effective in vivo, this approach should have a profound effect
`on the ability to lower plasma lipid levelsitherapeutically.
`
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`Gregg, R. E. (1992) Science 258, 999-1001.
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`U!
`
`U!
`
`PENN EX. 2152
`CFAD V. UPENN
`IPR2015—0‘1835
`
`5 of 5
`
`PENN EX. 2152
`CFAD V. UPENN
`IPR2015-01835