# Patent

## Title
LXR ligand testing method

## Abstract

A method of easily measuring whether or not an LXR ligand has the function of effecting, e.g., increasing, plasma triglyceride concentration and/or LDL cholesterol concentration in a mammal by using the binding activity between LXR and a coactivator, and a method of identifying LXR ligands that do not have the function of effecting, e.g., increasing, plasma triglyceride concentration and/or LDL cholesterol concentration by using the binding activity between LXR and a coactivator.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the United States national phase application of International Application PCT/US2006/024945 filed Jun. 26, 2006. This application claims the benefit of priority under 35 USC 119(e) for U.S. provisional application Ser. No. 60/694,806 filed Jun. 28, 2005.


BACKGROUND OF THE INVENTION
Description of the Related Art
Plasma triglycerides and LDL cholesterol are known to be risk factors for arteriosclerosis. Hyperlipidemia, in which blood concentrations of triglycerides and LDL cholesterol are elevated, is a disease that not only causes impairment of vascular endothelial cells, but also causes deposition of cholesterol on vascular walls.
Since ATP binding transporter A1 (ABCA1) has a function that removes cholesterol deposited on vascular walls, it is believed that increasing the expressed amount of ABCA1 would make it possible to prevent progression of or improve arteriosclerosis (Bodzioch, M. et al. Nat. Genet., 22, 347-351, 1999).
The nuclear receptor, Liver X receptor (LXR) controls transcription of the regulatory gene of cholesterol and lipid metabolism. Since LXR agonists have the ability of increasing expression of ABCA1, LXR agonists are expected to be useful as novel anti-arteriosclerotic agents.
LXR is known to have two isoforms consisting of LXRα and LXRβ. LXRα is highly expressed in the liver, intestine, fat cells and kidney, and only slightly expressed in the adrenal, muscle and hematopoietic cells. On the other hand, LXRβ is universally ubiquitously expressed.
When a pan-LXR agonist was administered to wild-type mice, LXRα-deficient mice or LXRβ-deficient mice, the pan-LXR agonist elevated plasma triglyceride levels in wild-type mice and LXRα-deficient mice, whereas the pan-LXR agonist did not affect plasma triglycerides levels. From these results, a process has been disclosed for acquiring LXRβ-selective agonists (US2003/0073614A1).
When a ligand binds to a nuclear receptor, the three-dimensional structure of the nuclear receptor is changed, and these conformational changes are known to occur in the binding between the nuclear receptor and transcription associating factor, namely a coactivator or co-repressor protein. The type of coactivator that binds to the nuclear receptor varies according to the type of cell and tissue. In addition, if the ligand that binds with the nuclear receptor differs, then the change in the three-dimensional structure of the nuclear receptor also differs, and as a result, the types and numbers of coactivators that bind with the nuclear receptor also differ.
Although known examples of coactivator proteins for LXR include PGC-1α (Homo sapiens peroxisome proliferative activated receptor, gamma, coactivator 1, alpha: Proc Natl Acad Sci USA. 100, 5419-24, 2003), TIF-2(Homo sapiens nuclear receptor coactivator 2) (EMBO J., 23, 2083-2091, 2004), ASC-2 (Activating signal cointegrator 2) (Mol. Cell Biol., 23, 3583-3592, 2003), SRC-1 (Human steroid receptor coactivator-1) (Arterioscler Thromb. Vasc. Biol., 24, 703-708, 2004), DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital (AHC) critical region on the X chromosome, gene 1) (Mol. Endocrinol., 17, 994-1004, 2003), PNRC (proline-rich nuclear receptor coregulatory protein) (Mol. Endocrinol., 14, 986-998, 2000), TRAP220 (thyroid hormone receptor-associated protein 220) (J. Biol. Chem., 280, 1625-1633, 2005), PERC (Peroxisome proliferator-activated receptor gamma coactivator-1 beta) (J. Biol. Chem., 281, 14537-14546, 2006), ACTR (steroid receptor coactivator-3) (J. Biol. Chem., 281, 14787-14795, 2006), research on LXR ligands has yet to be conducted in consideration of the interaction between these coactivators and LXR as well as LXR ligands.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of identifying LXR ligands that do not cause an effect, e.g., increase, in LDL cholesterol and/or plasma triglyceride concentrations in a mammal.
Moreover, another object of the present invention is to provide a method of easily measuring the effects of LXR ligands on plasma lipids.
Moreover, another object of the present invention is to provide a kit that can be used to identify LXR ligands that do not cause an effect, e.g., increase, in LDL cholesterol concentration and/or plasma triglycerides in a mammal.
As a result of conducting extensive studies to solve the aforementioned problems, the inventors of the present invention found that among LXR agonists, certain LXR agonists have the function of increasing low density lipoprotein (LDL) cholesterol concentration, and that LXR ligands for which there is low binding activity between LXRα and a specific coactivator are not observed or only observed to slightly increase plasma triglyceride and/or LDL cholesterol concentrations.
The inventors of the present invention also discovered a method of easily measuring whether an LXR ligand has the function of increasing plasma triglyceride concentration and/or LDL cholesterol concentration by utilizing the binding activity between LXR and a coactivator.
Moreover, the inventors of the present invention discovered a method of identifying LXR ligands that do not have the function of effecting, e.g., increasing, plasma triglyceride concentration and/or LDL cholesterol concentration in a mammal by utilizing the binding activity between LXR and a coactivator, thereby leading to completion of the present invention.
The present invention provides a method for easily measuring whether or not an LXR ligand has the function of effecting, e.g., increasing, plasma triglyceride concentration and/or LDL cholesterol concentration in a mammal.
Moreover, the present invention provides a method for identifying LXR ligands that do not have the function of effecting, e.g., increasing, plasma triglyceride concentration and/or LDL cholesterol concentration in a mammal.
Moreover, the present invention provides a kit that can be used in a method for identifying LXR ligands that do not cause a significant effect, e.g., increase, in LDL cholesterol concentration and/or plasma triglyceride concentration in a mammal.
These inventions are as follows:
1) A method of identifying a therapeutic or preventive agent that affects LDL cholesterol and/or plasma triglyceride concentration in a mammal, the method comprising:
(i) providing a heterodimer comprising LXRα and RXRα;
(ii) contacting a test substance with the heterodimer in the presence of an LXR coactivator;
(iii) measuring the amount of coactivator bound to the heterodimer;
(iv) comparing the amount of the coactivator measured in step (iii) with the amount of the coactivator bound to the heterodimer in a control; and
(v) correlating the difference between the amount of bound coactivator and the amount of bound coactivator in the control as indicative of the activity of the test substance to significantly affect, e.g., increase, LDL cholesterol and/or plasma triglyceride concentration in a mammal.

2) The method according to 1), wherein the method is to identify a therapeutic or preventive agent that does not cause an increase in LDL cholesterol and/or plasma in a mammal.

3) The method according to 1), wherein the test substance is an LXR ligand.

4) The method according to 1) wherein the therapeutic or preventive agent is employed to treat or prevent a disease selected from the group consisting of arteriosclerosis, atherosclerosis, hyperlipidemia, lipid related diseases, an inflammatory disease mediated by inflammatory cytokines, autoimmune diseases, cardiovascular disease, cerebrovascular disease, renal disease, diabetes mellitus, diabetic complications, obesity, nephritis, hepatitis, and alzheimer's disease.

5) The method according to 1), wherein the coactivator is selected from the group consisting of PGC-1α (homo sapiens peroxisome proliferative activated receptor, gamma coactivator 1, alpha), TIF-2 (homo sapiens nuclear receptor coactivator 2), ASC-2 (activating signal cointegrator 2), SRC-1 (human steroid receptor coactivator-1), DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital (AHC) critical region on the X chromosome, gene 1), PNRC (proline-rich nuclear receptor coregulatory protein), TRAP220 (thyroid hormone receptor-associated protein 220), PERC (peroxisome proliferator-activated receptor gamma coactivator-1 beta) and ACTR (steroid receptor coactivator-3).

6) The method according to 1), wherein the coactivator is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and variants thereof.

7) The method according to 1), wherein the LXRα is a human full-length LXRα polypeptide having the amino acid sequence of SEQ ID NO: 2 or a variant thereof which has at least 80% identity to SEQ ID NO: 2, or a fused protein containing said polypeptide.

8) The method according to 1), wherein the LXRα is a ligand binding site of human full-length LXRα having the amino acid sequence of amino acid nos. 164 to 447 of SEQ ID NO: 2 or a variant thereof which has at least 80% identity to amino acid nos. 164 to 447 of SEQ ID NO: 2, or a fused protein containing said polypeptide.

9) The method according to 1), wherein the RXRα is a human full-length RXRα polypeptide having the amino acid sequence of SEQ ID NO: 4 or a variant thereof which has at least 80% identity to SEQ ID NO: 4, or a fused protein containing said polypeptide.

10) The method according to 1), wherein the RXRα is a ligand binding site of human full-length RXRα having the amino acid sequence of amino acid nos. 201 to 462 of SEQ ID NO: 4 or a variant thereof which has at least 80% identity to amino acid nos. 201 to 462 of SEQ ID NO: 4, or a fused protein containing said polypeptide.

11) The method according to 1) wherein the amount of the coactivator bound to the heterodimer is measured using a FRET assay.

12) The method according to 1), wherein the LXRα and/or the RXRα is provided by using cells that express LXRα and/or RXRα.

13) The method according to 1), wherein the LXRα and/or the RXRα is provided by using cells that express LXRα and/or RXRα as an exogenous protein.

14) The method according to 1) wherein the LXRα and/or the RXRα is provide by using cells that express LXRα and/or RXRα as an endogenous protein.

15) A kit which is used for any one of 1) to 14), the kit comprising one or more of the components selected from the group consisting of [A] to [L] below:

[A] a human full-length LXRα polypeptide, a human full-length RXRα polypeptide, and a coactivator selected from the group consisting of PGC-1α (homo sapiens peroxisome proliferative activated receptor gamma coactivator 1, alpha), TIF-2 (homo sapiens nuclear receptor coactivator 2), ASC-2 (activating signal cointegrator 2), SRC-1 (human steroid receptor coactivator-1), DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital (AHC) critical region on the X chromosome, gene 1), PNRC (proline-rich nuclear receptor coregulatory protein), TRAP220 (thyroid hormone receptor-associated protein 220), PERC (peroxisome proliferator-activated receptor gamma coactivator-1 beta) and ACTR (steroid receptor coactivator-3),

wherein the human full-length LXRα polypeptide has the amino acid sequence of SEQ ID NO: 2 or a variant thereof which has at least 80% identity to SEQ ID NO: 2, and the human full-length RXRα polypeptide has the amino acid sequence of SEQ ID NO: 4 or a variant thereof which has at least 80% identity to SEQ ID NO: 4;
[B] a ligand binding site of a polypeptide described in [A], and any one of the coactivators set forth in [A]; and wherein the ligand binding site of a human full-length LXRα polypeptide has the amino acid sequence of amino acid nos. 164 to 447 of SEQ ID NO: 2 or a variant thereof which has at least 80% identity to amino acid nos. 164 to 447 of SEQ ID NO: 2, and a ligand binding site of human full-length RXRα polypeptide has the amino acid sequence of amino acid nos. 201 to 462 of SEQ ID NO: 4 or a variant thereof which has at least 80% identity to amino acid nos. 201 to 462 of SEQ ID NO: 4;

[C] a fused polypeptide containing a ligand binding site set forth in [B], and any one of the coactivators set forth in [A];

[D] a polypeptide set forth in any one of [A] to [C], and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and variants thereof;

[E] a polynucleotide encoding a polypeptide described in [A], and any one of the coactivators set forth in [A];

[F] a polynucleotide encoding a polypeptide described in [B], and any one of the coactivators set forth in [A];

[G] a polynucleotide encoding a polypeptide described in [C], and any one of the coactivators set forth in [A];

[H] a polynucleotide set forth in any one of [E] to [G], and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and variants thereof;

[I] a recombinant vector containing a polynucleotide set forth in any one of [E] to [H], and any one of the coactivators set forth in [A];

[J] a recombinant vector set forth in [I] that is an expression vector, and any one of the coactivators set forth in [A];

[K] host cells transformed with a recombinant vector set forth in [I] or [J], and any one of the coactivators set forth in [A]; and

[L] host cells set forth in [K] that are mammalian cells, and any one of the coactivators set forth in [A];

16) A method of diagnosing a disease state in a mammal comprising

(i) collecting a biological sample, e.g., blood, plasma, liver, intestine, fat, kidney, adrenal gland, muscle or cells of the hematopoetic system, from the mammal;
(ii) contacting the biological sample with a heterodimer comprising LXRα and RXRα and a test substance in the presence of an LXR coactivator;
(iii) repeating said contacting step (i) with a control sample;
(iv) measuring the amount of the coactivator bound to the heterodimer;
(v) comparing the amount of the coactivator bound to the heterodimer in the biological sample collected from the mammal and the bound coactivator to the heterodimer in the control sample; and
(vi) determining whether the mammal is in a disease state, when the amount of the coactivator in the biological sample is greater than the amount of the coactivator bound to the heterodimer in the control, which is indicative of an increase in LDL cholesterol and/or plasma concentration levels in the mammal.
17) The method according to 16), wherein the disease state is selected from the group consisting of (a) arteriosclerosis, (b) atherosclerosis, (c) hyperlipidemia, (d) a lipid-related disease, (e) an inflammatory disease mediated by an inflammatory cytokine, (f) an autoimmune disease, (g) a cardiovascular disease, (h) a cerebrovascular disease, (i) a renal disease, (j) diabetes mellitus, (k) a diabetic complication, (l) obesity, (m) nephritis, (n) hepatitis, (o) a tumor, (p) Alzheimer's disease and (q) arteriosclerosis caused by one or more of the diseases (c) to (o).

18) The method according to 17), wherein the mammal is a human.

19) The method according to 18), wherein the biological sample is a blood sample.

20) The method according to 19), wherein the coactivator is an LXR coactivator.

21) The method according to 20), wherein the method according to claim 19, wherein the coactivator is selected from the group consisting of PGC-1α (homo-sapiens peroxisome proliferative activated receptor, gamma coactivator 1, alpha), TIF-2 (homo sapiens nuclear receptor coactivator 2), ASC-2 (activating signal cointegrator 2), SRC-1 (human steroid receptor coactivator-1), DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital (AHC) critical region on the X chromosome, gene 1), PNRC (praline-rich nuclear receptor coregulatory protein), TRAP220 (thyroid hormone receptor-associated protein 220), PERC (peroxisome proliferator-activated receptor gamma coactivator-1 beta) and ACTR (steroid receptor coactivator-3).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Explanation of Terms
The term “affect plasma triglyceride concentration” as used herein is intended to mean the use of LXR ligand as a therapeutic or preventive agent in regulating/monitoring the LDL cholesterol and plasma triglyceride concentration levels in a mammal, thereby inhibiting, preventing, ameliorating or reducing the risk of occurrence of a metabolic disease condition, such as atherosclerosis or an atherosclerotic disease event.
In the present specification, the term “arteriosclerosis-related diseases” refers to diseases that are present with symptoms of arteriosclerosis during the course of the disease from the time of onset, or diseases caused by arteriosclerosis. In addition, in the present specification, arteriosclerosis-related diseases refer to diseases for which all or a portion of the symptoms are improved by suppressing exacerbation of arteriosclerosis symptoms, improving arteriosclerosis symptoms, curing arteriosclerosis symptoms, preventing the appearance of arteriosclerosis symptoms or treating the causative disease.
Examples of arteriosclerosis-related diseases include arteriosclerosis, atherosclerosis, hyperlipidemia, lipid-related diseases, inflammatory diseases mediated by inflammatory cytokines, autoimmune diseases, cardiovascular diseases, cerebrovascular diseases.
Arteriosclerosis caused by one or more diseases selected from hyperlipidemia, lipid-related diseases, inflammatory diseases mediated by inflammatory cytokines, autoimmune diseases, cardiovascular diseases, cerebrovascular diseases, renal diseases, diabetes mellitus, diabetic complications, obesity is also included in the arteriosclerosis-related diseases of the present invention.
2. LXR Ligands
Although examples of LXR ligands include the compounds indicated below, there are no particular limitations on such compounds provided they are LXR ligands. For example, substances identified as LXR ligands based on the function of promoting expression of ABCA1 mRNA, the amount of cholesterol effluxed, cholesterol efflux activity or by the method described in a co-transfection assay, for example (WO2003/106435, Test Example 3) can also be used as LXR ligands in the present invention.
Examples of LXR ligands include: Compound 12 described on page 55 of International Publication WO2000/054759 (N-(2,2,2-trifluoroethyl)-N-{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl}benzene sulfonamide), 3-chloro-4-(3-(2-propyl-3-trifluoromethyl-6-benz-[4,5]-isooxazoloxy)propylthio)phenylacetic acid described in Example 20 on page 70 of WO1997/028137, 1-(2-Methoxyethyl)-4-[(4-methoxyphenyl)amino]-3-phenyl-5-thioxo-1,5-dihydro-2H-pyrrol-2-one described on page 41 of International Publication WO2005/005416, 2-methyl-N-{5-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]-1,3-thiazol-2yl}propanamide described on page 37 of WO03/090869, 1-[(6-fluoro(2H,4H-benzo[e]1,3-dioxin-8-yl))methoxy]-2-nitrobenzene described on page 27 of WO02/46181, 1-[1-(4-cyclohexylbenzoyl)-4-phenylpiperidin-4-yl]ethanone described on page 20 of WO2004/076418, 2-(3-{3-[[2-Chloro-3-(trifluoromethyl)benzyl](2,2-diphenylethyl)amino]propoxy}-phenyl)acetic acid described on page 46 of WO02/24632, methyl-4-amino-1-(2-chloro-6-fluorobenzyl)-2-piperidin-1-yl-1H-imidazole-5-carboxylate described on page 59 of WO2004/009091, 2,4-dihydroxy-3-propyl-1′, 1′,1′-trifluoroacetophenone described on page 24 of WO03/045382, 3-chloro-4-(3-(3-ethyl-7-propyl-6-benz-[4,5]-isoxazoloxy)propylthio)phenylacetic acid described on page 42 of WO97/28137, 3-[({2-[(2,2-dimethylpropanoyl)thio]methyl}-N-(4-methoxylbenzyl)benzamide described on page 24 of WO2004/026816, N-(2-{[6-chloro-1,3-benzodioxol-5-yl]methyl}thiophenyl)-2-2-2-trifluoroacetamide described on page 29 of WO03/059874, 2-(3-{3-[(2-Chloro-3-(trifluoromethyl)-benzyl)-(2,2-diphenyl)-amino]-propoxy}phenyl)-1-morpholin-4-yl-ethanone hydrochloride salt described on page 45 of WO2004/043939, (R)-2-(3-{3-[[Chloro-3-(trifluoromethyl)benzyl](2,2-diphenylethyl)amino]-2-methyl-propoxy}-phenyl)acetic acid hydroxychloride salt described on page 43 of WO03/082802, 2-(3-{3-[[2-Chloro-3-(trifluoromethyl)benzyl](2,2-diphenyethyl)amino]propoxy}-phenyl)acetic acid, N-oxide described on page 55 of WO03/082205, N-(4-{1-Hydroxy-1-[1-(2-methoxyethyl)-1H-pyrrol-2-yl]-ethyl}-phenyl)-N-methyl-benzenesulfonamide described on page 33 of WO03/063796, N-Methyl-N-[2-methyl-4-(2,2,2-trifluoro-1-hydroxy-1-phenethyl)]-benzenesulfonamide described on page 29 of WO03/063576, [4-({3-[3-Benzyl-8-(trifluoromethyl)quinolin-4-yl]phenoxy}methyl)phenyl]acetic acid described on page 86 of WO05/058834, [4-({3-[2-Methyl-7-(trifluoromethyl)-2H-indazol-3-yl)phenoxy)methyl)phenyl]acetic acid described on page 251 of WO06/017384.
Examples of LXR ligands also include compounds having structure (1) to (165) shown in “Recent Patents on Cardiovascular Drug Discovery, 2006, 1, 21-46.”
3. Preparation of LXRα, LXRβ and RXRα
Human LXRα, LXRβ and RXRα are not limited to their full-length proteins, but rather may also be partial peptides comprising of their partial sequences provided they contain the ligand binding domain. A human full-length LXRα has an amino acid sequence of SEQ ID NO: 2 or a variant thereof which has the ligand binding ability. A human full-length RXRα has an amino acid sequence of SEQ ID NO: 4 or a variant thereof which has the ligand binding ability. A human full-length LXRβ has an amino acid sequence of SEQ ID NO: 6 or a variant thereof which has the ligand binding ability. Suitably the degree of identity of polypeptide variants to SEQ ID NO: 2 or 4 or 6 is at least 80%, at least 90% or at least 95% or 100%. The degree of identity of a variant is preferably assessed by computer software, such as the BLAST program which uses an algorithm for performing homology searches. In addition, they may also be naturally-occurring proteins acquired from human-derived cells, and may also be proteins acquired from gene-recombinant cells designed to express said protein by a gene that has been cloned by PCR and so forth. In addition, these proteins may be purified or only partially purified.
Moreover, fused proteins, in which other amino acid sequences have been added to human LXRα, human LXRβ and human RXRα or their partial peptides, are also included in human LXRα, human LXRβ, human RXRα and their partial peptides. Examples of fused proteins include, but are not limited to, histidine tag fused proteins, FLAG fused proteins, and GFP and other fluorescent fused proteins.
Human LXRα gene is registered in GenBank as Accession No. U22662 (see P. J. Willy et al., Genes Dev. 9 (9), 1033-1045, 1995, nucleotide numbers 597 to 1379).
Human LXRβ gene is registered in GenBank as Accession No. U07132 (see P. J. Willy et al., Genes Dev. 9 (9), 1033-1045, 1995).
Human RXRα gene is registered in GenBank as Accession No. X52773.
4. Preparation of the Coactivator
Examples of LXR coactivators include PGC-1α (Homo sapiens peroxisome proliferative activated receptor, gamma, coactivator 1, alpha), TIF-2 (homo sapiens nuclear receptor coactivator 2), ASC-2 (activating signal cointegrator 2), SRC-1 (human steroid receptor coactivator-1), DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital (AHC) critical region on the X chromosome, gene 1), PNRC (proline-rich nuclear receptor coregulatory protein), TRAP220 (thyroid hormone receptor-associated protein 220), PERC (peroxisome proliferator-activated receptor gamma coactivator-1 beta), ACTR (steroid receptor coactivator-3). Coactivators are not limited to full-length proteins, but rather partial peptides containing an LXXLL motif (where L represents leucine and X represents an arbitrary amino acid) can also be used. The peptide selected from the group having an amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and variants thereof which contain an LXXLL motif can also be used as a coactivator. Suitably the degree of identity of polypeptide variants to SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28 is at least 80%, at least 90% or at least 95% or 100%. These proteins may also be naturally-occurring proteins acquired from human-derived cells, and may also be proteins acquired from gene-recombinant cells designed to express said protein by a gene that has been cloned by PCR and so forth. In addition, chemically synthesized proteins may be used.
The nucleotide sequence of PGC-1α is registered in GenBank as Accession No. NM—013261. The nucleotide sequence of TIF-2 is registered in GenBank as Accession No. NM—006540. The nucleotide sequence of ASC-2 is registered in GenBank as Accession No. AF177388. The nucleotide sequence of SRC-1 is registered in GenBank as Accession No. U90661. The nucleotide sequence of DAX1 is registered in GenBank as Accession No. NP—000466. The nucleotide sequence of PNRC is registered in GenBank as Accession No. NM—000044. The nucleotide sequence of TRAP220 is registered in GenBank as Accession No. NM—004774. The nucleotide sequence of PERC is registered in GenBank as Accession No. NM—133263. The nucleotide sequence of ACTR is registered in GenBank as Accession No. AF012108.
5. Method of Assessing Function of Increasing LDL Cholesterol and/or Plasma Triglyceride Levels by LXR Ligands
Whether or not an LXR ligand has the function of increasing plasma LDL cholesterol and/or plasma triglyceride concentrations can be assessed by whether or not said LXR ligand increases the amount of binding between LXRα and at least one coactivator selected from PGC-1α TIF-2, ASC-2, SRC-1, DAX1, PNRC, TRAP220, PERC and ACTR. More specifically, this method contains the steps of 1) or 2) below:
1)
i) a step of contacting a heterodimer comprising of LXRα and RXRα with an LXR coactivator and a test substance;
ii) a step of measuring the amount of coactivator bound to the heterodimer; and,
iii) a step of comparing the amount of coactivator measured in step ii) with the amount of coactivator bound to the heterodimer measured in the case of not contacting the heterodimer with the test substance.
2)
i) a step of contacting a heterodimer comprising of LXRα and RXRα with an LXR coactivator and a test substance;
ii) a step of measuring the amount of coactivator bound to the heterodimer;
iii) a step of comparing the amount of coactivator measured in step ii) with the amount of coactivator bound to the heterodimer measured in the case of not contacting the heterodimer with the test substance; and,
iv) a step of judging the LXR ligand to have the function of increasing plasma LDL cholesterol concentration and/or plasma triglyceride concentration in the case the amount of coactivator measured in step ii) increases in comparison with the amount of coactivator bound to the heterodimer measured in the case of not contacting the heterodimer with the test substance.
The following provides an explanation of each step.
1)
Step 1)-i):
A heterodimer comprising of LXRα and RXRα can be obtained by mixing LXRα and RXRα acquired according to the method described in the aforementioned section entitled “3. Preparation of LXRα, LXRβ and RXRα”.
In addition, a heterodimer of LXRα and RXRα can be acquired by, for example, producing a vector that co-expresses a fused protein of the ligand binding domain (amino acid nos. 164 to 447 of SEQ ID NO: 2 of the Sequence Listing) of human LXRα (SEQ ID NO: 2) and His tag, and a fused protein of the ligand binding domain (amino acid nos. 201 to 462 of SEQ ID NO: 4 of the Sequence Listing) of RXRα (SEQ ID NO: 4) and FLAG, and purifying the protein expressed by a recombinant transformed with said expression vector.
Furthermore, a heterodimer comprising of LXRβ and RXRα used for a comparative experiment can be acquired by, for example, producing a vector that co-expresses a fused protein of the ligand binding domain (amino acid nos. 155 to 461 of SEQ ID NO: 6 of the Sequence Listing) of human LXRβ (SEQ ID NO: 6) and His tag, and a fused protein of the ligand binding domain (amino acid nos. 201 to 462 of SEQ ID NO: 4 of the Sequence Listing) of RXRα (SEQ ID NO: 4) and FLAG, and purifying the protein expressed by a recombinant transformed with said expression vector.
The LXR coactivators explained in the section entitled “4. Preparation of Coactivator” can be used for the LXR coactivator.
The substances described in the section entitled “2. LXR Ligands” can be used for the test substance.
In addition, a buffer for controlling the pH, or antibody for detecting the fused protein can be added to the reaction solution as necessary.
These materials are then mixed and subjected to the reaction, for example, described below.
Temperature conditions: 0° C. to 40° C., and preferably 4° C.
Reaction solution pH: 6 to 9, and preferably 7.4
Reaction time: 1 minute to 48 hours, and preferably 17 hours
The reaction can be carried out using, for example, a 384-well assay plate.
Step 1)-ii):
An example of a method for measuring the amount of coactivator bound to the heterodimer is described below.
In the case of carrying out the reaction in an assay plate using a coactivator in which the N-terminal has been biotinylated, the plate is subjected to excitation light at 337 nm with a fluorescent plate reader following completion of the reaction, the fluorescent intensity (A) at 665 nm and the fluorescent intensity (B) at 620 nm are determined, and the measured value at 655 nm is divided by the measured value at 620 nm followed by multiplying the resulting value by 1000 to determine the (C).

C=(A/B)×1000

A: Fluorescent intensity at 665 nm
B: Fluorescent intensity at 620 nm
Step 1)-iii):
The above value of (C) is divided by the value (C′) obtained in the same experiment except for not adding the test substance, and that value is multiplied by 100 to determine the relative activity with respect to the control (R: % of control). The calculation formula is as shown below.

C′=(A′/B′)×1000

R=C/C′×100

A′: Fluorescent intensity at 665 nm in the case of carrying out the reaction without adding the test substance
B′: Fluorescent intensity at 620 nm in the case of carrying out the reaction without adding the test substance
This means that the higher the relative activity (R), the greater the bound amount of coactivator as compared with the control to which LXR ligand is not added.
In the case relative activity (R) is greater than 100, it can be judged that the amount of coactivator increases as a result of contacting the heterodimer with the test substance. A test substance can be judged to have less of a function that increases LDL cholesterol concentration and/or plasma triglyceride concentration the closer its value of relative activity is to 100.
In the case the relative activity of a test substance is higher than the relative activity determined for an LXR ligand for which the function of increasing LDL cholesterol and/or plasma triglyceride concentration is being investigated (referred to as “Compound X”), the function of increasing LDL cholesterol and/or plasma triglyceride concentration of the test substance can be judged to be stronger than that of Compound X.
In the case the relative activity of a test substance is lower than the relative activity determined for Compound X, the function of increasing LDL cholesterol and/or plasma triglyceride concentration of the test substance can be judged to be weaker than that of Compound X.
If the relative activities determined for a plurality of test substances are compared, a test substance can be judged to have a weaker function of increasing LDL cholesterol and/or plasma triglyceride concentration the lower its relative activity.
2)
Steps 2)-i), 2)-ii) and 2)-iii) are the same as the aforementioned steps 1)-i), 1)-ii) and 1)-iii).
Step 2)-iv):
As was explained in the aforementioned 1)-iii), a test substance can be judged to be a substance having less function that increases LDL cholesterol and/or plasma triglyceride concentration the closer its value of relative activity is to 100. Conversely, in the case its relative activity is greater than 100, the test substance can be judged to have a function that increases LDL cholesterol and/or plasma triglyceride concentration.
In the case the relative activity of a test substance is higher than the relative activity determined for an LXR ligand for which the function of increasing LDL cholesterol and/or plasma triglyceride concentration is being investigated (referred to as “Compound X”), the function of increasing LDL cholesterol and/or plasma triglyceride concentration of the test substance can be judged to be stronger than that of Compound X.
In the case the relative activity of a test substance is lower than the relative activity determined for Compound X, the function of increasing LDL cholesterol and/or plasma triglyceride concentration of the test substance can be judged to be weaker than that of Compound X.
If the relative activities determined for a plurality of test substances are compared, a test substance can be judged to have a weaker function of increasing LDL cholesterol and/or plasma triglyceride concentration the lower its relative activity.
In addition, in the aforementioned 1) and 2), in addition to being based on relative activity with respect to a single coactivator, relative activity can also be comprehensively assessed for a plurality of coactivators to judge the function of increasing plasma LDL cholesterol and/or plasma triglyceride concentration of a test substance.
6. Method for Identifying Substances Having Little or No Function of Increasing LDL Cholesterol and/or Plasma Triglyceride Concentration of an LXR Ligand
The following steps make it possible to acquire an LXR ligand having little or no function of increasing LDL cholesterol and/or plasma triglyceride concentration:
i) a step of contacting a heterodimer comprising of LXRα and RXRα with an LXR coactivator and a test substance;
ii) a step of measuring the amount of coactivator bound to the heterodimer;
iii) a step of comparing the amount of coactivator measured in step ii) with the amount of coactivator bound to the heterodimer measured in the case of not contacting the heterodimer with the test substance; and,
iv) a step of judging the LXR ligand to have the function of increasing plasma LDL concentration and/or plasma triglyceride concentration in the case the amount of coactivator measured in step ii) does not increase in comparison with the amount of coactivator bound to the heterodimer measured in the case of not contacting the heterodimer with the test substance.
These steps can be carried out by the same method as the method described in the aforementioned section entitled “5. Method of Assessing Function of Increasing LDL Cholesterol and/or Plasma Triglyceride Levels by LXR Ligands”.
The phrase “the amount of coactivator measured in step ii) does not increase” refers to at least one of the following conditions: a) relative activity is about 100, b) relative activity is demonstrated that is roughly equal to or less than the relative activity determined for an LXR ligand which is known to have little or no effect of increasing plasma LDL cholesterol and/or plasma triglyceride concentration.
According to this method, a substance identified to have little or no function of increasing LDL cholesterol and/or plasma triglyceride concentration can be a therapeutic or preventive agent of one or more of the diseases selected from the diseases of (a) to (q) indicated below.
(a) arteriosclerosis;
(b) atherosclerosis;
(c) hyperlipidemia;
(d) lipid-related diseases;
(e) inflammatory diseases mediated by inflammatory cytokines;
(f) autoimmune diseases;
(g) cardiovascular diseases;
(h) cerebrovascular diseases;
(i) renal diseases;
(j) diabetes mellitus;
(k) diabetic complications;
(l) obesity;
(m) nephritis;
(n) hepatitis;
(o) tumor;
(p) Alzheimer's disease; and
(q) arteriosclerosis caused by one or more of the diseases selected from (c) to (o).
7. A Kit for Identifying LXR Ligands
The kit indicated below can be used to identify LXR ligands that do not increase LDL cholesterol and/or plasma triglyceride concentration in a mammal, which comprises one or more of the components selected from the group consisting of [A] to [L] below:
[A] a human full-length LXRα polypeptide, a human full-length RXRα polypeptide, and a coactivator selected from the group consisting of PGC-1α (homo sapiens peroxisome proliferative activated receptor, gamma coactivator 1, alpha), TIF-2 (homo sapiens nuclear receptor coactivator 2), ASC-2 (activating signal cointegrator 2), SCR-1 (human steroid receptor coactivator-1), DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital (AHC) critical region on the X chromosome, gene 1), PNRC (proline-rich nuclear receptor coregulatory protein), TRAP220 (thyroid hormone receptor-associated protein 220), PERC (peroxisome proliferator-activated receptor gamma coactivator-1 beta) and ACTR (steroid receptor coactivator-3),
wherein the human full-length LXRα polypeptide has the amino acid sequence of SEQ ID NO: 2 or a variant thereof which has at least 80% identity to SEQ ID NO: 2, and the human full-length RXRα polypeptide has at least 80% identity to SEQ ID NO: 4;
[B] a ligand binding site of a polypeptide described in [A], and any one of the coactivators set forth in [A], wherein the ligand binding site of a human full-length LXRα polypeptide has the amino acid sequence of amino acid nos. 164 to 447 of SEQ ID NO: 2 or a variant thereof which has at least 80% identity to amino acid nos. 164 to 447 of SEQ ID NO: 2, and a ligand binding site of human full-length RXRα polypeptide has the amino acid sequence of amino acid nos. 201 to 462 of SEQ ID NO: 4 or a variant thereof which has at least 80% identity to amino acid nos. 201 to 462 of SEQ ID NO: 4;

[C] a fused polypeptide containing a ligand binding site set forth in [B], and any one of the coactivators set forth in [A];

[D] a polypeptide set forth in any one of [A] to [C], and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID No: 28 and variants thereof;

[E] a polynucleotide encoding a polypeptide described in [A], and any one of the coactivators set forth in [A];

[F] a polynucleotide encoding a polypeptide described in [B], and any one of the coactivators set forth in [A];

[G] a polynucleotide encoding a polypeptide described in [C], and any one of the coactivators set forth in [A];

[H] a polynucleotide set forth in any one of [E] to [G], and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and variants thereof;

[I] a recombinant vector containing a polynucleotide set forth in any one of [E] to [H], and any one of the coactivators set forth in [A];

[J] a recombinant vector set forth in [I] that is an expression vector, and any one of the coactivators set forth in [A];

[K] host cells transformed with a recombinant vector set forth in [I] or [J], and any one of the coactivators set forth in [A]; and

[L] host cells set forth in [K] that are mammalian cells, and any one of the coactivators set forth in [A].

EXAMPLES
Although the following provides a more detailed explanation of the present invention through its test examples and examples, the present invention is not limited thereto.
Test Example 1
Measurement of LXR Ligand Cholesterol Efflux Activity (Cholesterol Efflux Assay)
The cholesterol efflux activity of two types of LXR ligands (Compound A (N-(2,2,2-trifluoroethyl)-N-{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl}benzenesulfonamide and Compound B (3-chloro-4-(3-(2-propyl-3-trifluoromethyl-6-benz-[4,5]-isoxazoloxy)propylthio)phenylacetic acid) was measured using the method described below.
3×105 THP-1 cells (ATCC No.: TIB-202) were disseminated in a 96-well white plate (Costar) followed by the addition of 200 nM Phorbol 12-Myristate 13-Acetate (Sigma) and culturing for 24 hours at 37° C. in a CO2 incubator.
Next, 125 μl aliquots of medium (RPMI 1640 (Invitrogen)+1% fetal bovine serum (hereinafter referred to as “FBS”, (Invitrogen)) containing 0.2 μCi/ml 4-14C-Cholesterol (Perkin-Elmer) were added to each well followed by additionally culturing for 48 hours at 37° C. in a CO2 incubator.
Following completion of culturing, 100 μl of PBS containing 0.2% bovine serum albumin (hereinafter referred to as “BSA”, (Sigma Chemical)) were added to each well to wash the cells. Next, 100 μl of RPMI 1640 containing Apolipoprotein A1 (hereinafter referred to as “ApoA1”, (Biogenesis)) at a final concentration of 10 μg/ml, or RPMI 1640 not containing ApoA1, were added to each well. Next, a DMSO solution was added to the wells so that the final concentration of Compound A (N-(2,2,2-trifluoroethyl)-N-{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl}benzenesulfonamide or Compound B (3-chloro-4-(3-(2-propyl-3-trifluoromethyl-6-benz-[4,5]-isoxazoloxy)propylthio)phenylacetic acid) was 0.01 μM, 0.1 μM and 1 μM each and the final concentration of DMSO was 1%, followed by culturing for 24 hours at 37° C. in a CO2 incubator.
Following completion of culturing, the plate was centrifuged for 5 minutes at 1,000 rpm and 4° C. 75 μl of medium in the form of the centrifuged supernatant were transferred to a 96-well Luma plate (Packard) and allowed to dry. On the other hand, 100 μl of PBS were added to each well of the plate in which the cells had settled to wash the cells.
250 μl aliquots of Micro Scinti 20 (Packard) were added to each well of the plate containing the washed cells and allowed to stand overnight, followed by measuring the radioactivity of the medium and cells with a scintillation counter (Top Count, Packard). Percent (%) efflux was calculated from the measured values using the calculation formula shown below.

% efflux=(Specific radioactivity of medium(CPM)/75×100)/(Specific radioactivity of medium(CPM)+specific radioactivity of cells(CPM))×100

The value of % efflux obtained from Compound A at a concentration of 1 μM was assigned a value of 100, and the other results were converted on the basis of this value to determine relative activity (Table 1). Compound A and Compound B demonstrated cholesterol efflux activity of about 50% at a final concentration of 0.01 μM, demonstrated efflux activity of about 100% at 1 μM, and LXR ligands in the form of Compound A and Compound B were clearly determined to have equal cholesterol efflux activity.











TABLE 1






Compound final






concentration
0.01 μM
0.1 μM
1 μM








Compound A
49%
86%
100%



Compound B
54%
90%
101%









Example 1
Acquisition of Histidine Tag Fused Proteins
(1) Construction of Expression Plasmid of Histidine Tag Fused Human LXRα Protein
Oligonucleotides comprising of the following nucleotide sequences:
gccatatgcgggaggagtgtgtcctgtc (LXRα-F: SEQ ID NO: 7 of the Sequence Listing),
ctggatccttcgtgcacatcccagatct (LXRα-R: SEQ ID NO: 8 of the Sequence Listing)
were synthesized with a DNA synthesizer for use as PCR primers, PCR was carried out by using as template a human liver cDNA library (see P. J. Willy et al., Genes Dev. 9 (9), 1033-1045 (1995)), and a DNA fragment was amplified in which restriction enzyme NdeI and BamHI sites were introduced at the ligand binding domain (hereinafter referred to as “LBD”: amino acid nos. 164 to 447 of SEQ ID NO: 2 of Sequence Listing) of human LXRα (SEQ ID NO: 2). The DNA fragment was digested with restriction enzyme NdeI and BamHI, and ligated to His tag fused protein expression plasmid pET15b (Novagen) digested with NdeI and BamHI. The resulting Histidine tag fused human LXRα protein expression plasmid was designated as pET15b-LXRα.

(2) Construction of Expression Plasmid of Histidine Tag Fused Human LXRβ Protein

Oligonucleotides comprising of the following nucleotide sequences:
gccatatgagggagcagtgcgtcctttc (LXRβ-F: SEQ ID NO: 9 of the Sequence Listing)
ctggatccctcgtggacgtcccagatct (LXRβ-R: SEQ ID NO: 10 of the Sequence Listing)
were used as PCR primers, PCR was carried out by using as template a human liver cDNA library, and a DNA fragment was amplified in which restriction enzyme NdeI and BamHI sites were introduced at the ligand binding domain (LBD: amino acid nos. 155 to 461 of SEQ ID NO: 6) of human LXRβ (SEQ ID NO: 6). The amplified DNA fragment was digested with restrictases NdeI and BamHI, and ligated to His tag fused protein expression plasmid pET15b (Novagen) digested with NdeI and BamHI. The resulting histidine tag fused human LXRβ protein expression plasmid was designated as pET15b-LXRβ.

(3) Construction of His-LXRα, FLAG-RXRα Co-Expression Plasmid and His-LXRβ, FLAG-RXRα Co-Expression Plasmid

Oligonucleotides comprising of the following nucleotide sequences:
ccagatctaagcgggaagccgtgcagga (RXRα-F: SEQ ID NO: 11 of the Sequence Listing)
ccagatctagtcatttggtgcggcgcct (RXR-R: SEQ ID NO: 12 of the Sequence Listing)
were used as PCR primers, PCR was carried out by using as template human liver cDNA (Clontech), and a DNA fragment was amplified in which a Bg1II site was introduced at the ligand binding domain (amino acid nos. 201 to 462 of SEQ ID NO: 4) of human RXRα (GenBank Accession No. X52773; SEQ ID NO: 4). The amplified DNA fragment was digested with restriction enzyme Bg1II, and ligated to pET15b-LXRα digested with BamHI to construct a plasmid that co-expresses His-LXRα (SEQ ID NO: 13) and FLAG-RXRα (SEQ ID NO: 14 of the Sequence Listing) designated as pET15b-LXRα/FLAG-RXRα.
In addition, the aforementioned DNA fragment obtained by PCR was digested with Bg1II and incorporated at the BamHI site of pET15b-LXRβ digested with BamHI to construct a plasmid that co-expresses His-LXRβ (SEQ ID NO: 15) and FLAG-RXRα designated as pET15b-LXRβ/FLAG-RXRα.
(4) Acquisition of His-LXRα/FLAG-RXRα and His-LXRβ/FLAG-RXRα
Escherichia coli strain BL21(DE3) was transformed using pET15b-LXRα/FLAG-RXRα or pET15b-LXRβ/FLAG-RXRα. Each of the resulting transformants were shake cultured for 4 hours at 37° C. in 20 ml of L-broth medium (containing 10 g of tryptone (Difco), 5 g of yeast extract (Difco), 5 g of sodium chloride each in a 1 L aqueous solution) containing 100 μg/ml of ampicillin. Next, the transformants were inoculated at 5.0% (v/v) into 400 ml of L-broth medium containing 100 μg/ml of ampicillin and shake cultured for 4 hours at 37° C. Subsequently, 0.1 mM isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as “IPTG”) was added followed by shaking culture for 17 hours at 25° C.
Following completion of the reaction, the microbial cells were collected by centrifugal separation for 10 minutes at 8,000×g, and then suspended in 40 ml of lysis buffer (Table 2). Subsequently, the cells were disrupted by a ultrasonic homogenizer, and after removing the insoluble fraction by centrifugal separation (11,000×g, 20 minutes), 2 ml of Ni2+ resin (Probond Resin, Invitrogen) were added followed by shaking gently for 1.5 hours on ice. After washing the gel seven times with 20 ml of wash buffer (Table 3), the gel was eluted four times using 1 ml of elution buffer (Table 4) according to the batch method to obtain 4 ml each of His-LXRα/FLAG-RXRα and His-LXRβ/FLAG-RXRα. After carrying out 12.5% SDS polyacrylamide gel electrophoresis (hereinafter referred to as “SDS-PAGE”), the purified proteins were confirmed to be present at the locations corresponding to the predicted molecular weights of the fused protein of 35,500 for His-LXRα, 37,200 for His-LXRβ and 30,900 for FLAG-RXRα, and the protein concentrations were determined according to the Bradford method. 4 ml of storage solution (Table 5) was added to the resulting protein solutions after which they were stored at −20° C.







TABLE 2





Lysis buffer (pH 8.0)



















NaH2PO4
50
mM



NaCl
300
mM



MgCl2
5
mM



Tween 20
0.05%
(v/v)



Glycerol
10%
(v/v)



Imidazole
10
mM
















TABLE 3





Wash buffer (pH 8.0)



















NaH2PO4
50
mM



NaCl
300
mM



MgCl2
5
mM



Tween 20
0.05%
(v/v)



Glycerol
10%
(v/v)



Imidazole
20
mM
















TABLE 4





Elution buffer (pH 8.0)



















NaH2PO4
50
mM



NaCl
300
mM



MgCl2
5
mM



Tween 20
0.05%
(v/v)



Glycerol
10%
(v/v)



Imidazole
250
mM
















TABLE 5





Srorage solution



















Glycerol
90%
(v/v)



EDTA
2
mM



(±)-Dithiothreitol
20
mM



PMSF
2
mM



β-Mercaptoethanol
10
mM



Protease inhibitor









Example 2
Selection of LXR Ligands Based on the Binding Capacity to LXRα Using Fluorescence Resonance Energy Transfer (Hereinafter Referred to as “FRET”) Assay
8 μl of LXRα reaction solution (0.05 μl of 4 μM His-LXRα and FLAG-RXRα mixed solution, 1.00 μl of 10×PBS (Sigma Chemical), 2.50 μl of 2 M KF (Wako Pure Chemical Industries), 0.10 μl of 10% NP40 (Sigma Chemical), 0.15 μl of anti-His tag antibody (CIS Bio International) and 4.20 μl of H2O) containing the His-LXRα and FLAG-RXRα prepared according to Example 1 were placed in a 384-well assay plate (Greiner Bio-One).
Next, 2 μl of a solution of Compound A (N-(2,2,2-trifluoroethyl)-N-{4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl}benzene sulfonamide (Compound 12 described on page 55 of International Publication WO2000/054759) or Compound B (3-chloro-4-(3-(2-propyl-3-trifluoromethyl-6-benz-[4,5]-isooxazoloxy)propylthio)phenylacetic acid (a compound described in Example 20 on page 70 of International Publication WO1997/028137), dissolved to a concentration of 10 μM in dimethyl sulfoxide (hereinafter referred to as “DMSO”), were added to each well of a 384-well assay plate followed by incubating for 1 hour at room temperature.
Next, 10 μl of peptide reaction solution (1.00 μl of 5 μM peptide, 1.00 μl of 10×PBS, 2.50 μl of 2 M KF, 0.10 μl of 10% NP40, 0.20 μl of streptavidin (CIS Bio International) and 5.20 μl of H2O) were added to each well of the 384-well assay plate and incubated for 17 hours at 4° C. The peptide added here was comprising of any of the amino acid sequences of (a) to (g) below, and the amino acid of the N-terminal was biotinylated.
(a) PGC-1α: DGTPPPQEAEEPSLLKKLLLAPANT (aa 130-154) (NM—013261) (SEQ ID NO: 16 of the Sequence Listing)
(b) TIF2: HGTSLKEKHKILHRLLQDSSSPVDL (aa 679-703)(NM—006540) (SEQ ID NO: 17 of the Sequence Listing)
(c) ASC-2 NR-1: NKDVTLTSPLLVNLLQSDISAGHFGVNNKQ (LXXLL motif on the N-terminal side of ASC-2) (aa 887-906) (AF177388)(SEQ ID NO: 18 of the Sequence Listing)
(d) ASC-2 NR-2: SPAMREAPTSLSQLLDNSGAPNVTIKPPGL (LXXLL motif on the C-terminal side of ASC-2) (aa 1481-1510) (SEQ ID NO: 19 of the Sequence Listing)
(e) SRC-1-2: CPSSHSSLTERHKILHRLLQEGSPS (the second LXXLL motif from the N-terminal side of SRC-1) (aa 676-700) (U90661) (SEQ ID NO: 20 of the Sequence Listing)
(f) SRC-1-3: KESKDHQLLRYLLDKDEKDL (the third LXXLL motif from the N-terminal side of SRC-1) (aa 741-760) (SEQ ID NO: 21 of the Sequence Listing)
(g) SRC-1-4: QKPTSGPQTPQAQQKSLLQQLLTE (the fourth LXXLL motif from the N-terminal side of SRC-1) (aa 1418-1441)(SEQ ID NO: 22 of the Sequence Listing)
Following completion of incubation, the assay plate was subjected to excitation light at 337 nm using a fluorescent plate reader (Envision, Perkin-Elmer), and fluorescent absorbance at 665 nm and 620 nm was measured to determine the binding capacity between the protein and the peptide.
The value obtained by multiplying 1000 by the value resulting from dividing the measured value at 665 nm when applying excitation light at 337 nm by the measured value at 620 nm was determined. This value was then divided by the value determined by the same method when only DMSO was added without adding LXR ligand (Compound A or Compound B), and this value was then multiplied by 100 and indicated as the percentage relative to the control (% of control) as shown in Table 6.
The value for % of control determined here is the value, represented by a percentage, that indicates the ratio of the increase in the amount of coactivator bound to LXRα that results from adding LXR ligand, and the larger this value, the larger the amount of coactivator that binds to LXRα.
According to the present Example, Compound A was identified as a compound that increases the amount of coactivator bound to LXRα, while Compound B was identified as a compound that does not increase the amount bound.
Although Compound A and Compound B demonstrated equal cholesterol efflux activity based on the results of Test Example 1, the amount of coactivator bound to LXRα was greater for Compound A than Compound B.
Example 3
Selection of LXR Ligands Based on Binding Capacity to LXRβ Using Fluorescence Resonance Energy Transfer (Hereinafter Referred to as “FRET”) Assay (1)
8 μl of LXRβ reaction solution (0.025 μl of 8 μM His-LXRβ and FLAG-RXRα mixed solution, 1.00 μl of 10×PBS, 2.50 μl of 2 M KF, 0.10 μl of 10% NP40, 0.15 μl of anti-His tag antibody and 4.23 μl of H2O) containing the His-LXRβ and FLAG-RXRα prepared according to Example 1 were placed in a 384-well assay plate (Greiner Bio-One) and measured according to the same method as Example 2 to determine the % of control (Table 6). There was no significant difference observed in the resulting values between Compound A and Compound B.









TABLE 6








LXRα (% of control)
LXRβ (% of control)












Compound
Compound
Compound
Compound


Peptide
A
B
A
B





PGC-1α
361.7
207.1
191.0
195.0


ASC2 NR-1
155.8
116.0
114.5
115.1


ASC2 NR-2
254.0
137.2
120.4
116.3


SRC1-2
267.0
182.1
120.4
119.4


SRC1-3
181.3
111.6
110.6
108.4


SRC1-4
207.3
128.3
154.6
143.9


TIF2
266.7
157.1
107.9
114.4









Example 4
Selection of LXR Ligands Based on the Binding Capacity to LXRα and LXRβ Using Fluorescence Resonance Energy Transfer (Hereinafter Referred to as “FRET”) Assay
(1) According to the same method as Example 2) (using the following peptides (h) to (m), instead of the peptides (a) to (g) in Example 2), the amount of coactivator bound to the heterodimer LXRα and RXRα were determined.
The amount of the coactivator bound to the heterodimer comprising LXRα and RXRα was greater for Compound A than for Compound B (Table 7).
(h) DAX1: CCFCGEDHPRQGSILYSLLTSSKQT (aa 132-156) (NP—000466) (SEQ ID NO: 23 of the Sequence Listing)
(i) PNRC: KNPTSCSRRFYQLTKLLDSVQPIAR (aa 848-872) (NM—000044) (SEQ ID NO: 24 of the Sequence Listing)
(j) TIF2 NRB2: KQEPVSPKKKENALLRYLLDKDDTK (aa 731-755) (NM—006540) (SEQ ID NO: 25 of the Sequence Listing)
(k) TRAP220: GHGEDFSKVSQNPILTSLLQITGNG (aa 590-614) (NM—004774) (SEQ ID NO: 26 of the Sequence Listing)
(l) PERC NRB2: HSKASWAEFSILRELLAQDVLCD (aa 332-354) (NM—133263) (SEQ ID NO: 27 of the Sequence Listing)
(m) ACTR NRB3: SPKKKENNALLRYLLDRDDPSDALSK (aa 728-753) (AF012108) (SEQ ID NO: 28 of the Sequence Listing)
(2) LXR5: According to the same method in Example 3) (using the above peptides (h) to (m), instead of peptides (a) to (g) in Example 3), the amount of the coactivator bound to the heterodimer comprising LXRβ and RXRα were determined. There was no significant difference observed in the resulting values between Compound A and Compound B (Table 7).









TABLE 7








LXRα (% of control)
LXRβ (% of control)












Compound
Compound
Compound
Compound


Peptide
A
B
A
B














DAX1
158
107
116
120


PNRC
140
110
109
107


TIF2 NRB2
268
127
125
124


TRAP220
131
107
111
116


PERC NRB2
127
104
104
115


ACTR NRB3
167
92
129
103









Test Example 2
Cynomolgus Monkey Consecutive Daily Administration Study
Five to seven year old, male cynomolgus monkeys in groups of 5 animals each were force fed only an administration base (Propylene glycol (Wako Pure Chemical Industries)/Tween 80 (Kao) (volume ratio: 4/1, hereinafter referred to as “PG/Tween”)) (hereinafter referred to as “the control group”), or Compound A or Compound B dissolved in PG/Tween in an amount of 3 mg/kg or 10 mg/kg once a day for 7 days between the hours of 8:00 and 10:00 AM. After fasting for 16 hours starting at 5:00 PM on the 7th day of administration, 1 mL of blood was collected from the cephalic vein with a heparinized syringe followed by centrifuging for 15 minutes at 4° C. and 5000 rpm to obtain plasma.
The levels of LDL cholesterol and triglycerides in the plasma were measured with an auto analyzer (Hitachi Model 7170) followed by calculation of the % of control group (Tables 8 and 9).
Compound A, which was identified in Example 2 as being a compound that increases the amount of coactivator bound to LXRα, was clearly determined to increase LDL cholesterol concentration and triglyceride concentration in the plasma as compared with a compound identified as a compound that does not increase the amount of coactivator bound to LXRα.
Namely, whether or not an LXR ligand has the function of increasing plasma LDL cholesterol concentration and triglyceride concentration was clearly determined to be able to be assessed simply by measuring the amount of coactivator bound to a heteroprotein of LXRα and RXRα at the time of addition of LXR ligand without having to conduct an animal study.







TABLE 8







Relative value of LDL Cholesterol in Plasma










Daily Dosage of Compound











3 mg/kg
10 mg/kg














Compound A
137%
207%



Compound B
111%
150%
















TABLE 9







Relative value of TG in Plasma










Daily Dosage of Compound











3 mg/kg
10 mg/kg














Compound A
429%
661%



Compound B
 73%
120%

## Claims

1. A method of identifying a therapeutic or preventive agent that affects LDL cholesterol concentration and/or plasma triglyceride concentration in a mammal, the method comprising:
(i) providing a heterodimer comprising LXRα and RXRα;
(ii) contacting a test substance with the heterodimer in the presence of an LXR coactivator;
(iii) measuring the amount of the coactivator bound to the heterodimer;
(iv) comparing the amount of the coactivator measured in step (iii) with the amount of the coactivator bound to the heterodimer in a control;
(v) correlating the difference between the amount of bound coactivator and the amount of bound coactivator in the control as indicative of the activity of the test substance to affect the LDL cholesterol concentration and/or the plasma triglyceride concentration in a mammal;
wherein the activity of the test substance to affect the LDL cholesterol concentration and/or the plasma triglyceride concentration is greater when the amount of the coactivator bound to the heterodimer is higher than the amount of the coactivator bound to the heterodimer in the control.

2. The method according to claim 1, wherein the method is to identify a therapeutic or preventive agent that does not cause an increase in LDL cholesterol and/or plasma triglyceride concentration in a mammal.

3. The method according to claim 1, wherein the test substance is a LXR ligand.

4. The method according to claim 1, wherein the therapeutic or preventive agent is to treat or prevent a disease selected from the group consisting of arteriosclerosis, atherosclerosis, hyperlipidemia, lipid related diseases, inflammatory disease mediated by inflammatory cytokines, autoimmune diseases, cardiovascular disease, cerebrovascular disease, renal disease, diabetes mellitus, diabetic complications, obesity, nephritis, hepatitis and alzheimer's disease.

5. The method according to claim 1, wherein the coactivator is selected from the group consisting of PGC-1α (homo sapiens peroxisome proliferative activated receptor, gamma coactivator 1, alpha), TIF-2 (homo sapiens nuclear receptor coactivator 2), ASC-2 (activating signal cointegrator 2), SRC-1 (human steroid receptor coactivator-1), DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital (AHC) critical region on the X chromosome, gene 1), PNRC (proline-rich nuclear receptor coregulatory protein), TRAP220 (thyroid hormone receptor-associated protein 220), PERC (peroxisome proliferator-activated receptor gamma coactivator-1 beta) and ACTR (steroid receptor coactivator-3).

6. The method according to claim 1, wherein the coactivator is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and variants thereof.

7. The method according to claim 1, wherein the LXRα is a human full-length LXRα polypeptide having an amino acid sequence of SEQ ID NO: 2 or a variant thereof which has at least 80% identity to SEQ ID NO: 2, or a fused protein containing said polypeptide.

8. The method according to claim 1, wherein the LXRα is a ligand binding site of human full-length LXRα having an amino acid sequence of amino acid nos. 164 to 447 of SEQ ID NO: 2 or a variant thereof which has at least 80% identity to amino acid nos. 164 to 447 of SEQ ID NO: 2, or a fused protein containing said polypeptide.

9. The method according to claim 1, wherein the RXRα is a human full-length RXRα polypeptide having an amino acid sequence of SEQ ID NO: 4 or a variant thereof which has at least 80% identity to SEQ ID NO: 4, or a fused protein containing said polypeptide.

10. The method according to claim 1, wherein the RXRα is a ligand binding site of human full-length RXRα having an amino acid sequence of amino acid nos. 201 to 462 of SEQ ID NO: 4 or a variant thereof which has at least 80% identity to amino acid nos. 201 to 462 of SEQ ID NO: 4, or a fused protein containing said polypeptide.

11. The method according to claim 1, wherein the amount of the coactivator bound to the heterodimer is measured using a FRET assay.

12. The method according to claim 1, wherein the LXRα and/or the RXRα is provided by using cells that express LXRα and/or RXRα.

13. The method according to claim 1, wherein the LXRα and/or the RXRα is provided by using cells that express LXRα and/or RXRα as an exogenous protein.

14. The method according to claim 1, wherein the LXRα and/or the RXRα is provided by using cells that express LXRα and/or RXRα as an endogenous protein.