Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Reexamination Certificate
2000-01-06
2002-10-15
Whisenant, Ethan C. (Department: 1634)
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
C435S006120, C435S007100, C530S358000
Reexamination Certificate
active
06465258
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods and compounds for the modulation of cholesterol metabolism in a mammal and methods for screening to identify candidate therapeutic agents for modulation of cholesterol levels.
2. Background
Atherosclerosis is a leading cause of death, myocardial infarctions, stroke, peripheral vascular disease and cardiovascular disease (Libby, in Chapter 242 of
Harrison's Principles of Internal Medicine
, 14th edition (1998) (Fauci et al., eds.); Witztum, in Chapter 36 of Goodman & Gilman's
The Pharmacological Basis of Therapeutics
, 9th edition (1996) (Hardman et al., eds.)). One of the major contributing factors to atherosclerosis is hypercholesterolemia. Hypercholesterolemia is currently treated with a combination of dietary and pharmaceutical therapies. Often more than a single pharmaceutical agent and a dietary regimen are necessary to decrease total cholesterol and LDL cholesterol levels to the desired level. Drugs such as bile acid sequestrants, niacin and the statins are commonly used to treat hypercholesterolemia and atherosclerosis. The use of niacin, however, is limited by the high incidence (>50%) of numerous side effects that are experienced in patients. Thus, a need for therapeutic agents that would decrease cholesterol levels still exists.
Cholesterol homeostasis in mammals is maintained through the coordinate regulation of three major pathways in the liver. Two pathways supply cholesterol to cells and include an endogenous biosynthetic pathway in which acetate is converted into cholesterol and an exogenous pathway in which members of the low-density lipoprotein receptor family bind and internalize cholesterol-carrying particles from the blood. A third pathway involves the conversion of cholesterol into hydrophilic bile acids.
The conversion of cholesterol to bile acids involves a minimum of fourteen enzymes of the bile acid biosynthetic pathway. The first and rate-limiting enzymatic reaction of this pathway is catalyzed by the enzyme cholesterol 7&agr;-hydrolase. The cholesterol 7&agr;-hydrolase gene, also known as cyp7A, belong to the cytochrome P-450 family that contains many microsomal enzymes involved in liver metabolism. It has been shown that expression of the cyp7A gene is tightly regulated. This gene is expressed exclusively in the liver and its expression can be induced by dietary cholesterol and suppressed by bile acids. An alternative pathway involves the cyp7B gene, which is expressed in the brain as well as other tissues. It has been shown that cholesterol catabolism plays a central role in cholesterol homeostasis. For instance, treatment of laboratory animals with cholestid or cholestyramine, two bile acid-binding resins, decreases serum cholesterol levels. Moreover, overexpression of the cyp7 gene in hamsters reduces both total cholesterol levels and LDL cholesterol levels. As such, cholesterol 7&agr;-hydrolase is a potential therapeutic target for the identification of cholesterol reducing compounds and, thus, understanding the mechanism by which expression of the cyp7 genes are regulated is of particular importance.
Nuclear receptors form a large family of ligand-activated transcription factors that modify the expression of target genes by binding to specific cis-acting sequences (Laudet et al. (1992)
EMBO J
. 11: 1003-1013; Lopes da Silva et al. (1995)
Trends Neurosci
. 18: 542-548; Mangelsdorf et al. (1995)
Cell
83: 835-839; Mangelsdorf and Evans (1995)
Cell
83: 841-850). Nuclear receptors include those that remain sequestered in the cytoplasm in the absence of their cognate ligands (e.g., steroid hormone receptors). Upon binding of the ligand, the steroid hormone receptors are translocated to the nucleus where they bind to hormone response elements, typically as homodimers.
Most of the nuclear receptors, conversely, are not sequestered in the cytoplasm in the absence of their ligands but rather remain in the nucleus. These receptors, which include the thyroid hormone, retinoid, fatty acid, and eicosanoid receptors, typically bind to their cognate response elements as heterodimers with a 9-cis-retinoic acid receptor (RXR). Often, binding of a nuclear receptor to a response element occurs in the absence of the cognate ligand.
One such nuclear receptor is the farnesoid X receptor (FXR; also known as “RIP 14” and “NR1H4”) (Forman et al. (1995)
Cell
81: 687; Seol et al. (1995)
Mol. Endocrinol
. 9: 72; Mangelsdorf and Evans, supra.). FXR functions as a heterodimer with RXR, and several isoprenoid lipids can weakly activate FXR at supraphysiological concentration; however, these compounds do not activate all species of FXR and do not bind as ligands (Forman et al., supra.; Zavacki et al. (1997)
Proc. Nat'l. Acad. Sci
. USA 94: 7909). Thus, the identity and physiologic function of FXR ligands have remained unknown.
The lack of understanding of physiological mechanisms that regulate cholesterol levels has hampered the discovery of improved methods of treating hypercholesteremia. Thus, a need exists for characterization of regulatory mechanisms of cholesterol hemostasis, and for screening methods to identify better compounds for treating hypercholesteremia. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The present invention is based in part on the discovery that the orphan nuclear receptor FXR (farnesoid X receptor) is involved in the suppression of the human cyp7 genes. More particularly, it has been discovered that FXR functions as a bile acid receptor/sensor that mediates cyp7 expression in a bile-acid dependent manner. As such, in one aspect, the present invention provides a method for increasing cholesterol metabolism in a cell by contacting the cell with a compound that modulates the binding of an FXR to a ligand for FXR. In one embodiment, the ligand is a bile acid. In another embodiment, the compound modulates the binding of the FXR to an RXR or other coactivator or corepressor.
The invention provides methods for prescreening to identify a candidate therapeutic compound suitable for testing for ability to modulate cholesterol metabolism in a cell. These methods involve:
providing a reaction mixture that comprises:
a) a polypeptide that comprises a ligand binding domain of an FXR;
b) a ligand for FXR; and
c) a test compound; and
determining whether the amount of binding of the FXR ligand binding domain to the ligand for FXR is increased or decreased in the presence of the test compound compared to the amount of binding in the absence of the test compound. A test compound that causes an increase or decrease in binding is a candidate therapeutic agent for modulation of cholesterol metabolism. In some embodiments, the methods further involve administering the candidate therapeutic agent to a cell to determine whether the candidate therapeutic agent modulates cholesterol metabolism in the cell.
The invention also provides methods for screening to identify a compound that modulates cholesterol metabolism in a cell using a gene expression assay. For example, these methods can involve contacting a cell with a test compound, wherein said cell includes:
a) a polynucleotide that encodes a polypeptide comprising: 1) a DNA binding domain of a receptor which binds to DNA; and 2) a ligand binding domain that is substantially identical to a ligand binding domain of a FXR;
b) a ligand for FXR; and
c) a reporter gene construct which comprises a response element to which said DNA binding domain can bind, wherein said response element is operably linked to a promoter that is operative in the cell and said promoter is operably linked to a reporter gene.
By determining whether the reporter gene is expressed at a higher or lower level in the presence of said test compound compared to said reporter gene expression level in the absence of said test compound, one can identify a test compound that can modulate cholesterol metabolism in a cell. The cells used in the methods of the invention are typically mammalian cells. In a presently p
Okamoto Arthur Y
Shan Bei
Townsend and Townsend / and Crew LLP
Tularik Inc.
Whisenant Ethan C.
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