Influence of HDL-cholesterol-elevating drugs on the in vitro activity of the HDL receptor SR-BI

Treatment of atherosclerotic disease often focuses on reducing plasma LDL-cholesterol or increasing plasma HDL-cholesterol. We examined in vitro the effects on HDL receptor [scavenger receptor class B type I (SR-BI)] activity of three classes of clinical and experimental plasma HDL-cholesterol-elevating compounds: niacin, fibrates, and HDL376. Fenofibrate (FF) and HDL376 were potent (IC50 ∼ 1 μM), direct inhibitors of SR-BI-mediated lipid transport in cells and in liposomes reconstituted with purified SR-BI. FF, a prodrug, was a more potent inhibitor of SR-BI than an activator of peroxisome proliferator-activated receptor α, a target of its active fenofibric acid (FFA) derivative. Nevertheless, FFA, four other fibrates (clofibrate, gemfibrozil, ciprofibrate, and bezafibrate), and niacin had little, if any, effect on SR-BI, suggesting that they do not directly target SR-BI in vivo. However, similarities of HDL376 treatment and SR-BI gene knockout on HDL metabolism in vivo (increased HDL-cholesterol and HDL particle sizes) and structure-activity relationship analysis suggest that SR-BI may be a target of HDL376 in vivo. HDL376 and other inhibitors may help elucidate SR-BI function in diverse mammalian models and determine the therapeutic potential of SR-BI-directed pharmaceuticals. Treatment of atherosclerotic disease often focuses on reducing plasma LDL-cholesterol or increasing plasma HDL-cholesterol. We examined in vitro the effects on HDL receptor [scavenger receptor class B type I (SR-BI)] activity of three classes of clinical and experimental plasma HDL-cholesterol-elevating compounds: niacin, fibrates, and HDL376. Fenofibrate (FF) and HDL376 were potent (IC50 ∼ 1 μM), direct inhibitors of SR-BI-mediated lipid transport in cells and in liposomes reconstituted with purified SR-BI. FF, a prodrug, was a more potent inhibitor of SR-BI than an activator of peroxisome proliferator-activated receptor α, a target of its active fenofibric acid (FFA) derivative. Nevertheless, FFA, four other fibrates (clofibrate, gemfibrozil, ciprofibrate, and bezafibrate), and niacin had little, if any, effect on SR-BI, suggesting that they do not directly target SR-BI in vivo. However, similarities of HDL376 treatment and SR-BI gene knockout on HDL metabolism in vivo (increased HDL-cholesterol and HDL particle sizes) and structure-activity relationship analysis suggest that SR-BI may be a target of HDL376 in vivo. HDL376 and other inhibitors may help elucidate SR-BI function in diverse mammalian models and determine the therapeutic potential of SR-BI-directed pharmaceuticals. adrenocorticotropic hormone blocker of lipid transport-1 cholesteryl oleyl ether cholesteryl ester transfer protein 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate fatty acid-free fenofibrate fenofibric acid PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 peroxisome proliferator-activated receptor α standard error of the mean scavenger receptor class B type I Cardiovascular disease is the principal killer in Western industrialized societies. In the United States, ∼76% of cardiovascular disease is attributed to coronary heart disease, stroke, and heart failure (1.Thom T. Haase N. Rosamond W. Howard V.J. Rumsfeld J. Manolio T. Zheng Z.J. Flegal K. O'Donnell C. Kittner S. et al.Heart disease and stroke statistics—2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.Circulation. 2006; 113: e85-e151Crossref PubMed Scopus (2677) Google Scholar). Occlusive and unstable atherosclerosis is a major cause of cardiovascular disease. Epidemiologic and interventional studies have clearly established the critical role of lipoprotein metabolism in atherosclerotic disease, in which risk increases with plasma levels of LDL-cholesterol and is inversely proportional to HDL-cholesterol (2.Gordon D.J. Rifkind B.M. High-density lipoprotein—the clinical implications of recent studies.N. Engl. J. Med. 1989; 321: 1311-1316Crossref PubMed Scopus (1412) Google Scholar). Understanding receptor-mediated LDL metabolism (3.Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Scopus (4383) Google Scholar) has led to profound insights into basic biological mechanisms and key clinical advances, such as the introduction of HMG-CoA reductase inhibitor (statin) therapy (4.Hebert P.R. Gaziano J.M. Chan K.S. Hennekens C.H. Cholesterol lowering with statin drugs, risk of stroke, and total mortality. An overview of randomized trials.J. Am. Med. Assoc. 1997; 278: 313-321Crossref PubMed Google Scholar). The success of LDL-cholesterol-lowering therapy has stimulated increased interest in HDL-cholesterol-directed approaches. Several HDL-cholesterol-elevating drugs are currently available or under development (5.Clark R.W. Raising high-density lipoprotein with cholesteryl ester transfer protein inhibitors.Curr. Opin. Pharmacol. 2006; 6: 162-168Crossref PubMed Scopus (30) Google Scholar, 6.Duffy D. Rader D.J. Emerging therapies targeting high-density lipoprotein metabolism and reverse cholesterol transport.Circulation. 2006; 113: 1140-1150Crossref PubMed Scopus (98) Google Scholar). Some of the most widely used HDL-cholesterol-elevating drugs in the clinic include statins (∼5–10% increase), fibrates [peroxisome proliferator-activated receptor α (PPARα) agonists; ∼5–20% increase], and nicotinic acid or niacin (uncertain mechanism; ∼15–35% increase). Unfortunately, results in recent clinical trials of another HDL-cholesterol-elevating agent, the cholesteryl ester transfer protein (CETP) inhibitor torcetrapib, were disappointing (7.Nissen S.E. Tardif J.C. Nicholls S.J. Revkin J.H. Shear C.L. Duggan W.T. Ruzyllo W. Bachinsky W.B. Lasala G.P. Tuzcu E.M. Effect of torcetrapib on the progression of coronary atherosclerosis.N. Engl. J. Med. 2007; 356: 1304-1316Crossref PubMed Scopus (894) Google Scholar – 9.Honey K. Drug designed to raise HDL levels falls down.J. Clin. Invest. 2007; 117: 282Crossref PubMed Scopus (8) Google Scholar), casting uncertainty on the future of CETP as a target for pharmacotherapy. The identification of the scavenger receptor class B type I (SR-BI) as an HDL receptor (10.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar, 11.Rigotti A. Miettinen H.E. Krieger M. The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues.Endocr. Rev. 2003; 24: 357-387Crossref PubMed Scopus (350) Google Scholar) has raised the possibility that agents modulating SR-BI activity or expression levels might represent attractive candidates for HDL-cholesterol-directed therapies (11.Rigotti A. Miettinen H.E. Krieger M. The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues.Endocr. Rev. 2003; 24: 357-387Crossref PubMed Scopus (350) Google Scholar – 13.Nieland T.J. Penman M. Dori L. Krieger M. Kirchhausen T. Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI.Proc. Natl. Acad. Sci. USA. 2002; 99: 15422-15427Crossref PubMed Scopus (182) Google Scholar). In addition to its HDL receptor activity, SR-BI can function as a receptor for other lipoproteins, including LDL (14.Acton S.L. Scherer P.E. Lodish H.F. Krieger M. Expression cloning of SR-BI, a CD36-related class B scavenger receptor.J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar) and chylomicrons (15.Out R. Kruijt J.K. Rensen P.C. Hildebrand R.B. Vos P.de Eck M.Van Van Berkel T.J. Scavenger receptor BI plays a role in facilitating chylomicron metabolism.J. Biol. Chem. 2004; 279: 18401-18406Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16.Out R. Hoekstra M. de Jager S.C. Vos P.de van der Westhuyzen D.R. Webb N.R. Eck M.Van Biessen E.A. Van Berkel T.J. Adenovirus-mediated hepatic overexpression of scavenger receptor class B type I accelerates chylomicron metabolism in C57BL/6J mice.J. Lipid Res. 2005; 46: 1172-1181Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). SR-BI binds HDL primarily via HDL's main protein component, apolipoprotein A-I (17.Xu S. Laccotripe M. Huang X. Rigotti A. Zannis V.I. Krieger M. Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake.J. Lipid Res. 1997; 38: 1289-1298Abstract Full Text PDF PubMed Google Scholar). SR-BI-mediated delivery to cells of HDL-cholesterol occurs via selective lipid uptake, a two-step process involving HDL binding followed by a lipid transfer step (18.Gu X. Trigatti B. Xu S. Acton S. Babitt J. Krieger M. The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.J. Biol. Chem. 1998; 273 ([Erratum. 1998. 273: 35388.]): 26338-26348Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar – 21.Stein Y. Dabach Y. Hollander G. Halperin G. Stein O. Metabolism of HDL-cholesteryl ester in the rat, studied with a nonhydrolyzable analog, cholesteryl linoleyl ether.Biochim. Biophys. Acta. 1983; 752: 98-105Crossref PubMed Scopus (138) Google Scholar) that differs from classic LDL receptor-mediated endocytosis via clathrin-coated pits (3.Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Scopus (4383) Google Scholar). After HDL binds to SR-BI, HDL-cholesterol (primarily cholesteryl ester but also unesterified cholesterol) is transferred to the cells, and then the lipid-depleted HDL particles dissociate and reenter the extracellular space. SR-BI can also mediate unesterified cholesterol efflux from cells to HDL (22.Ji Y. Jian B. Wang N. Sun Y. Moya M.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux.J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). We have previously identified a set of small molecules, called blockers of lipid transport (BLTs), that inhibit both cellular SR-BI-mediated selective lipid uptake from HDL and cholesterol efflux to HDL yet increase the affinity of HDL binding to SR-BI (12.Nieland T.J. Chroni A. Fitzgerald M.L. Maliga Z. Zannis V.I. Kirchhausen T. Krieger M. Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide.J. Lipid Res. 2004; 45: 1256-1265Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 13.Nieland T.J. Penman M. Dori L. Krieger M. Kirchhausen T. Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI.Proc. Natl. Acad. Sci. USA. 2002; 99: 15422-15427Crossref PubMed Scopus (182) Google Scholar). At a fixed HDL concentration below the dissociation constant (Kd) the increase in affinity is readily observed as an increase in net HDL binding. The molecular target(s) of the BLTs has not yet been reported. BLTs are currently being used to explore the detailed mechanism underlying SR-BI's multiple activities (12.Nieland T.J. Chroni A. Fitzgerald M.L. Maliga Z. Zannis V.I. Kirchhausen T. Krieger M. Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide.J. 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Numerous genetic studies in mice have established that SR-BI plays a key role in murine HDL metabolism (11.Rigotti A. Miettinen H.E. Krieger M. The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues.Endocr. Rev. 2003; 24: 357-387Crossref PubMed Scopus (350) Google Scholar). For example, adenovirus-mediated hepatic overexpression of SR-BI dramatically decreases plasma HDL-cholesterol (41.Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Overexpression of the HDL receptor SR-BI alters plasma HDL and bile cholesterol levels.Nature. 1997; 387: 414-417Crossref PubMed Scopus (631) Google Scholar). Complete loss of SR-BI expression by homozygous gene inactivation increases both plasma HDL-cholesterol level (∼2-fold) and HDL particle size (42.Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism.Proc. Natl. Acad. Sci. USA. 1997; 94: 12610-12615Crossref PubMed Scopus (761) Google Scholar). A tissue-specific, ∼95% reduction in hepatic SR-BI protein level attributable to targeted disruption of the gene encoding a cytoplasmic adaptor protein, PDZK1, which binds to the C terminus of SR-BI (43.Ikemoto M. Arai H. Feng D. Tanaka K. Aoki J. Dohmae N. Takio K. Adachi H. Tsujimoto M. Inoue K. Identification of a PDZ-domain-containing protein that interacts with the scavenger receptor class B type I.Proc. Natl. Acad. Sci. USA. 2000; 97: 6538-6543Crossref PubMed Scopus (141) Google Scholar), increases both HDL-cholesterol level (∼1.5- to 1.7-fold) and HDL particle size (44.Kocher O. Yesilaltay A. Cirovic C. Pal R. Rigotti A. Krieger M. Targeted disruption of the PDZK1 gene in mice causes tissue-specific depletion of the high density lipoprotein receptor scavenger receptor class B type I and altered lipoprotein metabolism.J. Biol. Chem. 2003; 278: 52820-52825Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The influence of SR-BI on murine atherosclerosis has been examined in both the LDL receptor and apoE knockout models. Hepatic overexpression of SR-BI suppresses and loss of SR-BI expression enhances atherosclerosis in these models (45.Huby T. Doucet C. Dachet C. Ouzilleau B. Ueda Y. Afzal V. Rubin E. Chapman M.J. Lesnik P. Knockdown expression and hepatic deficiency reveal an atheroprotective role for SR-BI in liver and peripheral tissues.J. Clin. Invest. 2006; 116: 2767-2776Crossref PubMed Scopus (94) Google Scholar – 55.Arai T. Wang N. Bezouevski M. Welch C. Tall A.R. Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BI transgene.J. Biol. Chem. 1999; 274: 2366-2371Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The atheroprotective activity of SR-BI, even though its expression decreases plasma HDL-cholesterol levels, suggests that SR-BI's role in reverse cholesterol transport (the movement of cholesterol from peripheral tissues to the liver and then excretion in the bile) may contribute to its protective activity in mice. The physiologic role of SR-BI in HDL metabolism is probably conserved among mammals; however, supporting data in species less genetically tractable than mice are very limited. Thus, it is not known whether the atheroprotective effects of SR-BI in mice, associated with reduced HDL-cholesterol levels, would be exhibited in humans, in which HDL metabolism differs and reduced HDL-cholesterol levels are associated with increased risk (56.Wilson P.W. Abbott R.D. Castelli W.P. High density lipoprotein cholesterol and mortality. The Framingham Heart Study.Arteriosclerosis. 1988; 8: 737-741Crossref PubMed Google Scholar). In this study, we explored in cultured cells and in an in vitro system of highly purified SR-BI reconstituted in liposomes the possibility that some of the in vivo plasma HDL-cholesterol-elevating activities of three classes of drugs might be attributable to their ability to inhibit SR-BI directly. These drugs included niacin (Fig. 1A) and members of the fibrate family (Fig. 1B– G) such as gemfibrozil, drugs currently in use in humans as indicated above. Our results suggest that SR-BI is not a direct target of these drugs in vivo. Although the prodrug fenofibrate (FF) does inhibit SR-BI in vitro, it is rapidly hydrolyzed to fenofibric acid (FFA) in vivo (reviewed in Ref. 57.Despres J.P. Increasing high-density lipoprotein cholesterol: an update on fenofibrate.Am. J. Cardiol. 2001; 88: 30N-36NAbstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Because we show here that FFA does not inhibit SR-BI in vitro, it is highly unlikely that the in vivo HDL-cholesterol-elevating activity of FF involves the inhibition of SR-BI. We also studied an experimental compound called HDL376 [N-(5-chloro-2-methyphenyl)-N′-(1-methylethyl)-thiourea] (58.Coppola G.M. Damon R.E. Eskesen J.B. France D.S. Paterniti Jr, J.R. Biological evaluation of 1-alkyl-3-phenylthioureas as orally active HDL-elevating agents.Bioorg. Med. Chem. Lett. 2006; 16: 113-117Crossref PubMed Scopus (5) Google Scholar, 59.Coppola G.M. Damon R.E. Paterniti Jr., J.R. Weinstein D.B. France D.S. Eskesen J.B. Hughes T.E. Discovery of HDL376, a novel thiourea-based HDL elevating agent.in: 5th Annual HDL Cholesterol. Knowledge Foundation, Cambridge, MA2004: 1-15Google Scholar) (Fig. 1H). HDL376 was discovered in an in vivo screen designed to identify novel drugs that increase plasma HDL-cholesterol levels in rats (58.Coppola G.M. Damon R.E. Eskesen J.B. France D.S. Paterniti Jr, J.R. Biological evaluation of 1-alkyl-3-phenylthioureas as orally active HDL-elevating agents.Bioorg. Med. Chem. Lett. 2006; 16: 113-117Crossref PubMed Scopus (5) Google Scholar, 59.Coppola G.M. Damon R.E. Paterniti Jr., J.R. Weinstein D.B. France D.S. Eskesen J.B. Hughes T.E. Discovery of HDL376, a novel thiourea-based HDL elevating agent.in: 5th Annual HDL Cholesterol. Knowledge Foundation, Cambridge, MA2004: 1-15Google Scholar). It also increases plasma HDL-cholesterol levels in hamsters and dogs, two species not sensitive to gemfibrozil, and in rhesus and cynomolgus monkeys (58.Coppola G.M. Damon R.E. Eskesen J.B. France D.S. Paterniti Jr, J.R. Biological evaluation of 1-alkyl-3-phenylthioureas as orally active HDL-elevating agents.Bioorg. Med. Chem. Lett. 2006; 16: 113-117Crossref PubMed Scopus (5) Google Scholar, 59.Coppola G.M. Damon R.E. Paterniti Jr., J.R. Weinstein D.B. France D.S. Eskesen J.B. Hughes T.E. Discovery of HDL376, a novel thiourea-based HDL elevating agent.in: 5th Annual HDL Cholesterol. Knowledge Foundation, Cambridge, MA2004: 1-15Google Scholar). The HDL376-induced percentage increases in HDL-cholesterol varied from 15% (chow-fed hamsters at 64 mg/kg/day) to 100% (chow-fed cynomolgus monkeys at 12 mg/kg/day) (58.Coppola G.M. Damon R.E. Eskesen J.B. France D.S. Paterniti Jr, J.R. Biological evaluation of 1-alkyl-3-phenylthioureas as orally active HDL-elevating agents.Bioorg. Med. Chem. Lett. 2006; 16: 113-117Crossref PubMed Scopus (5) Google Scholar). Furthermore, it increases HDL particle size in rhesus monkeys (59.Coppola G.M. Damon R.E. Paterniti Jr., J.R. Weinstein D.B. France D.S. Eskesen J.B. Hughes T.E. Discovery of HDL376, a novel thiourea-based HDL elevating agent.in: 5th Annual HDL Cholesterol. Knowledge Foundation, Cambridge, MA2004: 1-15Google Scholar), and a similar compound (Fig. 1J, compound 2) has the same effect in rats (58.Coppola G.M. Damon R.E. Eskesen J.B. France D.S. Paterniti Jr, J.R. Biological evaluation of 1-alkyl-3-phenylthioureas as orally active HDL-elevating agents.Bioorg. Med. Chem. Lett. 2006; 16: 113-117Crossref PubMed Scopus (5) Google Scholar). Importantly, HDL376 has been reported to increase serum HDL-cholesterol levels in healthy humans [∼13% after 7 days of treatment at 100 mg/day (59.Coppola G.M. Damon R.E. Paterniti Jr., J.R. Weinstein D.B. France D.S. Eskesen J.B. Hughes T.E. Discovery of HDL376, a novel thiourea-based HDL elevating agent.in: 5th Annual HDL Cholesterol. Knowledge Foundation, Cambridge, MA2004: 1-15Google Scholar)] to about the same extent as seen with statins and the fibrates (see above). These data, together with the report that HDL376 does not appear to act as a PPARα or PPARδ agonist (58.Coppola G.M. Damon R.E. Eskesen J.B. France D.S. Paterniti Jr, J.R. Biological evaluation of 1-alkyl-3-phenylthioureas as orally active HDL-elevating agents.Bioorg. Med. Chem. Lett. 2006; 16: 113-117Crossref PubMed Scopus (5) Google Scholar), indicate that the mechanism by which HDL376 increases plasma HDL-cholesterol, which has been undefined to date, differs from that of the fibrates. The similarities of the effects on plasma HDL of either HDL376 treatment or dramatically decreasing hepatic SR-BI activity by genetic manipulation (SR-BI or PDZK1 knockout mice) led us to hypothesize that it might be an SR-BI inhibitor; thus, we included HDL376 in this study. We found that HDL376 does inhibit SR-BI-mediated lipid transport in cells and in liposomes containing purified SR-BI and that HDL376 behaves as a BLT-like drug. This raises the possibility that some of its in vivo HDL-cholesterol-elevating activity may be attributable to inhibition of SR-BI. Our results underscore the potential usefulness of HDL376 for the in vivo study of SR-BI function in animals that are not easily amenable to genetic studies, such as targeted gene inactivation, and raise the possibility that modulators of SR-BI activity in vivo may lead to the development of novel HDL-elevating therapeutics. FF, clofibrate, ciprofibrate, gemfibrozil, bezafibrate, and niacin were obtained from Sigma. FFA was obtained from Biofine International (Vancouver, Canada). HDL376 and HDL376U were synthesized as described below. Stock solutions of all compounds were dissolved in DMSO at concentrations ranging from 25 to 500 mM. Isobutylamine (0.110 ml, 1.09 mmol) was added to a solution of 5-chloro-2-methylphenyl isothiocyanate (0.200 g, 1.09 mmol) in dichloromethane (6 ml) and stirred for 1 h, all at room temperature. The mixture was filtered to yield the product as a white solid (0.263 g, 94%): 1H NMR (300 MHz, CDCl3) δ 7.99 (br s, 1H), 7.24–7.28 (m, 3H), 5.70 (br s, 1H), 3.41–3.45 (m, 2H), 2.27 (s, 3H), 1.84–1.93 (m, 1H), 0.88 (d, J = 6.6, 6H); 13C NMR (75 MHz, CDCl3) δ 181.1, 135.5, 134.2, 132.7, 132.5, 128.5, 127.4, 52.8, 28.1, 20.1, 17.3; IR (thin film) 3,320, 2,975, 2,870, 1,632, 1,566, 1,479, 1,407, 1,238, 1,082, 814 cm−1; reverse-phase LC-MS m/z for C12H17ClN2S (M + H)+ 258.9, retention time = 1.76 min. LC-MS (Waters 2795/Micromass LCT) data for HDL376 and HDL376U were collected using a 5:95 to 95:5 acetonitrile-water (0.01% formic acid) solvent gradient on an XTerra column using electrospray ionization. Isobutylamine (0.600 ml, 5.95 mmol) was added to a solution of 5-chloro-2-methylphenyl isocyanate (1.00 g, 5.95 mmol) in n-hexane (50 ml) and stirred for 1 h, all at room temperature. The mixture was filtered to yield the product as a white solid (1.33 g, 93%): 1H NMR (300 MHz, acetone-d6) δ 8.26 (d, J = 2.1, 1H), 7.33 (br s, 1H), 7.11 (d, J = 8.1, 1H), 6.87 (dd, J = 2.4, 8.1, 1H), 6.27 (br s, 1H), 3.06 (dd, J = 6, 6.6, 2H), 2.19 (s, 3H), 1.71–1.84 (m, 1H), 0.92 (d, J = 6.6, 6H); 13C NMR (75 MHz, acetone-d6) δ 155.8, 140.7, 132.1, 131.9, 125.3, 122.0, 120.2, 47.9, 29.7, 20.3, 17.5; IR (thin film) 3,222, 2,956, 2,869, 1,530, 1,483, 1,407, 1,363, 1,270, 1,206, 1,149, 1,124, 1,084, 1,036, 995, 912, 809, 733 cm−1; reverse-phase LC-MS m/z for C12H17ClN2O (M + H)+ 242.9, retention time = 1.76 min. Human HDL (density of ∼1.09–1.16 g/ml) was isolated and labeled either with 125I to label its protein constituents (125I-HDL) or with [3H]cholesteryl oleyl ether (CE; [3H]CE-HDL) as described previously (10.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar, 14.Acton S.L. Scherer P.E. Lodish H.F. Krieger M. Expression cloning of SR-BI, a CD36-related class B scavenger receptor.J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 18.Gu X. Trigatti B. Xu S. Acton S. Babitt J. Krieger M. The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.J. Biol. Chem. 1998; 273 ([Erratum. 1998. 273: 35388.]): 26338-26348Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 60.Gu X. Kozarsky K. Krieger M. Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor.J. Biol. Chem. 2000; 275: 29993-30001Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Most experiments using intact cells were conducted using two stable cell lines, ldlA-7 and ldlA[mSR-BI] cells. The ldlA-7 cells are LDL receptor-deficient Chinese hamster ovary cells that express very low levels of endogenous SR-BI (61.Kingsley D.M. Krieger M. Receptor-mediated endocytosis of low density lipoprotein: somatic cell mutants define multiple genes required for expression of surface-receptor activity.Proc. Natl. Acad. Sci. USA. 1984; 81: 5454-5458Crossref PubMed Scopus (132) Google Scholar), and ldlA[mSR-BI] cells are ldlA-7 cells stably transfected with an expression vector in which murine SR-BI (mSR-BI) cDNA is under the control of a cytomegalovirus promoter, which results in the expression of high levels of mSR-BI (10.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar). These cells were maintained in tissue culture medium (Ham's F12 supplemented with 5% fetal bovine serum, 2 mM l-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, and, for ldlA[mSR-BI] cells only, 0.25 mg/ml G418) (10.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar). In a few control experiments, we examined the effects of FF and HDL376 on the HDL lipid uptake activity using Y1-BS1 cells, in which endogenous, rather than recombinant, SR-BI is expressed. Y1-BS1 cells are cultured murine adenocortical cells that express high levels of endogenous SR-BI on stimulation with adrenocorticotropic hormone (ACTH) (62.Rigotti A. Edelman E.R. Seifert P. Iqbal S.N. DeMattos R.B. Temel R.E. Krieger M. Williams D.L. Regulation by adrenocorticotropic hormone of the in vivo expression of scavenger receptor class B type I (SR-BI), a high density lipoprotein receptor, in steroidogenic cells of the murine adrenal gland.J. Biol. Chem. 1996; 271: 33545-33549Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 63.Temel R.E. Trigatti B. DeMattos R.B. Azhar S. Krieger M. Williams D.L. Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells.Proc. Natl. Acad. Sci. USA. 1997; 94: 13600-13605Crossref PubMed Scopus (210) Google Scholar). All experiments with cultured cells were conducted at 37°C. 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