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• W2106548780 abstract "Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles. Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles. Extensive epidemiological and preclinical mechanistic and intervention studies suggest that low HDL-C 5The abbreviations used are: HDL-CHDL cholesterolCETPcholesteryl ester transfer proteinTHQtetrahydroquinolineLDL-CLDL-cholesterolVLDLvery low-density lipoprotein. levels, in addition to high LDL-C levels, are powerful predictors of cardiovascular risks and that increasing plasma HDL-C levels may be beneficial (1Bruckert E. Hansel B. HDL-c is a powerful lipid predictor of cardiovascular diseases.Int. J. Clin. Pract. 2007; 61: 1905-1913Crossref PubMed Scopus (38) Google Scholar). By shuttling cholesteryl ester from HDL to LDL, CETP decreases plasma HDL-C levels and increases VLDL cholesterol (VLDL-C) and subsequently LDL cholesterol (LDL-C) levels (2Barter P.J. Brewer Jr., H.B. Chapman M.J. Hennekens C.H. Rader D.J. Tall A.R. Cholesteryl ester transfer protein: a novel target for raising HDL and inhibiting atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 160-167Crossref PubMed Scopus (708) Google Scholar, 3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). In addition to cholesteryl esters, CETP also transfers triglycerides and phospholipids between plasma lipoproteins. The suggestion that inhibition of CETP could provide a lipid profile with reduced atherosclerotic risk first arose >20 years ago (4Brown M.L. Inazu A. Hesler C.B. Agellon L.B. Mann C. Whitlock M.E. Marcel Y.L. Milne R.W. Koizumi J. Mabuchi H. Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins.Nature. 1989; 342: 448-451Crossref PubMed Scopus (410) Google Scholar). Epidemiological studies as well as transgenic animal models strongly suggest a role of CETP in the development of atherosclerosis and that blocking CETP could be beneficial (5Inazu A. Brown M.L. Hesler C.B. Agellon L.B. Koizumi J. Takata K. Maruhama Y. Mabuchi H. Tall A.R. Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation.N. Engl. J. Med. 1990; 323: 1234-1238Crossref PubMed Scopus (705) Google Scholar, 6Agellon L.B. Walsh A. Hayek T. Moulin P. Jiang X.C. Shelanski S.A. Breslow J.L. Tall A.R. Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice.J. Biol. Chem. 1991; 266: 10796-10801Abstract Full Text PDF PubMed Google Scholar, 7Sugano M. Makino N. Sawada S. Otsuka S. Watanabe M. Okamoto H. Kamada M. Mizushima A. Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits.J. Biol. Chem. 1998; 273: 5033-5036Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). For example, populations with a mutation that leads to CETP deficiency have a lower incidence of cardiovascular diseases (5Inazu A. Brown M.L. Hesler C.B. Agellon L.B. Koizumi J. Takata K. Maruhama Y. Mabuchi H. Tall A.R. Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation.N. Engl. J. Med. 1990; 323: 1234-1238Crossref PubMed Scopus (705) Google Scholar). HDL cholesterol cholesteryl ester transfer protein tetrahydroquinoline LDL-cholesterol very low-density lipoprotein. Inhibition of CETP by small molecule drugs to raise human plasma HDL-C levels has been actively pursued as an approach to prevent cardiovascular diseases (8Linsel-Nitschke P. Tall A.R. HDL as a target in the treatment of atherosclerotic cardiovascular disease.Nat. Rev. Drug. Discov. 2005; 4: 193-205Crossref PubMed Scopus (411) Google Scholar, 9Dullaart R.P. Dallinga-Thie G.M. Wolffenbuttel B.H. van Tol A. CETP inhibition in cardiovascular risk management: a critical appraisal.Eur. J. Clin. Invest. 2007; 37: 90-98Crossref PubMed Scopus (48) Google Scholar, 10Joy T. Hegele R.A. Is raising HDL a futile strategy for atheroprotection?.Nat. Rev. Drug. Discov. 2008; 7: 143-155Crossref PubMed Scopus (125) Google Scholar, 11Cannon C.P. High-density lipoprotein cholesterol as the Holy Grail.Jama. 2011; 306: 2153-2155Crossref PubMed Scopus (19) Google Scholar). Despite the success of statins, which lower LDL-C levels significantly, cardiovascular disease is still the leading cause of mortality worldwide claiming ∼17 million lives each year (12World Health OrganizationThe Atlas of Heart Disease and Stroke. World Health Organization, Geneva, Switzerland2008Google Scholar). Many CETP inhibitors have been developed, with four having reached late stage clinical trials (see Fig. 1): torcetrapib (1) (13Ruggeri R.B. Cholesteryl ester transfer protein: pharmacological inhibition for the modulation of plasma cholesterol levels and promising target for the prevention of atherosclerosis.Curr. Top Med. Chem. 2005; 5: 257-264Crossref PubMed Scopus (23) Google Scholar), anacetrapib (3, MK-859) (14Krishna R. Anderson M.S. Bergman A.J. Jin B. Fallon M. Cote J. Rosko K. Chavez-Eng C. Lutz R. Bloomfield D.M. Gutierrez M. Doherty J. Bieberdorf F. Chodakewitz J. Gottesdiener K.M. Wagner J.A. Effect of the cholesteryl ester transfer protein inhibitor, anacetrapib, on lipoproteins in patients with dyslipidaemia and on 24-h ambulatory blood pressure in healthy individuals: two double-blind, randomised placebo-controlled phase I studies.Lancet. 2007; 370: 1907-1914Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 15Ali, A., Napolitano, J. M., Deng, Q., Lu, Z., Sinclair, P. J., Taylor, G. E., Thompson, C. F., Quraishi, N., (July 1, 2005) U. S. Patent 2006/0040999 A1Google Scholar), evacetrapib (4, LY2484595) (16Cao G. Beyer T.P. Zhang Y. Schmidt R.J. Chen Y.Q. Cockerham S.L. Zimmerman K.M. Karathanasis S.K. Cannady E.A. Fields T. Mantlo N.B. Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure.J. Lipid Res. 2011; 52: 2169-2176Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and dalcetrapib (5, R1658, also known as JTT-705) (17Okamoto H. Yonemori F. Wakitani K. Minowa T. Maeda K. Shinkai H. A cholesteryl ester transfer protein inhibitor attenuates atherosclerosis in rabbits.Nature. 2000; 406: 203-207Crossref PubMed Scopus (509) Google Scholar). Torcetrapib, the first CETP inhibitor to advance to late stage clinical trials, showed a substantial effect on plasma lipoprotein levels, raising HDL-C and lowering LDL-C. However, torcetrapib also exhibited adverse effects in a cardiovascular events trial in combination with atorvastatin and was found to increase the risk of mortality and morbidity over atorvastatin alone (18Barter P.J. Caulfield M. Eriksson M. Grundy S.M. Kastelein J.J. Komajda M. Lopez-Sendon J. Mosca L. Tardif J.C. Waters D.D. Shear C.L. Revkin J.H. Buhr K.A. Fisher M.R. Tall A.R. Brewer B. Effects of torcetrapib in patients at high risk for coronary events.N. Engl. J. Med. 2007; 357: 2109-2122Crossref PubMed Scopus (2605) Google Scholar, 19Nissen 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. Lasala G.P. Tuzcu E.M. ILLUSTRATE InvestigatorsEffect of torcetrapib on the progression of coronary atherosclerosis.N. Engl. J. Med. 2007; 356: 1304-1316Crossref PubMed Scopus (891) Google Scholar, 20Kastelein J.J. van Leuven S.I. Burgess L. Evans G.W. Kuivenhoven J.A. Barter P.J. Revkin J.H. Grobbee D.E. Riley W.A. Shear C.L. Duggan W.T. Bots M.L. Effect of torcetrapib on carotid atherosclerosis in familial hypercholesterolemia.N. Engl. J. Med. 2007; 356: 1620-1630Crossref PubMed Scopus (606) Google Scholar, 21Bots M.L. Visseren F.L. Evans G.W. Riley W.A. Revkin J.H. Tegeler C.H. Shear C.L. Duggan W.T. Vicari R.M. Grobbee D.E. Kastelein J.J. Torcetrapib and carotid intima-media thickness in mixed dyslipidaemia (RADIANCE 2 study): a randomized, double-blind trial.Lancet. 2007; 370: 153-160Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). Dalcetrapib did not show the same adverse effects but was not potent enough to demonstrate clinical efficacy (22Hooper A.J. Burnett J.R. Dalcetrapib, a cholesteryl ester transfer protein modulator.Expert Opin. Investig. Drugs. 2012; 21: 1427-1432Crossref PubMed Scopus (19) Google Scholar). The other CETP inhibitors named in Fig. 1 are being actively tested in later stage clinical trials (23Vergeer M. Kastelein J.J. Anacetrapib: new hope for cholesteryl ester transfer protein inhibitors in the treatment of dyslipidemia.Nat. Clin. Pract. Cardiovasc Med. 2008; 5: 302-303Crossref PubMed Scopus (7) Google Scholar, 24Stein E.A. Roth E.M. Rhyne J.M. Burgess T. Kallend D. Robinson J.G. Safety and tolerability of dalcetrapib (RO4607381/JTT-705): results from a 48-week trial.Eur. Heart. J. 2010; 31: 480-488Crossref PubMed Scopus (105) Google Scholar, 25Nicholls S.J. Brewer H.B. Kastelein J.J. Krueger K.A. Wang M.D. Shao M. Hu B. McErlean E. Nissen S.E. Effects of the CETP inhibitor evacetrapib administered as monotherapy or in combination with statins on HDL and LDL cholesterol: a randomized controlled trial.Jama. 2011; 306: 2099-2109Crossref PubMed Scopus (368) Google Scholar); their effects on disease outcome remain unknown (26Tall A.R. Yvan-Charvet L. Wang N. The failure of torcetrapib: was it the molecule or the mechanism?.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 257-260Crossref PubMed Scopus (269) Google Scholar, 27Tall A.R. CETP inhibitors to increase HDL cholesterol levels.N. Engl. J. Med. 2007; 356: 1364-1366Crossref PubMed Scopus (111) Google Scholar, 28Joy T. Hegele R.A. The end of the road for CETP inhibitors after torcetrapib?.Curr. Opin. Cardiol. 2009; 24: 364-371Crossref PubMed Scopus (39) Google Scholar, 29Hamilton J.A. Deckelbaum R.J. Crystal structure of CETP: new hopes for raising HDL to decrease risk of cardiovascular disease?.Nat. Struct. Mol. Biol. 2007; 14: 95-97Crossref PubMed Scopus (12) Google Scholar). Torcetrapib (1) is a representative of the tetrahydroquinoline (THQ, Fig. 1) series of inhibitors with a very potent CETP IC50 of 50 nm, measured in whole human plasma (30Clark R.W. Ruggeri R.B. Cunningham D. Bamberger M.J. Description of the torcetrapib series of cholesteryl ester transfer protein inhibitors, including mechanism of action.J. Lipid Res. 2006; 47: 537-552Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Anacetrapib (3) contains the triad of trifluoromethyl groups found in torcetrapib but also has a distinct biaryl moiety (Fig. 1). Evacetrapib (4) contains a homologated core of torcetrapib and the 3,5-bis-trifluoromethylbenzyl group but also a methyl tetrazole and cyclohexane carboxylic acid side chain. Dalcetrapib (5) is more distinct from the fluorine-containing structures above, with an ortho-thio-anilide core (Fig. 1) and only modest CETP inhibitory activity in the human plasma assay (IC50, 9 μm), although it relies on forming a covalent disulfide linkage with CETP rather than reversible binding as in the above compounds. In a phase III clinical trial, torcetrapib demonstrated an increase of 72.1% in HDL-C and a decrease of 24.9% in LDL-C (18Barter P.J. Caulfield M. Eriksson M. Grundy S.M. Kastelein J.J. Komajda M. Lopez-Sendon J. Mosca L. Tardif J.C. Waters D.D. Shear C.L. Revkin J.H. Buhr K.A. Fisher M.R. Tall A.R. Brewer B. Effects of torcetrapib in patients at high risk for coronary events.N. Engl. J. Med. 2007; 357: 2109-2122Crossref PubMed Scopus (2605) Google Scholar). In the same clinical trial, torcetrapib was also shown to increase systolic blood pressure and serum bicarbonate and decrease serum potassium at a daily dose of 60 mg (18Barter P.J. Caulfield M. Eriksson M. Grundy S.M. Kastelein J.J. Komajda M. Lopez-Sendon J. Mosca L. Tardif J.C. Waters D.D. Shear C.L. Revkin J.H. Buhr K.A. Fisher M.R. Tall A.R. Brewer B. Effects of torcetrapib in patients at high risk for coronary events.N. Engl. J. Med. 2007; 357: 2109-2122Crossref PubMed Scopus (2605) Google Scholar). It is still not clear whether the increased risk associated with torcetrapib is due to its off-target effects. On the other hand, anacetrapib, dalcetrapib, and evacetrapib have been shown to increase HDL-C and lower LDL-C without increasing blood pressure (14Krishna R. Anderson M.S. Bergman A.J. Jin B. Fallon M. Cote J. Rosko K. Chavez-Eng C. Lutz R. Bloomfield D.M. Gutierrez M. Doherty J. Bieberdorf F. Chodakewitz J. Gottesdiener K.M. Wagner J.A. Effect of the cholesteryl ester transfer protein inhibitor, anacetrapib, on lipoproteins in patients with dyslipidaemia and on 24-h ambulatory blood pressure in healthy individuals: two double-blind, randomised placebo-controlled phase I studies.Lancet. 2007; 370: 1907-1914Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 15Ali, A., Napolitano, J. M., Deng, Q., Lu, Z., Sinclair, P. J., Taylor, G. E., Thompson, C. F., Quraishi, N., (July 1, 2005) U. S. Patent 2006/0040999 A1Google Scholar, 16Cao G. Beyer T.P. Zhang Y. Schmidt R.J. Chen Y.Q. Cockerham S.L. Zimmerman K.M. Karathanasis S.K. Cannady E.A. Fields T. Mantlo N.B. Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure.J. Lipid Res. 2011; 52: 2169-2176Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 17Okamoto H. Yonemori F. Wakitani K. Minowa T. Maeda K. Shinkai H. A cholesteryl ester transfer protein inhibitor attenuates atherosclerosis in rabbits.Nature. 2000; 406: 203-207Crossref PubMed Scopus (509) Google Scholar, 23Vergeer M. Kastelein J.J. Anacetrapib: new hope for cholesteryl ester transfer protein inhibitors in the treatment of dyslipidemia.Nat. Clin. Pract. Cardiovasc Med. 2008; 5: 302-303Crossref PubMed Scopus (7) Google Scholar, 24Stein E.A. Roth E.M. Rhyne J.M. Burgess T. Kallend D. Robinson J.G. Safety and tolerability of dalcetrapib (RO4607381/JTT-705): results from a 48-week trial.Eur. Heart. J. 2010; 31: 480-488Crossref PubMed Scopus (105) Google Scholar, 25Nicholls S.J. Brewer H.B. Kastelein J.J. Krueger K.A. Wang M.D. Shao M. Hu B. McErlean E. Nissen S.E. Effects of the CETP inhibitor evacetrapib administered as monotherapy or in combination with statins on HDL and LDL cholesterol: a randomized controlled trial.Jama. 2011; 306: 2099-2109Crossref PubMed Scopus (368) Google Scholar). Illuminating the interactions of these small molecule compounds with CETP at the atomic level may further our understanding of how CETP facilitates lipid transfer, shed light onto how these compounds inhibit CETP, and provide insight into drug design. Previously, we reported the crystal structure of the holo-CETP structure (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). The holo-protein structure reveals a long hydrophobic tunnel in which four lipid molecules are found to bind. Based on the structure we proposed a mechanism by which the lipids transfer through this hydrophobic tunnel between HDL and LDL (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). A recent report of single particle electron microscopy imaging experiments indicate that CETP bridges a ternary complex with its N-terminal domain penetrating into HDL particles and its C-terminal domain interacting with LDL or VLDL particles, supporting the tunnel mechanism for neutral cholesteryl ester transfer and illuminating the direction of lipid transfer (31Zhang L. Yan F. Zhang S. Lei D. Charles M.A. Cavigiolio G. Oda M. Krauss R.M. Weisgraber K.H. Rye K.A. Pownall H.J. Qiu X. Ren G. Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein.Nat. Chem. Biol. 2012; 8: 342-349Crossref PubMed Scopus (107) Google Scholar). To study the molecular basis of CETP inhibition, holo-CETP crystals were produced using previously described methods (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar) and soaked with saturated solutions of torcetrapib or a structurally distinct inhibitor ((2R)-3-{[4-(4-chloro-3-ethylphenoxy)pyrimidin-2-yl][3-(1,1,2,2-tetrafluoroethoxy)benzyl]amino}-1,1,1-trifluoropropan-2-ol), herein called compound 2 (Fig. 1, IC50 of 37 nm in the whole human plasma inhibition assay) to produce complex crystals. The inhibitors were observed unambiguously in the omit (2Fo − Fc) and (Fo − Fc) electron density maps generated from x-ray diffraction data collected at synchrotron sources (Fig. 2). Both CETP-inhibitor complex structures have been fully refined, with the torcetrapib complex structure having the higher resolution of 2.6 Å (Table 1).TABLE 1Data collection and refinement statisticsTorcetrapibCompound 2Data collectionSpace groupP212121P212121Cell dimensionsa = 69.6, b = 69.3, and c = 188.2 Å; α, β, and γ = 90°a = 68.8, b = 69.9, c = 187.1 Å; α, β, and γ = 90°Resolution (Å)aStatistics in the highest resolution shell are shown in parentheses.50.0–2.6 (2.67–2.6)50–3.1 (3.21–3.1)Rmerge0.083 (0.34)0.111 (0.513)I/σ18.5 (2.4)12.1 (2.1)Completeness (%)79.2 (30.9)76.1 (30.6)Redundancy4.0 (1.6)5.0 (2.2)RefinementResolution (Å)50–2.650–3.1No. of reflections21,81011,592Rwork/Rfree0.213/0.2590.198/0.239No. of atomsProtein3,7403,700Ligand/ion239227Water11032r.m.s.d bond lengths (Å)br.m.s.d., root mean square deviation.0.0090.008r.m.s.d bond angles1.3°1.2°Ramachandran plot favored, allowed, & disallowed (%)95.7, 99.6, and 0.692.6, 99.6, and 0.4a Statistics in the highest resolution shell are shown in parentheses.b r.m.s.d., root mean square deviation. Open table in a new tab Detailed descriptions on DNA constructs, protein expression, and purifications have been reported previously (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). The human CETP construct (1–476, sequence numbering starts at the first amino acid of the mature protein after signal peptide removed) used for crystallization in this study contains five point mutations, C1A, N88D, C131A, N240D, and N341D, to eliminate heterogeneous post-translational modifications on the protein surface to facilitate protein crystallization (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). Protein expression was carried out in Chinese hamster ovary cell line DG44. CETP was purified through an immobilized monoclonal antibody column (the monoclonal antibody was immobilized on CNBr-activated Sepharose Fast Flow resin, GE Healthcare), a hydrophobic interaction column (Butyl-650 Toyopearl M) (Toshoh Haas, Montgomeryville, PA), and an anion exchange column (Q Sepharose Fast Flow, GE Healthcare) (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). Two CETP peaks eluted from the anion exchange column were further purified through the same hydrophobic interaction column described above (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). The purified mutant CETP has similar activity as the wild type protein. The typical protein yield after final purification is >10 mg protein per liter of expression media, with purity >95%. Crystallization of the holo-CETP has been reported previously (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). Briefly, the holo-CETP crystals were obtained by hanging drop vapor diffusion, using 10 mg ml−1 protein in a buffer of 20 mm Tris, pH 8.0, 250 mm NaCl, and 1 mm EDTA mixed 1:1 with a well solution of 0.1 m HEPES (pH 7.5), 0.2 m MgCl2, and 27–35% (w/v) PEG 400 at 4 °C. Note that neither cholesteryl ester nor phospholipid was added during purification and crystallization, but they were detected in purified protein sample using mass spectrum analysis and observed in the holo-CETP crystal structure (3Qiu X. Mistry A. Ammirati M.J. Chrunyk B.A. Clark R.W. Cong Y. Culp J.S. Danley D.E. Freeman T.B. Geoghegan K.F. Griffor M.C. Hawrylik S.J. Hayward C.M. Hensley P. Hoth L.R. Karam G.A. Lira M.E. Lloyd D.B. McGrath K.M. Stutzman-Engwall K.J. Subashi A.K. Subashi T.A. Thompson J.F. Wang I.K. Zhao H. Seddon A.P. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.Nat. Struct. Mol. Biol. 2007; 14: 106-113Crossref PubMed Scopus (213) Google Scholar). A large number of holo-CETP crystals were soaked at 4 °C in mother liquors containing saturate concentrations of inhibitors, 0.1% β-octylglucoside, 0.2 m MgCl2, 0.1 m HEPES buffer at pH 7.5 and 30% polyethylene glycol 400 for days. The crystals were then cooled directly in liquid nitrogen before data collection. Crystallographic data sets were collected at the 17-ID beamline of the Advanced Photon Source at the Argonne National Laboratory (Chicago, IL). Diffraction data were processed with the program suite HKL-2000 (32Otwinowski Z. Borek D. Majewski W. Minor W. Multiparametric scaling of diffraction intensities.Acta Crystallogr. A. 2003; 59: 228-234Crossref PubMed Scopus (662) Google Scholar), whereas the structure solution and refinement were carried out using the CCP4 program suite (33Collaborative Computational Project, Number 4The CCP4 suite: programs for protein crystallography.Acta Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar). The starting CETP model was derived from the holo-CETP structure (Protein Data Bank code 2OBD), excluding the bound lipids and solvent molecules, and the manual model building was carried out using program COOT (34Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17079) Google Scholar). The inhibitor-bound structures have been refined satisfactorily. The diffraction data collection and final refinement statistics are listed in Table 1. Probably due to prolonged soaking that was necessary to observe inhibitors, all crystals suffered from loss of resolution and anisotropic diffractions. The torcetrapib-CETP complex crystal has a higher resolution. Its data were complete to 2.8 Å, and partially complete between 2.6 to 2.8 Å due to anisotropic diffraction pattern (Table 1). Mutant CETP cDNAs were cloned into a modified version of pSecTag2/Hygro containing N-terminal His6 and V5 tags (Invitrogen). HEK293S cells were cultured and transfected with the cDNAs as described previously (35Lloyd D.B. Reynolds J.M. Cronan M.T. Williams S.P. Lira M.E. Wood L.S. Knight D.R. Thompson J.F. Novel variants in human and monkey CETP.Biochim. Biophys. Acta. 2005; 1737: 69-75Crossref PubMed Scopus (6) Google Scholar). Medium from transfected cells was collected and concentrated. With the aid of GeneTools software (Syngene)" @default.
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• W2106548780 title "Crystal Structures of Cholesteryl Ester Transfer Protein in Complex with Inhibitors" @default.
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