A Novel Principle for Partial Agonism of Liver X Receptor Ligands

Abstract

Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoR-dissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs. Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoR-dissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs. Nuclear receptors are a family of transcriptional regulators whose activity can be modulated by their binding to small molecule compounds, such as hormones and metabolites. For many members of the family, this property has allowed their use as drug targets (1Gronemeyer H. Gustafsson J.A. Laudet V. Nat. Rev. Drug Discov. 2004; 3: 950-964Crossref PubMed Scopus (946) Google Scholar). In most cases, however, full activation or inhibition of the receptor is not desired. Instead, agonists are required that only partially activate the receptor. Partial agonists can display tissue-specific activation or repression of nuclear receptors, as has been shown for the estrogen receptor partial agonist raloxifen (2Delmas P.D. Bjarnason N.H. Mitlak B.H. Ravoux A.C. Shah A.S. Huster W.J. Draper M. Christiansen C. N. Engl. J. Med. 1997; 337: 1641-1647Crossref PubMed Scopus (1606) Google Scholar). However, little is known about the molecular mechanisms leading to partial versus full agonism. Another example for nuclear receptors, which, if they are to be used as drug targets, require partial agonists, are the liver X receptors α (LXRα, 2The abbreviations used are: LXRα, liver X receptor α; FRET, fluorescence resonance energy transfer; LBD, ligand-binding domain; 22R-HC, 22(R)-hydroxycholesterol; NCoR, nuclear receptor corepressor. NR1H3) and β (LXRβ, NR1H2). They have been shown to play a central role in the transcriptional regulation of lipid and cholesterol homeostasis and inflammation (3Li A.C. Glass C.K. J. Lipid Res. 2004; 45: 2161-2173Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 4Lund E.G. Menke J.G. Sparrow C.P. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1169-1177Crossref PubMed Scopus (120) Google Scholar, 5Lala D.S. IDrugs. 2004; 7: 563-569PubMed Google Scholar, 6Steffensen K.R. Gustafsson J.A. Diabetes. 2004; 53: 36-42Crossref PubMed Google Scholar, 7Joseph S.B. Tontonoz P. Curr. Opin. Pharmacol. 2003; 3: 192-197Crossref PubMed Scopus (90) Google Scholar). Activation of LXR-dependent transcription leads to increased expression of cholesterol transporters (8Whitney K.D. Watson M.A. Goodwin B. Galardi C.M. Maglich J.M. Wilson J.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2001; 276: 43509-43515Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 9Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1151) Google Scholar, 10Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (376) Google Scholar, 11Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (848) Google Scholar) and has been shown to enhance the efflux of cholesterol from macrophages (10Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (376) Google Scholar, 11Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (848) Google Scholar, 12Sparrow C.P. Baffic J. Lam M.H. Lund E.G. Adams A.D. Fu X. Hayes N. Jones A.B. Macnaul K.L. Ondeyka J. Singh S. Wang J. Zhou G. Moller D.E. Wright S.D. Menke J.G. J. Biol. Chem. 2002; 277: 10021-10027Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), reducing the formation of atherosclerotic plaques (13Tangirala R.K. Bischoff E.D. Joseph S.B. Wagner B.L. Walczak R. Laffitte B.A. Daige C.L. Thomas D. Heyman R.A. Mangelsdorf D.J. Wang X. Lusis A.J. Tontonoz P. Schulman I.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11896-11901Crossref PubMed Scopus (375) Google Scholar, 14Joseph S.B. Castrillo A. Laffitte B.A. Mangelsdorf D.J. Tontonoz P. Nat. Med. 2003; 9: 213-219Crossref PubMed Scopus (1015) Google Scholar, 15Terasaka N. Hiroshima A. Koieyama T. Ubukata N. Morikawa Y. Nakai D. Inaba T. FEBS Lett. 2003; 536: 6-11Crossref PubMed Scopus (293) Google Scholar). This observation has raised hopes that the manipulation of LXR activity would be of therapeutic value in the treatment of lipid disorders and atherosclerosis. However, activation of LXRs by agonistic compounds induces the expression of enzymes involved in the synthesis of fatty acids in liver cells (16Yoshikawa T. Shimano H. Amemiya-Kudo M. Yahagi N. Hasty A.H. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Kimura S. Ishibashi S. Yamada N. Mol. Cell. Biol. 2001; 21: 2991-3000Crossref PubMed Scopus (435) Google Scholar, 17Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1423) Google Scholar, 18Joseph S.B. Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. J. Biol. Chem. 2002; 277: 11019-11025Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 19Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1404) Google Scholar). As a consequence, agonists for LXRs cause liver steatosis and elevated serum triglyceride levels in mice (19Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1404) Google Scholar, 20Chisholm J.W. Hong J. Mills S.A. Lawn R.M. J. Lipid Res. 2003; 44: 2039-2048Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Thus, to develop LXR ligands as drugs for the treatment of atherosclerosis, partial, selective activators of LXRs are needed that induce cholesterol efflux in macrophages but do not induce fatty acid synthesis in liver. Among the currently known ligands for LXRs, some compounds, such as the sulfonamide T0901317, appear to have a purely agonistic activity, whereas others, such as GW3965, have been reported to be more selective in their activation of LXR function (19Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1404) Google Scholar, 21Quinet E.M. Savio D.A. Halpern A.R. Chen L. Miller C.P. Nambi P. J. Lipid Res. 2004; 45: 1929-1942Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Based on structural data, it has been predicted that the cofactor interactions induced by T0901317 and a natural ligand would differ from each other (22Williams S. Bledsoe R.K. Collins J.L. Boggs S. Lambert M.H. Miller A.B. Moore J. McKee D.D. Moore L. Nichols J. Parks D. Watson M. Wisely B. Willson T.M. J. Biol. Chem. 2003; 278: 27138-27143Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). We present here a detailed study of the mechanisms of LXR activation by synthetic agonists, including a novel chemotype and a natural LXR ligand. We show that partial activation of LXRs can be brought about by inducing a receptor conformation that enhances the affinity for both activating and repressing cofactors at the same time. We show that the net effect of the activated receptor on transcription depends on the prevalence of coactivators and corepressors in the cell and propose a novel classification scheme for partial nuclear receptor agonists. Yeast Two-hybrid Assays—To measure the ligand-dependent binding of cofactor fragments to LXRs, proteins and protein fragments were expressed in GATEWAY-compatible versions of pGBT9 and pGAD424 in the haploid yeasts CG1945 and Y187 (Clontech), The protein fragments were acids acids acids NCoR, acids acids NCoR, acids acids acids acids acids In fragments from and were used that been in M. H. I. C. M. J. R. M. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). of the were in cells by of the were in in and selective fluorescence as M. H. I. C. M. J. R. M. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar) for T0901317 and for other compounds. from the fluorescence of LXR were by the of only the cofactors but an of the receptors from the of both cofactors and receptors in the of compounds. ligand-binding of expressed as protein with in cells the The expressed as a from a in were by and the proteins over to the were in a of in a in a serum of of of the receptor and of cofactor and molecular ligands for The for at and in a as and and as The and of the used are shown in The coactivator were from such that they a of for For we the affinity from the nuclear each on NCoR and of used in this study of acid to the data from in a new and of by of the cell and the were from the and were in with and with and with at in cells were differentiated macrophages by the of for and in the of to were in and were at cells were in at in serum and for cells were in at in serum and to treatment with LXR compounds, to serum for for and in serum in the of LXR compounds were in with the not the and with with and transcription the and by state levels were to expression The of and were as LXRα, were in cells from of and in with and with at in For cells were in in with serum and the at by were in in and the of the not were for and activities were in the same cell to R. Biochem. 2000; PubMed Scopus Google the The ligand-binding of and were expressed from a of as to the of the protein used as a The NCoR expression by and LXR Partial used to for novel compounds in a of Among the a of quinazolinone compounds, such as LN6500 that activated and activities in with an of and with the LXR agonist T0901317, the of LN6500 in is in the of for and for of LN6500 are to or than of the natural agonist activities are compounds are used in that have a LXR-dependent the expression of the not of in cofactor binding The ligand-dependent binding of a on a receptor from by for are S.D. from However, the same compounds are in a that the of to the in the of the compounds The natural agonist and the quinazolinone only in the of of GW3965 clearly only a partial activation of the receptors, and of the activity of T0901317 for and Thus, it that in the compounds as partial agonists, to This can be in and biochemical but to for in the of by LXR of nuclear receptors by small molecule ligands the formation of a binding for cofactor proteins on the LBD, In the of agonistic most coactivators to the of nuclear receptors a B. 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We the binding of of cofactor to the receptors at of As can be in the unliganded a affinity for corepressor for whereas it with the coactivator of T0901317 in this the increased affinity of the receptor for coactivator and a repression of corepressors the known of the In GW3965 a the affinity for coactivator increased as the binding to corepressors also enhanced with the data from the LN6500 and induced the binding of however, compounds induced coactivator binding more than T0901317 and binding of cofactor to the of LXRs at of ligands by The and of the ligand used is each of that the binding with the corepressor and is in the of the GW3965, and than in the of ligand. are S.D. of with the data, a affinity for corepressors in the of compounds with LXRα, the compounds display a with to the of coactivator and corepressor binding they induce. However, corepressor affinity is not enhanced over the unliganded receptor by of the compounds. by the induction of corepressor GW3965 to be more agonistic to than to LXRα, as in the shown in The behavior of receptors in the of the compounds can also be in in the is as shown in the full agonist T0901317 binding with the partial agonists or induce the binding to the corepressor. The of GW3965 on and are clearly whereas for LXRα, the binding to NCoR is induced over that of the unliganded it is for but to a than for We that T0901317 induces a conformation that only weak binding of corepressors and binding of coactivators, as for a pure agonist. In GW3965, and the natural ligand as partial agonists, as they induce a state of the is to an agonistic or cofactor to of the in an with the of the compounds in and an for the partial by GW3965, and We would to that the cofactor used here as biochemical and not be of the behavior of the cofactor of LXR to the the data, it be that the prevalence and of coactivators and corepressors in a cell would the net effect of a ligand on LXR-dependent that induce that an interaction with corepressors be more to increased of corepressors than compounds that only weak corepressor binding. This in As shown in the of partial agonists are more to of NCoR than of This effect is most for activity and weaker for is in with the cofactor in the with to the of LN6500 and are also more to an increased of NCoR in the cell than of data that the activities of the compounds to in the cofactor of cells and are to be cell we the of the activities of the synthetic ligands to corepressor would be in a cell by the induction of in the cell and in differentiated that their for LXRs compounds induced the cholesterol efflux and and the in cells to In T0901317 the expression of including involved in lipid and whereas GW3965 induces more is most for LN6500 to be in Thus, the of partial in the to be in the cell activity of the compounds. The activity of GW3965 in cells is in with on the of GW3965 and T0901317 on lipid in E.M. Savio D.A. Halpern A.R. Chen L. Miller C.P. Nambi P. J. Lipid Res. 2004; 45: 1929-1942Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, B. S. S. D. J. R. J. Lipid Res. 2004; 45: Full Text Full Text PDF PubMed Scopus Google Scholar). In a GW3965 reported to on the expression of in but to to induce triglyceride synthesis in the liver B. S. S. D. J. R. J. Lipid Res. 2004; 45: Full Text Full Text PDF PubMed Scopus Google Scholar). We would to propose that this selective effect is of the effect of GW3965 on the of coactivators and a new to LXR In the of drugs nuclear receptors, pure or are only the In most cases, partial and selective agonists are activate the receptor in a tissue-specific The for such selective nuclear receptor are and have been shown to activate the estrogen receptor in a tissue-specific in D.P. 2003; PubMed Scopus Google Scholar). to raloxifen and are to the of coactivators and corepressors in a such that the and can be by in the expression of coactivators and corepressors C.L. Mol. 1997; PubMed Scopus Google Scholar, Y. Brown M. Science. 2002; PubMed Scopus Google Scholar, M. M. A. A. T. R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. Y. S. Mol. 2004; PubMed Scopus Google Scholar). and other a for the behavior of estrogen receptor ligands has been that the of corepressors and coactivators in a cell will the agonistic or behavior of partial agonists C.L. Mol. 1997; PubMed Scopus Google Scholar). to the a of can be by the receptor the of a purely state and a purely agonistic state in C.L. Rev. 2004; PubMed Scopus Google Scholar). Partial agonists would induce that are to be in the on a Our clearly show that a for tissue-specific of LXR data that the by are with such a However, the present study also that the of that can a GW3965 induces binding of to coactivators comparably to T0901317, but dramatically in the effect on corepressor binding. GW3965 and on the other have on corepressor but LN6500 induces coactivator binding more Thus, we would to propose a classification of partial agonists to their cofactor as in In to pure agonists and pure of partial are full induction of corepressor as as coactivator partial induction of coactivator binding induction of corepressor partial induction of both coactivator and and inhibition of the binding of in as has been in mice B.L. G. Daige C.L. Bischoff E.D. M. K. Heyman R.A. Schulman I.G. Glass C.K. Mol. Cell. Biol. 2003; 23: PubMed Scopus Google Scholar). the for the binding of cofactors to the receptors can be it an to a this of receptor the can be at of ligand and is of the of the the of this classification of partial agonists, we have the of and on the that it is clearly that compounds to a for of a of For LXRα, however, the competitive induction of cofactor binding is for data that the nuclear receptor required for coactivator and binding are Lambert M.H. McKee D.D. Galardi C.M. K.D. Parks D.J. Moore Kliewer S.A. Willson T.M. 2002; PubMed Scopus Google Scholar). In the of and a binding coactivator the receptor an agonistic conformation (22Williams S. Bledsoe R.K. Collins J.L. Boggs S. Lambert M.H. Miller A.B. Moore J. McKee D.D. Moore L. Nichols J. Parks D. Watson M. Wisely B. Willson T.M. J. Biol. Chem. 2003; 278: 27138-27143Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, M. T. S. J. H. A. Gustafsson J.A. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus (137) Google Scholar, S. A. A. R.M. H. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). of in the of GW3965 have not been but data clearly show that GW3965 will enhance the binding to both activators and with the unliganded receptor. has to be that GW3965 a conformation in the receptor is for cofactor but is to The of a coactivator or corepressor will the receptor in an agonistic or This is in with the that T0901317 to in in a state that coactivators to to the receptor. GW3965, on the other not in M. T. S. J. H. A. Gustafsson J.A. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus (137) Google Scholar), is the for the affinity of the receptor for corepressors as shown Our data that the will be more for and be to to both of We would that it be to of agonistic and on the of cofactor of partial agonists on receptor α and Galardi C.M. Lambert M.H. J.A. Moore 2003; PubMed Scopus Google Scholar) and acid receptor α C. J. Res. 2002; PubMed Scopus Google Scholar) have been to data, the natural ligand is a partial agonist that not activate the transcriptional of In with data, S. T. M. C. K. D. K. D. L. J. 2003; PubMed Scopus Google Scholar), and structural data (22Williams S. Bledsoe R.K. Collins J.L. Boggs S. Lambert M.H. Miller A.B. Moore J. McKee D.D. Moore L. Nichols J. Parks D. Watson M. Wisely B. Willson T.M. J. Biol. Chem. 2003; 278: 27138-27143Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) have that the of by the natural ligands of LXR is than by This would explain of the receptor by more agonistic synthetic ligands is as has been shown C.P. Baffic J. Lam M.H. Lund E.G. Adams A.D. Fu X. Hayes N. Jones A.B. Macnaul K.L. Ondeyka J. Singh S. Wang J. Zhou G. Moller D.E. Wright S.D. Menke J.G. J. Biol. Chem. 2002; 277: 10021-10027Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). the of LXR activation by is to on the of coactivators and corepressors in a This would also for tissue-specific of LXR activity by in the cofactor of LXRs as drug targets has been by the that agonists not only the formation of atherosclerotic plaques but also serum and liver triglyceride GW3965 has been shown to have a effect on triglyceride levels in mice than T0901317 B. S. S. D. J. R. J. Lipid Res. 2004; 45: Full Text Full Text PDF PubMed Scopus Google Scholar), to the mechanisms of partial we However, it is that the are for drug and a drug will have to be more selective in Although we have shown that compounds such as LN6500 can be that display agonistic than GW3965, it to be can be in with increased is not at present a can be that not the and the receptor in of the of or the of partial in this not be the only to LXR activity, compounds to the of LXR the increased of partial of LXR and the of to of LXR activation the to further use of this drug We are to for with the