WO2003087140A1

WO2003087140A1 - NON-PRIMATE FXRb AS A LANOSTEROL SENSING NUCLEAR HORMONE RECEPTOR AND RELATED USES

Info

Publication number: WO2003087140A1
Application number: PCT/EP2003/002357
Authority: WO
Inventors: Ulrich Deuschle; David Jackson; Ingo Kober; Harald Kranz; Claus Kremoser; Kerstin Otte; Bettina Remmel
Original assignee: Lion Bioscience Ag
Priority date: 2002-04-13
Filing date: 2003-03-07
Publication date: 2003-10-23
Also published as: AU2003219028A1

Abstract

The present invention relates to a novel nuclear receptor called 'npFXRB' or also npFXR-β a homologue of the npFXR-α, a prototypical type 2 nuclear receptor. The invention also relates to the isolated nucleic acid sequence of npFXRB and the isolated protein thereof. The invention further relates to processes for isolating and/or producing the nucleic acid or the protein as well as methods of use of the receptor npFXRB.

Description

Lion bioscience AG, 69120 Heidelberg L30275PCT

Non-primate FXRb. as a lanpsterol sensing nuclear hormone receptor and related uses

BACKGROUND OF THE INVENTION

Nuclear hormone (NRs) receptors constitute a superfamily of ligand-induced transcription factors with important roles in processes as diverse as reproduction, development, and metabolism (Mangelsdorf, D.J., et al. The nuclear receptor superfamily: the second decade. Cell. 83, 835-839 (1995). A standardised nomenclature based on evolutionary sequence conservation has been proposed (A unified nomenclature system for the nuclear receptor superfamily. Cell 97,161-163 (1999)) and six subfamilies are currently recognised. The subgroup NR1H is formed by the Drosophila ecdysone receptor (EcR), with the metamorphosis steroid ecdysone as its natural ligand (Koelle, M.R., et al. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell. 61, 59-77 (1991)), and the vertebrate homologues FXRa, LXRa and LXRb. The latter receptors are involved in the regulation of cholesterol catabolism into bile acids through transcriptional regulation of key enzymes of this pathway (Goodwin, B., et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6, 517-526 (2000); Lu, T.T., et al. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell. 6, 507-515 (2000)).

Farnesoid X Receptor alpha (hereinafter FXR-α) is a prototypical type 2 nuclear receptor (US Pat. 6,005,086) which activates genes upon binding to promoter region of target genes in a heterodimeric fashion with Retinoid X Receptor (hereinafter RXR, Forman et al., Cell, 81, 687-93, 1995). The relevant physiological ligands of FXR-α seem to be bile acids (Maki- shima et al, Science, 284, 1362-65, 1999; Parks et al., Science, 284, 1365-68, 1999). The most potent is chenodeoxycholic acid, which regulates the expression of several genes that participate in bile acid homeostisis. Farnesoid, originally described to activate the rat ortholog at high concentration does not activate the human or mouse receptor. It is highly expressed in the liver, intestine and kidney. Like LXR-α FXR-α is involved in intracrine signalling.

Consequently, FXR-α is proposed to be a nuclear bile acid sensor. As a result, it modulates both, the synthetic output of bile acids in the liver and their recycling in the intestine (by regulating bile acid binding proteins). It is also activated by retoinic acid and TTNPB at su- praphisiological concentration. Furthermore, it regulates the conversion of dietary cholesterol into bile acids by regulation the metabolizing genes like CYP7-α. This is a feed back regulation since the receptor is activated by bile acids. - -

Through its regulatory function in cholesterol and bile acid metabolism, in general any FXR- α homologue could serve as a target for cholesterol lowering drugs and exert beneficial effects in diseases like artheriosclerosis and other metabolic disorders. Therefore, the identification of the human (primate) FXRb nuclear receptor seemed to be a promising approach for the of the elucidation and examination of the cholesterol related metabolism.

Recently, we and others (Robinson-Rechavi, M., Carpentier, A.S., Duffraisse, M., Laudet, N. How many nuclear hormone receptors are there in the human genome? Trends Genet 17, 554- 556 (2001); Maglich, J.M., et al. Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes. Genome Biol. 2, RESEARCH0029 (2001); Enmark, E., Gustafsson, J.A. Comparing nuclear receptors in worms, flies and humans. Trends Parmacol Sci 22, 611-615 (2001)) detected a novel member of the ΝR1H subgroup, termed FXRb (ΝR1H5) on the basis of its homology to FXRa. In addition to previously published results (Maglich, J.M., et al. Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes. Genome Biol. 2, RESEARCH0029 (2001)), we identified several splice variants of FXRb in RNA from human testis. The extremely low transcript levels as determined by nested RT-PCR and the presence of two stop codons and three frameshifts in the cDNA (Fig.la) strongly argue that FXRb is a pseudogene in the human lineage. Sequence analysis of the FXRb genes in primates including chimpanzee, gorilla, orang-utan, and rhesus monkey was performed. In all cases, stop codons and frame shifts were identified at similar positions as in the human mRN A and no transcripts could be detected by RT-PCR from liver and testis RNA sources (data not shown). Thus, FXRb seems to be a pseudogene in these primate species as well which did, cast a doubt on < the usefulness of this system for human diagnostic purposes. Therefore, new candidates were needed in order to further study FXRb function in a functional in vivo model.

It was thus an object of the present invention to provide for a novel nuclear receptor. In a preferred embodiment of the invention it was an object to provide for a non-primate homologue of FXR-α. It was an object of the present invention to provide for means of producing this receptor as well as means of screening for agonists and antagonists to the receptor. Further objects of the invention are outlined below.

SUMMARY OF THE INVENTION

The present invention is primarily directed to non-primate npFXRb as functional members of this family. In humans and primates, FXRb mRNA is detected at very low levels but puta- tively encodes a non-functional truncated protein due to stop codons and frame shifts present in the mRNA sequence. In contrast, FXRb mRNAs isolated from other mammalian species, including mouse, rat, rabbit and dog, have the potential to code for functional receptor proteins. In mice, FXRb is transcribed rather ubiquitously during embryonic development but is confined to liver and reproductive tissues in the adult. Ligand binding studies demonstrate that mouse FXRb binds lanosterol, an intermediate of the cholesterol biosynthetic pathway, with high affinity. These data suggest a role for npFXRb in cholesterol biosynthesis that is lost during evolution in the primate lineage. Identification of npFXRb as a novel functional receptor will further our understanding of species specific differences in cholesterol metabolism.

The present invention provides, inter alia, a novel nuclear receptor protein, together with several of its potential splice variants. In a preferred embodiment of the invention a novel FXRb homologue is provided for. Also provided is the nucleic acid sequence encoding this novel nuclear receptor protein, as well as compounds and methods for using this protein and its nucleic acid sequence.

The present invention provides a novel proteins, nucleic acids, and methods useful for developing and identifying compounds for the analysis of diseases and disorders as metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases and in preferred embodiments, high cholesterol and arteriosclerosis as well as other metabolic disorders. Furthermore, the differences between primates and non-primates will allow the species specific use of this target for agricultural applications as, e.g. pest control and/or regulation of the lanolin content in sheep.

The foregoing merely summarizes certain aspects of the present invention and is not intended, nor should it be construed, to limit the invention in any manner. All patents and other publications recited herein are hereby incorporated by reference in their entirety. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In an effort to identify novel nuclear hormone receptor proteins, after the publication of the human genome sequence, we performed exhaustive data base searches using the BLAST algorithm (Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs). As one result, we and others (Robinson-Rechavi, M., Carpentier, A.S., Duffraisse, M., Laudet, V. How many nuclear hormone receptors are there in the human genome? Trends Genet 17, 554-556 (2001); Maglich, J.M., et al. Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes. Genome Biol. 2, RESEARCH0029 (2001); Enmark, E., Gustafsson, J.A. Comparing nuclear reeptors in worms, flies and humans. Trends Parmacol Sci 22,. 611-615 (2001)) detected a novel member of the NR1H subgroup, termed npFXRb (NR1H5) on the basis of its homology to FXRa. In addition to previously published results (Maglich, J.M., et al. Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes. Genome Biol. 2, RESEARCH0029 (2001)), we identified several splice variants of FXRb in RNA from human testis. The extremely low transcript levels as determined by nested RT-PCR and the presence of two stop codons and three frameshifts in the cDNA (Fig.la) strongly argue that npFXRb is a pseudogene in the human lineage. Sequence analysis of the npFXRb genes in primates including chimpanzee, gorilla, orang-utan, and rhesus monkey was performed. In all cases, stop codons and frame shifts were identified at similar positions as in the human mRNA and no transcripts could be detected by RT-PCR from liver and testis RNA sources (data not shown). Thus, FXRb seems to be a pseudogene in these primate species as well.

Surprisingly, however, in non-primate mammals including mouse, rat, rabbit and dog, the corresponding npFXRb mRNA's that we identified, show continuous open reading frames and thus potentially encode functional nuclear receptor proteins.

As a first indication for functionality we observed negative selection pressure on non-primate npFXRb when calculating non-synonymous to synonymous substitution rates in the amino acid sequence (Fig. 2a). However, it is important to note, that the selection pressure on the FXRb LBD is more relaxed compared to other NRs. In primates, almost no selection pressure is observed, supporting the prediction for a non-functional FXRb protein in these species. All isolated FXRb sequences form a robust phylogenetic group well separated from the FXRa cluster (Fig. 2b) including the recently isolated FXRa orthologs from Xenopus leavis, FOR1 and 2 (Seo, Y. W., et al. FOR, a novel orphan nuclear receptor related to FXR. J Biol Chem. 26, 26 (2002)). The in silico identified Fugu rupies FXR2 appears to be ancestral to the duplication that led to the clusters of FXRa and FXRb. Mutation rates are higher for FXRb than FXRa, as observed by respective branch lengths on the phylogenetic tree (Fig. 2b).

To further investigate the potential functionality of the novel receptor we analysed the transcription of murine npFXRb (mnpFXRb or mFXRb) in embryonic and adult tissues. Several splice variants were isolated from adult liver and testis differing in their DBDs or LBDs (Fig. lb). mnpFXRb is ubiquitously and strongly expressed during embryonic development (Fig. 3). In the adult, however, its expression is more tissue specific and mainly restricted to liver, reproductive tissues and the heart. This pattern of expression suggests a critical function of mnpFXRb during embryonic development. In contrast, murine FXRa (mFXRa) is expressed more ubiquitously in both embryonic and adult tissues. In the analysed tissues of the embryo, FXRa is coexpressed with mnpFXRb except for brain and skin. In the adult, however, mFXRa transcripts are detected in many tissues with highest mRNA levels in liver followed by kidney (Fig. 3), a result differing from other reports that indicated a more restricted expression for rodent FXRa (Forman, B.M., et al. Identification of a nuclear receptor that is activated by farnesol metabolites. Cell. 81, 687-693. (1995); Lu, T.T., Repa, J.J., Mangelsdorf, DJ. Orphan nuclear receptors as eLiXiRs and FiXeRs of sterol metabolism. J Biol Chem. 276, 37735-37738 (2001)). Since FXRa is a transcription factor activated by bile acids (Wang, H., Chen, J., Hollister, K., Sowers, L.C., Forman, B.M. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell. 3, 543-553 (1999); Parks, D.J., et al. Bile acids: natural ligands for an orphan nuclear receptor. Science. 284, 1365-1368 (1999); Makishima, M., et al. Identification of a nuclear receptor for bile acids. Science 284 1362- 1365 (1999)), we tested intermediates of the bile acid and cholesterol biosynthetic pathways for FXRb binding. .

Lanosterol, an intermediate of the de novo cholesterol biosynthetic pathway, showed the highest affinity in a HTRF ligand sensing assay with an EC₅o of ~1 mM (Fig. 4a). Similar affinities were reported for the physiological ligands of other lipid receptors Janowski, B.A., Willy, P.J., Devi, T.R., Falck, J.R., Mangelsdorf, DJ. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature 383, 728-731 (1996); Lehmann, J.M., et al. Activation of the nuclear receptor LXR by oxysterols defines a new hormone response path- way. J Biol Chem. 272, 3137-3140 (1997); Willson, T.M., Brown, P.J., Sternbach, D.D., Henke, B.R. The PPARs: from orphan receptors to drug discovery. J Med Chem. 43, 527-550 (2000). Lanosterol is therefore a likely candidate to be an endogenous FXRb ligand that can induce coactivator recruitment resulting most likely in the activation of target gene transcription. Lanosterol displays a remarkable specificity for FXRb as it does neither bind to the closest relative of FXRb, FXRa (Fig. 4a), nor to the more distantly related LXRa (data not shown). In addition, the FXRa specific compound GW4064 has only a very poor affinity towards mFXRb. Interestingly, FXRb displays promiscuous ligand binding properties by interacting with a diverse spectrum of chemical structures with low affinity (Fig. 4b). These compounds again do not show affinity for FXRa (data not shown).

Considering available data, FXRb seems to represent the first and only member of the nuclear hormone receptor family present as a pseudogene in the primate lineage, but constituting a functional receptor protein in other mammalian species. In mice, both the expression of npFXRb in hepatic tissue and its binding of lanosterol, an intermediate in the cholesterol biosynthetic pathway, suggest a regulatory function for this novel receptor in cholesterol metabolism. Since primates are lacking a functional FXRb protein, the putative regulatory function of this receptor in cholesterol biosynthesis may have been overtaken by other members of the nuclear hormone receptor family. Alternatively, this layer of regulation in the cholesterol metabolism may have been modulated in the primate lineage and thereby contribute to the species specific differences observed in this pathway.

Our understanding of cholesterol metabolic pathways in humans that are in part deduced from pharmacological studies and compound testing in non-primate mammals may have to be revisited due to the presence of an additional nuclear receptor, npFXRb, in animal models that is a pseudogene in primates.

THE npFXRB PROTEIN AND NUCLEIC ACID:

The present invention comprises a novel member of the nuclear receptor superfamily which the inventors herein refer to as non-primate npFXRb. In contrast to the previously reported human FXRb, this protein is functional and not based on a pseudogene. Particularly preferred embodiments of the npFXRb receptor, are those having an amino acid sequence substantially the same as SEQ ID NO 25. Examination of the amino acid sequence confirms that the present protein is indeed a member of the nuclear receptor family (see also US 6,005,086) which is closely related to FXR. The carboxy-terminal ligand binding domain "LBD" of FXRB is a complex region encoding subdomains for ligand binding, often dimerization and transcriptional activation.

The present invention provides a novel proteins, nucleic acids, and methods useful for developing and identifying compounds for the treatment of such diseases and disorders as metabolic disorders, immύnological indications, hormonal dysfunctions, neurosystemic diseases and in preferred embodiments, high cholesterol and artheriosclerosis as well as other metabolic disorders. Furthermore, the differences between primates and non-primates will allow the species specific use of this target for agricultural applications as, e.g. pest control and/or regulation of the lanolin content in, e.g. sheep.

Identified and disclosed herein is the protein sequence for a novel nuclear receptor and the nucleic acid sequence encoding this non-primate nuclear receptor FXRB, which we call the npFXRB nuclear receptor (or simply "npFXRB") receptor, or also npFXR-β or npFXRb.

The importance of this discovery is manifested in the effects of npFXR-β to modulate genes involved in cellular functions like regulation of metabolism and cell homeostasis, cell proliferation and differentiation, pathological cellular aberrations, or cellular defence mechanisms including tumour development, i.e. cancer of non-primate model systems.

Thus, this npFXRB protein is useful for screening for npFXRB agonists and antagonist activity for controlling these conditions.

In one aspect of the present invention, we provide isolated nucleic acid sequences for a novel non-primate receptor, the npFXRB receptor. In particular, we provide the cDNA sequences, protein sequences as well as the genomic sequences encoding the npFXRB receptor, as well as the cDNA sequence, protein sequence and genomic sequence of the Mus musculus (mouse) receptor.

These nucleic acid sequences have a variety of uses. For example, they are useful for making vectors and for transforming cells, both of which are ultimately useful for production of the npFXRB protein. They are also useful as scientific research tools for developing nucleic acid probes for determining npFXRB expression levels, e.g., to identify diseased or otherwise abnormal states. They are useful for developing analytical tools such as antisense ohgonucleotides for selectively inhibiting expression of the npFXRB gene to determine physiological responses.

In another aspect of the present invention, we provide a homogenous composition comprising the npFXRB protein. The protein is useful for screening drugs for agonist and antagonist activity, and, therefore, for screening for drugs useful in regulating physiological responses associated with npFXRB. Specifically, antagonists to the npFXRB receptor could be used to treat diseases and disorders as metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases and in preferred embodiments, high cholesterol and arthe- riosclerosis as well as other metabolic disorders, whereas agonists could be used for the treatment of these conditions. The proteins are also useful for developing antibodies for detection of the protein. Furthermore, the differences between primates and non-primates will allow the species specific use of this target for agricultural applications as, e.g. pest control and/or regulation of the lanolin content in sheep.

Flowing from the foregoing are a number of other aspects of the invention, including (a) vectors, such as plasmids, comprising the npFXRB nuclear receptor nucleic acid sequence that may further comprise additional regulatory elements, e.g., promoters, (b) transformed cells that express the npFXRB, (c) nucleic acid probes, (d) antisense ohgonucleotides, (e) agonists, (f) antagonists, and (g) transgenic mammals. Further aspects of the invention comprise methods for making and using the foregoing compounds and compositions.

The nucleic acids according to the present invention may be present in various forms, i.e. as an RNA, DNA, cDNA or as genomic DNA.

As used herein, if reference to npFXRB, the npFXRB receptor, the nuclear receptor npFXRB or the npFXRB nuclear receptor is made it is meant as a reference to any protein having an amino acid sequence substantially the same as SEQ ID NO.:25.

According to one aspect, the present invention provides a nucleic acid molecule coding for the non-primate nuclear receptor npFXRb or splice variants thereof, which is selected from the group comprising: a) the nucleotide sequences set forth in SEQ ID NOs: 1 to 6 or the complements thereof; and b) a nucleic acid which hybridizes to a nucleic acid of SEQ ID NOs: 1 to 6 under conditions of high stringency. According to another aspect of the present invention, provided is a nucleic acid molecule according to the invention that encodes a splice variant of the nuclear receptor npFXRb.

Herein the "complement" refers to the complementary strand of the nucleic acid according to the invention, thus the strand that would hybridize to the nucleic acid according to the invention. In accordance with standard biological terminology all DNA sequences herein are however written in 5 '-3' orientation, thus the if a complement is mentioned (see also figures) it is actually a "reverse" complement (as also stated in the figures). For simplification purposes they may however sometimes be referred to simply as "complements".

As used herein, a protein "having an amino acid sequence substantially the same or similar as SEQ ID NO x" (where "x" is the number of one of the protein sequences recited in the Sequence Listing) means a protein whose amino acid sequence is the same as SEQ ID NO x or differs only in a way such that at least 50% of the residues compared in a sequence alignment with SEQ ID NO. x are identical, preferably 75% of the residues are identical, even more preferably 95% of the residues are identical and most preferably at least 98% of the residues are identical.

Those skilled in the art will appreciate that conservative substitutions of amino acids can be made without significantly diminishing the protein's affinity for interacting proteins, DNA binding sites, npFXRB receptor modulators, e.g. small molecular hydrophobic compounds, or RNA.

Other substitutions may be made that increase the protein's affinity for these compounds. Making and identifying such proteins is a routine matter given the teachings herein, and can be accomplished, for example, by altering the nucleic acid sequence encoding the protein (as disclosed herein), inserting it into a vector, transforming a cell, expressing the nucleic acid sequence, and measuring the binding affinity of the resulting protein, all as taught herein.

As used herein the term "a molecule having a nucleotide sequence substantially the same as SEQ ID NO y" (wherein "y" is the number of one of the protein-encoding nucleotide sequences listed in the Sequence Listing) means a nucleic acid encoding a protein "having an amino acid sequence substantially the same as SEQ ID NO y+1" (wherein "y+1" is the number of the amino acid sequence for which nucleotide sequence "y" codes) as defined above. This definition is intended to encompass natural allelic variations in the npFXRB sequence. Cloned nucleic acid provided by the present invention may encode npFXRB protein of any species of origin, including (but not limited to), for example, mouse, rat, rabbit, cat, sheep, lama and dog.

IDENTIFICATION OF VARIANTS AND HOMOLOGUES AS WELL AS USE OF PROBES:

Nucleic acid hybridization probes provided by the invention are nucleic acids consisting essentially of the nucleotide sequences complementary to any sequence depicted in SEQ 'ID NO. 1 to 6 or a part thereof and that are effective in nucleic acid hybridization

Nucleic acid hybridization probes provided by the invention are nucleic acids capable of de- tecting i.e. hybridizing to the gene encoding the polypeptides according to SEQ ID NO. 25.

Nucleic acid probes are useful for detecting npFXRB gene expression in cells and tissues using techniques well-known in the art, including, but not limited to, Northern blot hybridization, in situ hybridization, and Southern hybridization to reverse transcriptase - polymerase chain reaction product DNAs. The probes provided by the present invention, including oligonucleotide probes derived therefrom, are also useful for Southern hybridization of murine genomic DNA for screening for restriction fragment length polymorphism (RFLP) associated with certain genetic disorders. As used herein, the term complementary means a nucleic acid having a sequence that is sufficiently complementary in the Watson-Crick sense to a target nucleic acid to bind to the target under physiological conditions or experimental conditions those skilled in the art routinely use when employing probes.

It is understood in the art that a nucleic acid sequence will hybridize with a complementary nucleic acid sequence under high stringent conditions as defined herein, even though some mismatches may be present. Such closely matched, but not perfectly complementary sequences are also encompassed by the present invention. For example, differences may occur through genetic code degeneracy, or by naturally occurring or man made mutations and such mismatched sequences would still be encompassed by the present claimed invention. Preferably, the nucleotide sequence of the nuclear receptor npFXRB (SEQ ID NO. 1 or splice variants thereof) and/or their complements can be used to derive oligonucleotide fragments (probes) of various length. If the probe is used to detect a npFXRB sequence most preferably a complement of SEQ ID NO. 1 or its complement is used. If the probe is supposed to detect a mouse or a rodent sequence probes complementary to SEQ ID NO. 1 to 6, or their respective complements are preferred. Stretches of 17 to 30 nucleotides are used frequently but depending on the screening parameters longer sequences as 40, 50, 100, 150 up to the full length of the sequence may be used. Those probes can be synthesized chemically and are obtained readily from commercial oligonucleotide providers. Chemical synthesis has improved over the years and chemical synthesis of ohgonucleotides as long as 100-200 bases is possible. The field might advance further to allow chemical synthesis of even longer fragments. Alternatively, probes can also be obtained by biochemical de novo synthesis of single stranded DNA. In this case the nucleotide sequence of the nuclear receptor npFXRB or its complement (see figures) serve as a template and the corresponding complementary strand is synthesized. A variety of standard techniques such as nick translation or primer extension from specific primers or short random oligonucleotides can be used to synthesize the probe (Molecular Cloning: A Laboratory Manual (3 Volume Set) by Joseph Sambrook, David W. Russell, Joe Sambrook, 2100 pages 3rd edition (January 15, 2001; . Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, 1989)). Nucleic acid reproduction technologies exemplified by the polymerase chain reaction (Saiki, R.K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491 (1988)) are commonly applied to synthesize probes. In the case of techniques using specific primers the nucleic acid sequence of the nuclear receptor npFXRB or its complement is not only used as a template in the biochemical reaction but also to derive the specific primers which are needed to prime the reaction.

In some cases one might also consider to use the nucleic acid sequence of the cofactor or its complement as a template to synthesize an RNA probe. A promoter sequence for a DNA- dependent RNA polymerase has to be introduced at the 5 '-end of sequence. As an example this can be done by cloning the sequence into a vector which carries the respective promoter sequence. It is also possible to introduce the needed sequence by synthesizing a primer with the needed promoter in the form of a 5' "tail". The chemical synthesis of a RNA probe is another option. Appropriate means are available to detect the event of a hybridization. There is a wide variety of labels and detection systems, e.g. radioactive isotopes, fluorescent, or chemiluminescent molecules which can be linked to the probe. Furthermore, there are methods of introducing haptens which can be detected by antibodies or other ligands such as the avidin/biotin high affinity binding system.

Hybridization can take place in solution or on solid phase or in combinations of the two, e.g. hybridization in solution and subsequent capture of the hybridization product onto a solid phase by immobilized antibodies or by ligand coated magnetic beads.

Hybridization probes . act by forming selectively duplex molecules with complementary stretches of a sequence of a gene or a cDNA. The selectivity of the process can be controlled by varying the conditions of hybridization. To select sequences which are identical highly homologous to the sequence of interest stringent conditions for the hybridization will be used, e.g. low salt in the range of 0.02 M to 0.15 M salt and/or high temperatures in the range from 50°C degrees centigrade to 70°C degrees centigrade. Stringency can be further improved by the addition of formamide to the hybridization solution. The use of stringent conditions which means that only little mismatch or a complete match will lead to a hybridization product would be used to isolate closely related members of the same gene family. Thus, as used herein stringent hybridization conditions are those where between 0.02 M to 0.15 M salt and/or high temperatures in the range from 50°C degrees centigrade to 70°C degrees centigrade are applied.

The use of highly stringent conditions or conditions of "high stringency" means that only very little mismatch or a complete match which lead to a hybridization product would be used to isolate very closely related members of the same gene family. Thus, as used herein highly stringent hybridization conditions are those where between 0.02 - 0.3 M salt and 65°C degrees centigrade are applied for about 5 to 18 hours of hybridization time and additionally, the sample filters are washed twice for about 15 minutes each at between 60°C - 65°C degrees centigrade, wherein the first washing fluid contains about 0.1 M salt (NaCl and/or Sodium Citrate) and the second contains only about 0.02 M salt (NaCl and/or Sodium Citrate). In a preferred embodiment the following conditions are considered to be highly stringent: Hybridisation in a buffer containing 2 x SSC (0.03 M Sodium Citrate, 0.3 M NaCl) at 65°C - 68°C degrees centigrade for 12 hours, followed by a washing step for 15 minutes in 0.5 x SSC, 0.1% SDS, and a washing step for 15 minutes at 65°C degrees centigrade in.0.1 x SSC, 0.1% SDS.

Less stringent hybridization conditions, e.g. 0.15 M salt - 1 M salt and/or temperatures from 22°C degrees centigrade to 56°C degrees centigrade are applied in order to detect functionally equivalent genes in the same species or for orthologous sequences from other species.

Unspecific hybridization products are removed by washing the reaction products repeatedly in 2 x SSC solution and increasing the temperature.

DEGENERATE PCR AND CLONING OF HOMOLOGUES

The nucleotide sequence of the nuclear receptor npFXRB or its complement can be used to design primers for a polymerase chain reaction. Due to the degeneracy of the genetic code the respective amino acid sequence is used to design ohgonucleotides in which varying bases coding for the same amino acid are included. Numerous design rules for degenerate primers have been published (Compton et al, 1990). As in hybridization there are a number of factors known to vary the stringency of the PCR. The most important parameter is the annealing temperature. To allow annealing of primers with imperfect matches annealing temperatures are often much lower than the standard annealing temperature of 55°C, e.g. 35°C to 52°C degrees can be chosen. PCR reaction products can be cloned. Either the PCR product is cloned directly, with reagents and protocols from commercial manufacturers (e.g. from Invitrogen, San Diego, USA). Alternatively, restriction sites can be introduced into the PCR product via a 5'- tail of the PCR primers and used for cloning. Primers for the amplification of the entire or partial pieces of the npFXRB gene or mRNA, or for reverse transcription may be designed making use of the sequences according to the invention, i.e. those depicted in the figures below.

GENETIC VARIANTS

Fragments from the nucleotide sequence of the nuclear receptor npFXRB (SEQ ID NO. 1 to 6) or their complements can be used to cover the whole sequence with overlapping sets of PCR primers. Also the genomic sequences may be used (see figures for sequences). These primers are used to produce PCR products using genomic DNA from a human diversity panel of healthy individuals or genomic DNA from individuals which are phenotypically conspicu- ous. Also the genomic sequences may be used, i.e. that of the human clone as deposited by the applicant (deposit number DSM 14483 ) or that of the mouse according to SEQ ID NO. 1 (or the complement thereof). The PCR products can be screened for polymorphisms, for example by denaturing gradient gel electrophoresis, binding to proteins detecting mismatches or cleaving heteroduplices or by denaturing high-performance liquid chromatography. Products which display mutations need to be sequenced to identify the nature of the mutation. Alternatively, PCR products can be sequenced directly omitting the mutation screening step to identify genetic polymorphisms. If genetic variants are identified and are associated with a discrete phenotype, these genetic variations can be included in diagnostic assays. The normal variation of the, e.g. the murine population is of interest in designing screening assays as some variants might interact better or worse with a respective lead substance (a pharmacody- namic application). Polymorphisms or mutations which can be correlated to phenotypic outcome are a tool to extend the knowledge and the commercial applicability of the nucleotide sequence of the nuclear receptor npFXRB or its complement or their gene product, as variants might have a slightly different molecular behavior or desired properties. Disease-causing mutations or polymorphisms allow the replacement of this disease inducing gene copy with a wild-type copy by means of gene therapy approaches and/or the modulation of the activity of the gene product by drugs.

PREPARATION OF POLYNUCLEOTIDES:

DNA which encodes receptor npFXRB may be obtained, in view of the instant disclosure, by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by combinations of these procedures, as illustrated below.

Screening of mRNA or genomic DNA may be carried out with oligonucleotide probes generated from the npFXRB nucleotide sequences information provided herein.

Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with known procedures and used in conventional hybridization assays, as described in greater detail in the Examples below. Alternatively, the npFXRB nucleotide sequence may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the npFXRB nucleotide sequences provided herein. Upon purification or synthesis, the nucleic acid according to the invention may be labeled, e.g. for use as a probe.

As single and differential labeling agents and methods, any agents and methods which are known in the art can be used. For example, single and differential labels may consist of the group comprising enzymes such as β-galactosidase, alkaline phosphatase and peroxidase, enzyme substrates, coenzymes, dyes, chromophores, fluorescent, chemiluminescent and bio- luminescent labels such as FITC, Cy5, Cy5.5, Cy7, Texas-Red and IRD40(Chen et al. (1993), J. Chromatog. A 652: 355-360 and Kambara et al. (1992), Electrophoresis 13: 542-546), ligands or haptens such as biόtin, and radioactive isotopes such as ³H, ³⁵S, ³²P ¹²⁵I and ¹⁴C.

EXPRESSION OF THE npFXRB PROTEIN/POL YPETIDE:

The nuclear receptor npFXRB nucleic acid or polypeptide may be synthesized in host cells transformed with a recombinant expression construct comprising a nucleic acid encoding the nuclear receptor npFXRB. Such a recombinant expression construct can also be comprised of a vector that is a replicable DNA construct.

Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. See, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Press, New York, 1989).

An expression vector comprises a polynucleotide operatively linked to a prokaryotic promoter. Alternatively, an expression vector is a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter, and the expression vector further has a polyade- nylation signal that is positioned 3' of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide. A promoter is a region of a DNA molecule typically within about 500 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site). In general, a vector contains a replicon and control sequences which are derived from species compatible with the host cell. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phe- notypic selection in transformed cells. Another type of discrete transcription regulatory sequence element is an enhancer. An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a_. coding sequence in a cell.

As used herein, the phrase "enhancer-promoter" means a composite unit that contains both enhancer and promoter elements. An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product.

An enhancer-promoter used in a vector construct of the present invention may be any enhancer-promoter that, drives expression in a prokaryotic or eukaryotic cell to be trans- formed/transfected.

A coding sequence of an expression vector is operatively linked to a transcription terminating region. RNA polymerase transcribes an encoding DNA sequence through a site where poly- adenylation occurs.

An expression vector comprises a polynucleotide that encodes a nuclear receptor npFXRB polypeptide. Such a polynucleotide is meant to include a sequence of nucleotide bases encoding a nuclear receptor npFXRB polypeptide sufficient in length to distinguish said segment from a polynucleotide segment encoding a non- nuclear receptor npFXRB polypeptide.

A polypeptide of the invention may also encode biologically functional polypeptides or pep- tides which have variant amino acid sequences, such as with changes selected based on considerations such as the relative hydropathic score of the amino acids being exchanged.

These variant sequences are those isolated from natural sources or induced in the sequences disclosed herein using a mutagenic procedure such as site-directed mutagenesis.

Furthermore, an expression vector of the present invention may contain regulatory elements for optimized translation of the polypeptide in prokaryotic or eukaryotic systems. This sequences are operatively located around the transcription start site and are most likely similar to ribosome recognition sites like prokaryotic ribosome binding sites (RBS) or eukaryotic Kozak sequences as known in the art (Kozak M., Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187-208 (1999).

An expression vector of the present invention is useful both as a means for preparing quantities of the nuclear receptor npFXRB polypeptide-encoding DNA itself, and as a means for preparing the encoded nuclear receptor npFXRB polypeptide and peptides. It is contemplated that where nuclear receptor npFXRB polypeptides of the invention are made by recombinant means, one may employ either prokaryotic or eukaryotic expression vectors as shuttle systems.

Where expression of recombinant nuclear receptor npFXRB polypeptides is desired and a eukaryotic host is contemplated, it is most desirable to employ a vector such as a plasmid, that incorporates a eukaryotic origin of replication. Additionally, for the purposes of expression in eukaryotic systems, one desires to position the nuclear receptor npFXRB encoding sequence or if desired parts thereof (SEQ ID NO. 25) adjacent to and under the control of an effective eukaryotic promoter. To bring a coding sequence under control of a promoter, whether it is eukaryotic or prokaryotic, what is generally needed is to position the 5' end of the translation initiation site of the proper translational reading frame of the polypeptide between about 1 and about 2000 nucleotides 3' of or downstream with respect to the promoter chosen.

Furthermore, where eukaryotic expression is anticipated, one would typically desire to incorporate into the transcriptional unit which includes the nuclear receptor npFXRB polypeptide, an appropriate polyadenylation side. Furthermore, a genetic knock-out construct, comprising the nucleic acid molecule of present invention can be provided, in order to construct host-cells and/or transgenic non-primate animals that do not contain and/or express a functional npFXRb. The construction of such genetic constructs as well as the generation of transgenic knock-out animals are well known to the person skilled in the art.

The invention provides homogeneous compositions of mammalian nuclear receptor npFXRB polypeptide produced by transformed prokaryotic or eukaryotic cells as provided herein. Such homogeneous compositions are intended to be comprised of mammalian nuclear receptor npFXRB protein that comprises at least 90% of the protein in such homogenous composition. The invention also provides membrane preparation from cells expressing mammalian nuclear receptor npFXRB polypeptide as the result of transformation with a recombinant expression construct, as described here.

Within the scope of the present invention the terms recombinant protein or coding sequence both also include tagged versions of the protein depicted in SEQ ID NO. 25, and/or encoded by the nucleic acids according to the invention and fusion proteins of said proteins or parts thereof such as splice variants with any other recombinant protein. Tagged versions here means that small epitopes of 3-20 amino acids are added to the original protein by extending the coding sequence either at the 5 'or the 3 'terminus leading to N-terminal or C-terminal extended proteins respectively, or that such small epitopes are included elsewhere in the protein. The same applies for fusion proteins where the added sequences are coding for longer proteins, varying between 2 and 100 kDa. Tags and fusion proteins are usually used to facilitate purification of recombinant proteins by specific antibodies or affinity matrices or to increase solubility of recombinant proteins within the expression host. Fusion proteins are also of major use as essential parts of yeast two hybrid screens for interaction partners of recombinant proteins.

Tags used in the scope of the present invention may include but are not limited to the following: EEF (alpha Tubulin), B-tag (QYPALT), E tag (GAPVPYPDPLEPR) c-myc Tag (EQKLISEEDL), Flag epitope (DYKDDDDK, HA tag (YPYDVPDYA), 6 or 10 x His Tag, HSV (QPELAPEDPED), Pk-Tag (GKPIPNPLLGLDST), protein C (EDQVDPRLIDGK), T7 (MASMTGGQQMG), VSV-G (YTDIEMNRLGK), Fusion proteines may include Thiore- doxin, Glutathiontransferase (GST), Maltose binding Protein (MBP), Cellulose Binding protein (CBD), chitin binding protein, ubiquitin, the Fc part of Immunoglobulins, and the IgG binding domain of Staphylococcus aureus protein A. These examples of course are illustrative and not limiting and the standard amino acid one letter code was used above.

For expression of recombinant proteins in living cells or organisms, vector constructs harboring recombinant npFXRB nuclear receptor as set forth in SEQ ID NO. 1 to 6 are transformed or transfected into appropriate host cells. Preferably, a recombinant host cell of the present invention is transfected with a polynucleotide of SEQ ID NO. 1 to 6.

Means of transforming or transfecting cells with exogenous polynucleotide such as DNA molecules are well known in the art and include techniques such as calcium-phosphate- or DEAE-dextran-mediated transfection, protoplast fusion, electroporation, liposome mediated transfection, direct microinjection and virus infection (Sambrook et al., 1989).

The most frequently applied technique for transformation of prokaryotic cells is transformation of bacterial cells after treatment with calciumchloride to increase permeability (Dagert & Ehrlich, 1979), but a variety of other methods is also available for one skilled in the art.

The most widely used method for transfection of eukaryotic cells is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechanism remains obscure, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of cultured cells may be transfected at any one, time. Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for studies requiring transient expression of the foreign nucleic acid in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that integrate copies of the foreign DNA, which are usually arranged in head-to-tail tandem arrays into the host cell genome.

In the protoplast fusion method, protoplasts derived from bacteria carrying high numbers of copies of a plasmid of interest are mixed directly with cultured mammalian cells, After fusion of the cell membranes (usually with polyethylene glycol), the contents of the bacterium are delivered into the cytoplasm of the mammalian cells and the plasmid DNA is transported to the nucleus. Protoplast fusion is not as efficient as transfection for many of the cell lines that are commonly used for transient expression assays, but it is useful for cell lines in which endocytosis of DNA occurs inefficiently. Protoplast fusion frequently yields multiple copies of the plasmid DNA tandemly integrated into the host chromosome.

The application of brief, high-voltage electric pulses to a variety of mammalian and plant cells leads to the formation of nanometer sized pores in the plasma membrane. DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. Electroporation may be extremely efficient and may be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA. Liposome transfection involves encapsulation of DNA and RNA within liposomes, followed by fusion of the liposomes with the cell membrane. The mechanism of how DNA is delivered into the cell is unclear but transfection efficiencies may be as high as 90%.

Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to cellular compartments such as low-pH endosomes. Microinjection is therefore used primarily as a method to establish lines of cells that carry integrated copies of the DNA of interest.

The use of adenovirus as a vector for cell transfection is well known in the art. Adenovirus vector-mediated cell transfection has been reported for various cells (Stratford-Perricaudet et al., 1992).

A transfected cell may be prokaryotic or eukaryotic, transfection may be transient or stable.

In another aspect, the recombinant host cells of the present invention are prokaryotic host cells. In addition to prokaryotes, eukaryotic microbes, such as yeast may also be used illustrative examples for suitable cells and organisms for expression of recombinant proteins are belonging to but not limited to the following examples: Insect cells, such as Drosophila Sf21, SF9 cells or others, Expression strains of Escherichia coli, such as XLI blue, BRL21, M15, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenlua polymorpha and Pichia pastoris strains, immortalized mammalian cell lines such as AtT-20, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7. 293 and MDCK cells, BHK-21 cells, Att 20HeLa cells, HeK 294, T47 D cells and others.

In another aspect, the recombinant host cells of the present invention are host cells transfected with the vector, expression vector and/or genetic knock-out construct of the present invention. Preferred is a non-primate host cell that lacks a functional npFXRb and/or splice variants thereof.

Another aspect of the present invention is a method of producing a polypeptide comprising the step of culturing the host cell of the present invention in an appropriate culture medium to, thereby, produce the polypeptide. Expression of recombinant proteins within the scope of this invention can also be performed in vitro. This may occur by a two step procedure, thereby producing first mRNA by in vitro transcription of an apt polynucleotide construct followed by in vitro translation with convenient cellular extracts. These cellular extracts may be reticulocyte lysates but are not limited to this type. In vitro transcription may be performed by T7 or SP6 DNA polymerase or any other RNA polymerase which can recognize per se or with the help of accessory factors the promoter sequence contained in the recombinant DNA construct of choice. Alternatively one of the recently made available one step coupled transcription/translation systems may be used for in vitro translation of DNA coding for the proteins of this invention, e.g. from Roche Molecular Biochemicals. One illustrative but not limiting example for such a system is the TNT® T7 Quick System by Promega.

Expression of recombinant proteins in transfected ceil may occur constitutively or upon induction. Procedures depend on the Cell/vector combination used and are well known in the art. In all cases, transfected cells are maintained for a period of time sufficient for expression of the recombinant npFXRB nuclear receptor protein. A suitable maintenance time depends strongly on the cell type and organism used and is easily ascertainable by one skilled in the art. Typically, maintenance time is from about 2 hours to about 14 days. For the same reasons and for sake of protein stability and solubility incubation temperatures during maintenance time may vary from 20°C to 42 °C.

Recombinant proteins are recovered or collected either from the transfected cells or the medium in which those cells are cultured. Recovery comprises cell disruption, isolation and purification of the recombinant protein. Isolation and purification techniques for polypeptides are well-known in the art and include such procedures as precipitation, filtration, chromatography, electrophoresis and the like.

In a preferred embodiment, purification includes but is not limited to affinity purification of tagged or non-tagged recombinant proteins. This is a well established robust technique easily adapted to any tagged protein by one skilled in the art. For affinity purification of tagged proteins, small molecules such as gluthathione, maltose or chitin, specific proteins such as the IgG binding domain of Staphylococcus aureus protein A, antibodies or specific chelates which bind with high affinity to the tag of the recombinant protein are employed. For affinity purification of non-tagged proteins specific monoclonal or polyclonal antibodies, which were raised against said protein, can be used. Alternatively immobilized specific interactors of said protein may be employed for affinity purification. Interactors include native or recombinant proteins as well as native or artificial specific low molecular weight ligands.

CHEMICAL SYNTHESIS OF THE POLYPEPTIDE ACCORDING TO THE INVENTION: Alternatively, the protein itself may be produced using chemical methods to synthesize any of the amino acid sequences according to the invention or that is encoded by the nucleotide sequences according to the invention (SEQ ID NO. 1 to 6) and/or a portion thereof and/or splice variants thereof. For example, peptide synthesis can be performed using conventional Merri- fϊeld solid phase f-Moc or t-Boc chemistry or various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer). The newly synthesized peptide(s) may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra). Additionally, the amino acid sequence according to the invention, i.e. SEQ ID NO. 25 or the sequence that is encoded by SEQ ID NO. 1 to 6 or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

SCREENING ASSAYS

The invention also concerns a method for screening for agents which are capable of inhibiting the cellular function of the nuclear receptor npFXRB comprising the steps of contacting one or more candidate agents with a polypeptide according to the invention, removing unbound agent(s) and detecting whether the agent(s) interact with the polypeptide of the nuclear receptor.

The invention also concerns method for inhibiting the cellular function of the nuclear receptor npFXRB, comprising the steps of contacting a cell with a binding agent of a polypeptide previously identified as outlined herein whereby the cellular function of npFXRB is inhibited. Such a binding agent may be an antibody., RNA. an anti-sense oligonucleotide. a ribozyme or one of substances shown below or identified in a respective assay as disclosed herein.

In still a further embodiment, the present invention concerns a method for identifying new nuclear receptor inhibitory or stimulatory substances, which may be termed as "candidate substances". It is contemplated that this screening technique proves useful in the general identification of compounds that serve the purpose of inhibiting or stimulating nuclear receptor activity.

In one embodiment of the invention the following substances are disclosed as potential interactors of the nuclear receptor according to the invention:

Steroids: dexamethasone-t-butylacetate, RU486, progesterone, 17-alpha- hydroxyprogesterone, 1,16-alpha dimethylpregnenolone, 17-alpha-hydroxypregnenonlone, pregnenonlone, 5beta-pregnane-3,20-dione, pregnenonlone-16-carbonitrile, 5beta-pregnane- 3,20-dione, androstanol, corticosterone, dehydroepiandrosterone, dihydroxytestosterone, es- tradiol, cortisol, cortisone, dihydroxytestosterone.

Other substances: transnonachlor, chlordane, spironolactone, cyproterone acetate, rifampicin, nefipine, diethylstilbestrol, coumesterol, clotrimazole, lovastatin, phenoarbital, pthalic acid, nonylphenol, l,4-bis(2-(3,5-dichloropyridyloxyl))benzene,

This also includes the use of heteromultimeric complexes of the nuclear receptor with other proteins, such as heterodimeric complexes with RXR, or any other binding partner.

Accordingly, in screening assays to identify pharmaceuticals agents which affect nuclear receptor activity, it is proposed that compounds isolated from natural sources, such as fungal extracts, plant extracts, bacterial extracts, higher eukaryotic cell extracts, or even extracts from animal sources, or marine, forest or soil samples, may be assayed for the presence of potentially useful pharmaceutical agents.

It will be understood that that the pharmaceutical agents to be screened could also be derived from chemical compositions or man-made compounds. The candidate substances can could also include monoclonal or polyclonal antibodies, peptides or proteins, such as those derived from recombinant DNA technology or by other means, including chemical peptide synthesis. The active compounds may include fragments or parts or derivatives of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. We anticipate that such screens will in some cases lead to the isolation of agonists of nuclear receptors, in other cases to the isolation of antagonists. In other instances, substances will be identified that have mixed agonistic and antagonistic effects, or affect nuclear receptors in any other way.

CELL BASED ASSAYS

To identify a candidate substance capable of influencing npFXRB nuclear receptor activity, one first obtains a recombinant cell line. One designs the cell line in such a way that the activity of the nuclear receptor leads to the expression of a protein which has an easily detectable phenotype ( a reporter), such as luciferase, fluorescent proteins such as green or red fluorescent protein, beta-galactosidase, alpha-galactosidase, beta-lactamase, chloramphenicol- acetyl-transferase, beta-glucuronidase, or any protein which can be detected by a secondary reagent such as an antibody.

Methods for detecting proteins using antibodies, such as ELISA assays, are well known to those skilled in the art.

Here, the amount of reporter protein present reflects the transcriptional activity of the nuclear receptor. This recombinant cell line is then screened for the effect of substances on the expression of the reporters, thus measuring the effect of these substances on the activity of the nuclear receptor. These substances can be derived from natural sources, such as fungal extracts, plant extracts, bacterial extracts, higher eukaryotic cell extracts, or even extracts from animal sources, or marine, forest or soil samples, may be assayed for the presence of potentially useful pharmaceutical agents. It will be understood that that the pharmaceutical agents to be screened may be derived from chemical compositions or man-made compounds.

The candidate substances can also include monoclonal or polyclonal antibodies, peptides or proteins, such as those derived from recombinant DNA technology or by other means, including chemical peptide synthesis. The active compounds may include fragments or parts or derivatives of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. In general the assay comprises, contacting a suitable cell containing a reporter under the control of the npFXRB nuclear receptor with a test compound, monitoring said host_cell for the expression of the reporter gene, wherein expression of the reporter reflects the transcriptional activity of the nuclear receptor npFXRB, and therefore reflects effects of the compound on the nuclear receptor.

In other embodiments of the invention assays are included where measuring the activity of a dimer of the nuclear receptor npFXRB and another protein, such as RXR takes place. Further included are assays aiming at the identification of compounds which specifically influence only the monomeric, homodimeric or homomultimeric form of the nuclear receptor, or influencing only multimeric forms of the nuclear receptor. Such assays include measuring the effect of a compound on the nuclear receptor in the absence of a binding partner, and measuring the effect of a compound oii the nuclear receptor in the presence of a binding partner, such as RXR. One skilled in the art will find numerous more assays which are equally covered by the invention.

A cell line where the activity of a nuclear receptor determines the expression of a reporter can be obtained by creating a fusion gene driving the expression of a fusion protein consisting of the ligand-binding domain of the npFXRB nuclear receptor fused to the DNA binding domain of a transcription factor with a known specificity for a given DNA sequence (the binding site). This DNA sequence (the binding site) can then be inserted in one or multiple copies before (5') to the promoter driving the expression of the reporter. Transcription factors useful for this approach include bacterial proteins, such as lexA, yeast proteins, such as Gal4, mammalian proteins such as NFkappaB or NFAT, the glucocorticoid receptor, the estrogen receptor, or plant proteins. The binding sites for these proteins can all be used in combination with the appropriate transcription factor to generate a useful reporter assay.

Another way to screen for inhibitors is to identify binding sites on DNA for the npFXRB nuclear receptor, and operatively link this binding site to a promoter operatively linked to a reporter gene. Included among others are binding sites for heterodimers of the npFXRB nuclear receptor with another protein, such as RXR. Furthermore, transgenic animals described in the invention can be used to derive cell lines useful for cellular screening assays.

Cell lines useful for such an assay include many different kinds of cells, including prokaryotic, animal, fungal, plant and human cells. Yeast cells can be used in this assay, including Saccharomyces cerevisiae and Schizosaccharomyces pombe cells.

Another way to build cellular assays to measure the effect of compounds is the use of the yeast two hybrid system (see for example see, for example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; PCT Publication No. WO 94/10300, and U.S. Pat. No. 5,667,973), and or possible variants of the basic two hybrid system as discussed e.g in Vidal M, Legrain P, Nucleic Acids Res. 1999 Feb 15;27(4):919-29. Briefly, the two hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins. In particular, the method makes use of chimeric genes which express hybrid proteins. To illustrate, a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator fused in frame to the coding sequence for a TI polypeptide. The second hybrid protein encodes a transcriptional activation domain fused in frame to a sample gene from a cDNA library. If the bait and sample hybrid proteins are able to interact, e.g., form a Tl-dependent complex, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene can be detected and used to score for the interaction of the TI and sample proteins.

In such assays, one primarily measures the effect of a compound on a given interaction involving the npFXRB nuclear receptor and a binding protein. In a preferred embodiment of the invention systems using other hosts such as prokaryotes as E. coli, or eukaryotic mammalian cells are described.

Two hybrid systems using hybrid protein fusions with other proteins than transcription factors, including enzymes such as beta-galactosidase or dihydrofolate reductase may also be applied. These assays are useful both to monitor the effect of a compound, including peptides, proteins or nucleic acids on an interaction of a nuclear receptor with a given binding partner, as well as to identify novel proteins or nucleic acids interacting with the nuclear receptor.

Monitoring the influence of compounds on cells may be applied not only in basic drug screening, but also in clinical trials. In such clinical trials, the expression of a panel of genes may be used as a "read out" of a particular drug's therapeutic effect.

CELL-FREE ASSAYS

Recombinant forms of the polypeptide according to SEQ ID NO. 25 or as encoded by the nucleic acids according to the invention can be used in cell-free screening assays aiming at the isolation of compounds affecting the activity of nuclear receptors. In such an assay, the nuclear receptor polypeptide is brought into contact with a substance to test if the substance has an effect on the activity of the npFXRB receptor.

The detection of an interaction between an agent and a receptor may be accomplished through techniques well-known in the art. These techniques include but are not limited to centrifugation, chromatography, electrophoresis and spectroscopy. The use of isotopically labeled reagents in conjunction with these techniques or alone is also contemplated. Commonly used radioactive isotopes include ³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁵Ca, ⁶⁰Co, ¹²⁵I, and ¹³¹I. Commonly used stable isotopes include ²H, ¹³ C, ¹⁵N, ¹⁸O.

For example, if an agent binds to the receptor of the present invention, the binding may be detected by using radiolabeled agent or radiolabeled receptor. Briefly, if radiolabeled agent or radiolabeled receptor is utilized, the agent-receptor complex may be detected by liquid scintillation or by exposure to x-ray film or phosho-imaging devices.

One way to screen for substances affecting nuclear receptor activity is to measure the effect of the binding of nuclear receptors to ligands, such as cofactors, activators, repressors, DNA, RNA, proteins, antibodies, peptides or other substances, including chemical compounds known to affect receptor activity. Assays measuring the binding of a protein to a ligand are well known in the art, such as ELISA assays, FRET assays, bandshift assays, plasmon- resonance based assays, scintilllation proximity assays, fluorescence polarization assays. In one example, a mixture containing the npFXRB polypeptide, effector and candidate substance is allowed to incubate. The unbound effector is separable from any effector/receptor complex so formed. One then simply measures the amount of each (e.g., versus a control to which no candidate substance has. been added). This measurement may be made at various time points where velocity data is desired. From this, one determines the ability of the candidate substance to alter or modify the function of the receptor.

Numerous techniques are known for separating the effector from effector/receptor complex, and all such methods are intended to fall within the scope of the invention. This includes the use of thin layer chromatographic methods (TLC), HPLC, spectrophotometric, gas chroma- tographic/mass spectrophotometric or NMR analyses. Another method of separation is to immobilize one of the binding partners on a solid support, and to wash away, any unbound material. It is contemplated that any such technique may be employed so long as it is capable of differentiating between the effector and complex, and may be used to determine enzymatic function such as by identifying or quantifying the substrate and product.

A screening assay provides a npFXRB receptor under conditions suitable for the binding of an agent to the npFXRB receptor. These conditions include but are not limited to pH, temperature, tonicity, the presence of relevant cofactors, and relevant modifications to the polypeptide such as glycosylation or lipidation. It is contemplated that the receptor can be expressed and utilized in a prokaryotic or eukaryotic cell. The host cell expressing the npFXRB receptor can be used whole or the receptor can be isolated from the host cell. The npFXRB receptor can be membrane bound in the membrane of the host cell or it can be free in the cytosol of the host cell. The host cell can also be fractionated into sub-cellular fractions where the receptor can be found. For example, cells expressing the receptor can be fractionated into the nuclei, the endoplasmic reticulum, vesicles, or the membrane surfaces of the cell.

pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a value of about 6.8 to a value of about 7.8, and most preferably, about 7.4. In a preferred embodiment, temperature is from about 20°C degrees to about 50°C degrees more preferably, from about 30°C degrees to about 40°C degrees and even more preferably about 37°C degrees. Osmolality is preferably from about 5 milliosmols per liter (mosm/L) to about 400 mosm/1, and more preferably, from about 200 milliosmols per liter to about 400 mosm/1 and, even more preferably from about 290 mosm/L to about 310 mosm/L. The presence of cofac- tors can be required for the proper functioning of the npFXRB receptor. Typical cofactors include sodium, potassium, calcium, magnesium, and chloride. In addition, small, non-peptide molecules, known as prosthetic groups may. also be required. Other biological conditions needed for receptor function are well-known in the art.

It is well-known in the art that proteins can be reconstituted in artificial membranes, vesicles or liposomes. (Danboldt et al.,1990). The present invention contemplates that the receptor can be incorporated into artificial membranes, vesicles or liposomes. The reconstituted receptor can be utilized in screening assays.

It is further contemplated that a receptor of the present invention can be coupled to a solid support, e.g., to agarose beads, polyacrylamide beads, polyacrylic, sepharose beads or other solid matrices capable of being coupled to polypeptides. Well-known coupling agents include cyanogen bromide (CNBr), carbonyldiimidazole, tosyl chloride, diaminopimelimidate, and glutar aldehyde.

In a typical screening assay for identifying candidate substances, one employs the same recombinant expression host as the starting source for obtaining the receptor polypeptide, generally prepared in the form of a crude homogenate. Recombinant cells expressing the receptor are washed and homogenized to prepare a crude polypeptide homogenate in a desirable buffer such as disclosed herein. In a typical assay, an amount of polypeptide from the cell homogenate, is placed into a small volume of an appropriate assay buffer at an appropriate pH. Candidate substances, such as agonists and antagonists, are added to the admixture in convenient concentrations and the interaction between the candidate substance and the receptor polypeptide is monitored.

Where one uses an appropriate known substrate for the npFXRB receptor, one can, in the foregoing manner, obtain a baseline activity for the recombinantly produced npFXRB receptor. Then, to test for inhibitors or modifiers of the receptor function, one can incorporate into the admixture a candidate substance whose effect on the npFXRB receptor is unknown. By comparing reactions which are carried out in the presence or absence of the candidate substance, one can then obtain information regarding the effect of the candidate substance on the normal function of the receptor. Accordingly, this aspect of the present invention will provide those of skill in the art with methodology that allows for the identification of candidate substances having the ability to modify the action of nuclear receptor polypeptides in one or more manners.

Additionally, screening assays for the testing of candidate substances are designed to allow the determination of structure-activity relationships of agonists or antagonists with the receptors, e.g., comparisons of binding between naturally-occurring hormones or other substances capable of interacting with or otherwise modulating the receptor; or comparison of the activity caused by the binding of such molecules to the receptor.

In certain aspects, the polypeptides of the invention are crystallized in order to carry out x-ray crystallographic studies as a means of evaluating interactions with candidate substances or other molecules with the nuclear receptor polypeptide. For instance, the purified recombinant polypeptides of the invention, when crystallized in a suitable form, are amenable to detection of intra-molecular interactions by x-ray crystallography. In another aspect, the structure of the polypeptides can be determined using nuclear magnetic resonance.

PHARMACEUTICAL COMPOSITION:

This invention provides a pharmaceutical composition comprising an effective amount of a agonist or antagonist drug identified by the method described herein and a pharmaceutically acceptable carrier. Such drugs and carrier can be administered by various routes, for example oral, subcutaneous, intramuscular, intravenous or intracerebral. The preferred route of administration would be oral at daily doses of about 0.01 -100 mg/kg.

This invention provides a method of treating metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases wherein the abnormality is improved by reducing the activity of npFXRB receptor or blocking the binding of ligands to a -npFXRB receptor, which method comprises administering an effective amount of the antagonist- containing pharmaceutical composition described above to suppress the subject's appetite. Similarly, the invention also provides methods for treating diseases and conditions resulting from metabolic disorders, immunological indications, hormonal dysfunctions, neurosystemic diseases, which method comprises administering an effective amount of an agonist-containing pharmaceutical composition described above. TRANSFORMATION OF CELLS AND DRUG SCREENING :

The recombinant expression constructs of the present invention are useful in molecular biology to transform cells which do not ordinarily express npFXRB to thereafter express this receptor.

Such cells are useful as intermediates for making cellular preparations useful for receptor binding assays, which are in turn useful for drug screening. Drugs identified from such receptor assays can be used for the treatment of metabolic disorders, immunological indications, hormonal dysfunctions, and/or neurosystemic diseases.

The recombinant expression constructs of the present invention are also useful in gene therapy. Cloned genes of the present invention, or fragments thereof, may also be used in gene therapy carried out by homologous recombination or site-directed mutagenesis. See generally Thomas & Capecchi, Cell 51, 503-512 (1987); Bertling, Bioscience Reports 7, 107-112 (1987); Smithies et al., Nature 317, 230-234 (1985).

Ohgonucleotides of the present invention are useful as diagnostic tools for probing npFXRB expression in tissues. For example, tissues are probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiographic techniques, as explained in greater detail in the Examples below, to investigate native expression of this receptor or pathological conditions relating thereto. Further, chromosomes can be probed to investigate the presence or absence of the npFXRB, and potential pathological conditions related thereto, as also illustrated by the Examples below. Probes according to the invention should, generally be at least about 15 nucleotides in length to prevent binding to random sequences, but, under the appropriate circumstances may be smaller (see above for details on hybridization).

ANTIBODIES AGAINST THE npFXRB NUCLEAR RECEPTOR PROTEIN OR POLYPEPTIDE

Another aspect of the invention includes an antibody specifically reactive with the protein or any part of the protein according to the invention (SEQ ID NO. 25) and or a polypeptide encoded by the nucleotide sequence of the nuclear receptor npFXRB (see also figures). (The term „antibody" refers to intact molecules as well as fragments thereof, such as Fa, F(ab).sub.2, and Fv, which are capable of binding the epitopic determinant.) By using immu- nogens derived from the polypeptide according to the invention (SEQ ID NO. 25) and/or en- coded by the nucleic acids according to the invention, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (E. Howell & D. Lane. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory (1988)).

A polyclonal antibody is prepared by immunizing a mammal, such as a mouse, a hamster or rabbit with an immunogenic form of the polypeptide, i.e. the murine npFXRB polypeptide of the present invention, and collecting antisera from that immunized animal. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As an immunizing antigen, fusion proteins, intact polypeptides or fragments containing small peptides of interest can be used. They can be derived by expression from a cDNA transfected in a host cell with subsequent recovering of the protein/peptide or peptides can be synthesized chemically (e.g. oligopeptides with 10-15 residues in length). Important tools for monitoring the function of the gene according to the present invention, i.e. encoded by a sequence according to SEQ ID NO. 1 to 6 (or portions thereof or splice variants thereof) are antibodies against various domains of the protein according to the invention. Various Oligopeptides from the N- and C-terminal sequences and the DBD/hinge region of the protein can be used as antigens.

A given polypeptide or polynucleotide may vary in its immunogenicity. It is often necessary to couple the immunogen (e.g. the polypeptide) with a carrier. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal in the presence of an adjuvans, a non-specific stimulator of the immune response in order to enhance immunogenicity. The production of polyclonal antibodies is monitored by detection of antibody titers in plasma or serum at various time points following immunization. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. When a desired level of immunogenicity is obtained, the immunized animal may be bled and the serum isolated, stored and purified.

To produce monoclonal antibodies, antibody-producing cells (e.g. spleen cells) from an immunized animal (preferably mouse or rat) are fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Where the immunized animal is a mouse, a preferred myeloma cell is the murine NS-1 myeloma cell. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler & Milstein. Nature 256: 495-49.7.(1975)), the human B cell hybridoma technique (Kozbar et al. Immunology Today 4:72 (1983)), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al. Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, Inc. pp. 77-96 (1985)).

The fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells. Fused cells are separated from the mixture of non-fused parental cells, for example, by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media. This culturing provides a population of hy- bridomas from which. specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants for reactivity with an antigen-polypeptide. The selected clones may then be propagated indefinitely to provide the monoclonal antibody in convenient quantity.

The creation of antibodies which specifically bind the polypeptide according to the invention (SEQ ID NO. 25) and/or encoded by the nucleotide sequence of the nuclear receptor npFXRB or its complement (SEQ ID NO. 1 to 6) provides an important utility in immunolocalization studies, and may play an important role in the diagnosis and treatment of receptor disorders. The antibodies may be employed to identify tissues, organs, and cells which express or the nuclear receptor npFXRB. Antibodies can^' be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate nuclear receptor npFXRB protein levels in tissue or from cells in bodily fluid as part of a clinical testing procedure.

Monoclonal antibodies provided by the present invention are also produced by recombinant genetic methods well known to those of skill in the art, and the present invention encompasses antibodies made by such methods that are immunologically reactive with an epitope of a mammalian nuclear npFXRB receptor protein or peptide.

The present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a mammalian nuclear npFXRB receptor protein or peptide. Such fragments are produced by any number of methods, including but not limited to proteolytic cleav- age, chemical synthesis or preparation of such fragments by means of genetic engineering technology. The present invention also encompasses single-chain antibodies that are immunologically reactive with an epitope of a mammalian nuclear npFXRB receptor protein or peptide made by methods known to those of skilled in the art.

CHIMERIC ANTIBODIES AND OTHER TYPES OF ANTIBODIES:

The invention also includes chimeric antibodies, comprised of light chain and heavy chain peptides. immunologically reactive to an epitope that is a mammalian nuclear npFXRB receptor protein or peptide. The chimeric antibodies embodied in the present invention include those that are derived from naturally occurring antibodies as well as chimeric antibodies made by means of genetic engineering technology well known to those of skill in the art.

Also included are methods for the generation of antibodies against npFXRB which rely on the use of phage display systems and related systems, such as described in Hoogenboom HR, de Bruine AP, Hufton SE, Hoet RM, Arends JW, Roovers RC, Immunotechnology 1998 Jun;4(l):l-20, and references therein.

EPITOPES OF THE npFXRB NUCLEAR RECEPTOR

The present invention also encompasses an epitope of a non-primate nuclear npFXRB receptor protein or peptide that is comprised of sequences and/or a conformation of sequences present in the nuclear npFXRB receptor protein or peptide molecule. This epitope may be naturally occurring, or may be the result of proteolytic cleavage of the nuclear npFXRB receptor protein or peptide molecule and isolation of an epitope-containing peptide or may be obtained by synthesis of an epitope-containing peptide using method of genetic engineering technology and synthesized by genetically engineered prokaryotic or eukaryotic cells.

ANTISENSE OLIGONUCLEOTIDES AGAINST npFXRB

Antisense ohgonucleotides are short single stranded DNA or RNA molecules which may be used to block the availability of the npFXRB receptor messenger. Synthetic derivatives of ribonucleotides or deoxyribonucleotides and/or PNAs (see above) are equally possible.

The sequence of an antisense oligonucleotide is at least partially complementary to the sequence (or the gene) of interest. The complementarity of the sequence is in any case high enough to enable the antisense oligonucleotide to bind to the nucleic acid according to the invention or parts thereof. Many examples exist in which the binding of ohgonucleotides to the target sequence interfere with the biological function of the targeted sequence (Brysch W, Schlingensiepen KH, Design and application of antisense ohgonucleotides in cell culture, in vivo, and as therapeutic agents, Cell Mol Neurobiol 1994 Oct;14(5):557-68; Wagner RW, Gene inhibition using antisense oligodeoxynucleotides, Nature 1994 Nov 24;372(6504):333-5 or Brysch W, Magal E, Louis JC, Kunst M, Klinger I, Schlingensiepen R, Schlingensiepen KH Inhibition of pl85c-erbB-2 proto-oncogene expression by antisense oligodeoxynucleotides down-regulates pl85-associated tyrosine-kinase activity and strongly inhibits mammary tumor-cell proliferation, Cancer Gene Ther 1994 Jun;l(2).:99-105 or Monia BP, Johnston JF, Ecker DJ, Zounes MA, Lima WF, Freier SM Selective inhibition of mutant Ha-ras mRNA expression by antisense ohgonucleotides, J Biol Chem 1992 Oct 5;267(28): 19954-62 or Bertram J, Palfner K, Killian M, Brysch W, Schlingensiepen KH, Hiddemann W, Kneba M, Reversal of multiple drug resistance in vitro by phosphorothioate oligonucleotides and ribo- zymes, Anticancer Drugs 1995 Feb;6(l): 124-34)

This interference occurs in most instances at the level of translation, i.e. through the inhibition of the translational machinery by oligonucleotides that bind to mRNA, however, two other mechanisms of interference with a given gene's function by oligonucleotides can also be envisioned, (i) the functional interference with the transcription of a gene through formation of a triple helix at the level of genomic DNA and the interference of oligonucleotides with the function of RNA molecules that are executing at least part of their biological function in the untranslated form (Kochetkova M, Shannon MF, Triplex-forming oligonucleotides and their use in the analysis of gene transcription. Methods Mol Biol 2000;130:189-201 Rainer B. Lanzl, Neil J. McKennal, Sergio A. Onatel, Urs Albrecht2, Jiemin Wongl, Sophia Y. Tsail, Ming-Jer Tsail, and Bert W. O'Malley A Steroid Receptor Coactivator, SRA, Functions as an RNA and Is Present in an SRC-1 Complex Cell, Vol. 97, 17-27, April, 1999).

Antisense oligonucleotides can be conjugated to different other molecules in order to deliver them to the cell or tissue expressing npFXRB. For instance the antisense oligonucleotide can be conjugated to a carrier protein (e.g. ferritin) in order to direct the oligonucleotide towards the desired target tissue, i.e. in case of ferritin predominantly to the liver.

Antisense expression constructs are expression vector systems that allow the expression - either inducible or uninducible - of a complementary sequence to the npFXRB sequences ac- cording to the invention. The potential possibility of such an approach has been demonstrated in many different model systems (von Ruden T, Gilboa E, Inhibition of human T-cell leukemia virus type I replication in primary human T cells. that express antisense RNA, J. Virol 1989 Feb;63(2):677-82; Nemir M, Bhattacharyya D, Li X, Singh K, Mukherjee AB, Mukherjee BB, Targeted inhibition of osteopontin expression in the mammary gland causes abnormal morphogenesis and lactation deficiency, J Biol Chem 2000 Jan 14;275(2):969-76; Ma L, Gauville C, Berthois Y, Millot G, Johnson GR, Calvo F Antisense expression for am- phiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line, Oncogene 1999 Nov l l;18(47):6513-20; Refolo LM, Eckman C, Prada CM, Yager D, Samba- murti K, Mehta N, Hardy J, Younkin SG, Antisense-induced reduction of presenilin 1 expression selectively increases the production of amyloid beta42 in transfected cells, J Neurochem 1999 Dec;73(6):2383-8; Buckley NJ, Abogadie FC, Brown DA, Dayrell M, Caulfield MP, Delmas P, Haley JE, Use of antisense expression plasmids to attenuate G-protein expression in primary neurons, Methods Enzymol 2000;314:136-48).

According to the invention an antisense expression construct can be constructed with virtually any expression vector capable of fulfilling at least the basic requirements known to those skilled in the art. In one embodiment of the invention retroviral expression systems or tissue specific gene expression systems are preferred.

Current standard technologies for delivering antisense constructs are performed through a conjugation of constructs with liposomes and related, complex-forming compounds, which are transferred via electroporation techniques or via particle-mediated "gene gun" technologies into the cell. Other techniques may be envisioned by one skilled in the art.

Microinjection still plays a major role in most gene transfer techniques for the generation of germ-line mutants expressing foreign DNA (including antisense RNA constructs) and is preferred embodiment of the present invention.

RIBOZYMES DIRECTED AGAINST npFXRB

Ribozymes are either RNA molecules (Gibson SA, Pellenz C, Hutchison RE, Davey FR, Shillitoe EJ, Induction of apoptosis in oral cancer cells by an anti-bcl-2 ribozyme delivered by an adenovirus vector, Clin Cancer Res 2000 Jan;6(l):213-22; Folini M, Colella G, Villa R, Lualdi S, Daidone MG, Zaffaroni N, Inhibition of Telomerase Activity by a Hammerhead Ribozyme Targeting the RNA Component of Telomerase in Human Melanoma Cells, J Invest Dermatol 2000 Feb;114(2):259-267; Halatsch ME, Schmidt U, Botefur IC, Holland JF, Oh- numa T, Marked inhibition of glioblastoma target cell tumorigenicity in vitro by- retrovirus- mediated transfer of a hairpin ribozyme against deletion-mutant epidermal growth factor receptor messenger RNA, J Neurosurg 2000 Feb;92(2):297-305; Ohmichi T, Kool ET, The virtues of self-binding: high sequence specificity for RNA cleavage by self-processed hammerhead ribozymes, Nucleic Acids Res 2000 Feb l;28(3):776-783) or DNA molecules (Li J, Zheng W, Kwon AH, Lu Y, In vitro selection and characterization of a highly efficient Zn(II)- dependent RNA-cleaving deoxyribozyme; Nucleic Acids Res 2000 Jan 15;28(2):481-488) that have catalytic activity. The catalytic activity located in one part of the RNA (or DNA) molecule can be "targeted" to a specific sequence of interest by fusing the enzymatically active RNA molecule sequence with a short stretch of RNA (or DNA) sequence that is complementary to the npFXRB transcript. Such a construct will, when introduced into a cell either physically or via gene transfer of a ribozyme expression construct find the npFXRB sequence (our sequence of interest) and bind via its sequence-specific part to said sequence. The catalytic activity attached to the construct, usually associated with a special nucleic acid structure (people distinguish so called "hammerhead" structures and "hairpin" structures), will then cleave the targeted RNA. The targeted mRNA will be destroyed and cannot be translated efficiently, thus the protein encoded by the mRNA derived from npFXRB will not be expressed or at least will be expressed at significantly reduced amounts.

In a preferred embodiment the invention covers inducible ribozyme constructs (Koizumi M, Soukup GA, Kerr JN, Breaker RR, Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP, Nat Struct Biol 1999 Nov;6(l 1):1062-1071).

In a further preferred embodiment the invention concerns the use of "bivalent" ribozymes (multimers of catalytically active nucleic acids) as described -in (Hamada M, Kuwabara T, Warashina M, Nakayama A, Taira K, Specificity of novel allosterically trans- and cis- activated connected maxizymes that are designed to suppress BCR-ABL expression FEBS Lett 1999 Nov 12;461(l-2):77-85).

TRANSGENIC ANIMALS CARRYING THE npFXRB NUCLEAR RECEPTOR

Also provided by the present invention are non-human transgenic animals grown from germ cells transformed with the npFXRB nucleic acid sequence according to the invention and that express the npFXRB receptor according to the invention and offspring and descendants thereof. Also provided are transgenic non-human mammals comprising a homologous recombination knockout of the native npFXRB receptor, as well as transgenic non-human mammals grown from germ cells transformed with nucleic acid antisense to the npFXRB nucleic acid of the invention and offspring and descendants thereof. Further included as part of the present invention are transgenic animals which the native npFXRB receptor has been replaced with the human homolog. Of course, offspring and descendants of all of the foregoing transgenic animals are also encompassed by the invention.

Transgenic animals according to the invention can be made using well known techniques with the nucleic acids disclosed herein. E.g., Leder et al., U.S. Patent Nos.4,736,866 and 5,175,383; Hogan et al., Manipulating the Mouse Embryo, A Laboratory Manual (Cold Spring Harbor Laboratory (1986)); Capecchi, Science 244, 1288 (1989); Zirnmer and Gruss, Nature 338, 1,50 (1989); Kuhn et al., Science 269, 1427 (1995); Katsuki et al., Science 241, 593 (1988); Hasty et al., Nature 350, 243 (1991); Stacey et al., Mol. Cell Biol. 14, 1009 (1994); Hanks et al., Science 269, 679 (1995); and Marx, Science 269, 636 (1995). Such transgenic animals are useful for screening for and determining the physiological effects of npFXRB receptor agonists and antagonist.

Consequently, such transgenic animals are useful for developing drugs to regulate physiological activities in which npFXRB participates.

According to another aspect of the present invention, a transgenic animal, in particular a knock-out animal comprising the genetic knock-out construct of the present invention, can be used for the analysis of agents that modulate cholesterol, bile acids, and triglyceride synthesis. Thus, this model system can conveniently be used in order to obtain additional information with respect to properties of the compounds/agents to be examined with respect to, e.g., physiological properties, bioavailabilty and other important pharmaceutical parameters. The agents to be analyzed can be preferably selected from FXRa modulating agents, LXRa or LXRb modulating agents, HNF4a, HNF4b, HNF4g modulating agents, PPARa, PPARb, PPARg modulating agents SCAP modulating agents, ACAT modulating agents, HMG-CoA- reductase modulating agents, and the like. MODELLING OF THE STRUCTURE OF npFXRB

The novel nuclear receptor sequences disclosed herein may be used for various in silico, i.e. computer-supported analyses. Such analyses may be_for example nuclear receptor specific sequence alignments which permit the identification of domains and even new receptors. The novel domain sequences disclosed herein may be used in order to create domain specific hidden markov models (hmms) or simply as search sequences.

In a preferred embodiment this similarity search tool is the BLAST algorithm.(Altschul, Stephen F., Warren Gish, Webb Miller, Eugene W. Myers, and David J. Lipman (1990). Basic local alignment search tool. J. Mol. Biol. 215:403-10 and the sequence used is one of those disclosed herein.

Another search tool that may be used is FASTA (W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444- 2448, and W. R. Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA" Methods in Enzymology 183:63- 98).

The invention is not limited to one particular type of search tool. In one embodiment of the invention search tools are used that do not search by sequence similarity but by applying sequence profiles such as a profile generated when applying the Profile Hidden Markov Model.

Profile Hidden Markov Models also called „Hidden Markov Models", here abbreviated as HMM, are statistical models representing the consensus of the primary structure of a sequence family. The profiles use scores specific of the position of amino acids (or nucleotides) and position specific scores for the opening or the expansion of an insertion or deletion. Methods for the creation of profiles, starting from multiple alignments, have been introduced by Taylor (1986), Gribskov et al. (1987), Barton (1990) and Heinikoff (1996).

HMMs provide an utterly probabilistic description of profiles, i.e. Bayes' theory rules the positioning of all probability (evaluation) parameters (compare Krogh et al. 1994, Eddy 1996 and Eddy 1998). The central idea behind this is that a HMM is a finite model describing the probability distribution of an infinite number of possible sequences. The HMM consists of a number of states corresponding with the columns of a multiple alignment as it is usually depicted. Each state emits symbols (remainders) corresponding with the probability of the sym- bol emission (specific of the respective state), and the states are linked with each other by probabilities of the changing of states. Starting from one specific state, a succession of states is generated by. changing from one state to the other in accordance with the probability, of the changing of states, until a final state has been reached. Each state then emits symbols according to the probability distribution of emissions specific of this state, creating an observable sequence of symbols.

The attribute „hidden" has been derived from the fact that the underlying sequence of states cannot be observed. Only the sequence of symbols is visible. An assessment of the probabilities of changing of states and of emissions (the training of the model) is achieved by dynamic programming algorithms implemented in the HMMER package.

The sequences according to the invention may be aligned with other nuclear receptor sequences in order to create a multiple sequence alignment which is used as the basis for the creation of a HMM.

If an existing HMM and a sequence are given, the probability that the HMM could generate the sequence in question, can be calculated. The HMMER package provides a numerical quantity (the Score) in proportion to this probability, i.e. the information content of the sequence indicated as bits, measured according to the HMM.

See also Barton, G.J. (1990): Protein multiple alignment and flexible pattern matching, Methods Ezymol. 183: 403-427, Eddy, S.R. (1996): Hidden markov models. Curr. Opin. Strct. Biol. 6: 361-365, Eddy, S.R. (1998): Profile hidden markov models. Bioinformatics. 14: 755- 763, Gribskov, M. McLachlan, A.D. and Eisenberg D. (1987): Profile analysis: Detection of distantly related proteins. Proc. Natl. Acad. Sci. USA. 84: 4355-5358, Heinikoff, S. (1996): Scores for sequence searches and alignment, Curr. Opin. Strct. Biol. 6: 353-360, Krogh, A., Brown, M., Mian, I.S., Sjolander, K. and Haussler, D. (1994): Hidden markov models in computational biology: Applications to protein modelling. J. Mol. Biol. 235: 1501-1531, Taylor, W.R. (1986): Identification of protein sequence homology by consensus template alignment. J. Mol. Biol. 188: 233-258.

In general the sequence are selected such that a query using a search sequence returns a result consisting of sequences which are at least to a certain degree similar to the query sequence. In one embodiment of the invention amino acid sequences of the present invention are used to model the three-dimensional structure of the protein. Initially, this involves the comparison of the protein sequence with the sequence of related proteins where the structure is known, such as the human PPARα ligand-binding domain (Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV, Nature 1998 Sep 10;395(6698): 137-43). The three-dimensional structure can then be modelled using computer programs. From the three-dimensional structure, binding sites of potential inhibitors or activators can be predicted. It can further be predicted which kinds of molecule might bind there. The predicted substances can then be screened to test their effect on nuclear receptor activity.

The following examples are provided for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any manner. The invention will now be further described in the following examples with respect to the accompanying figures and the sequence protocol, in which

Figure 1 shows the cloning of npFXRb a, npFXRb genome locus. Murine npFXRb loci show similar exon/intron structures. The murine locus is displayed. It consists of at least 11 exons spanning 26kb of genomic DNA. Intron positions are conserved compared to FXRa. RT-PCR and RACE experiments verified 10 coding exons (striped boxes represent DBD, grey boxes LBD,* stop codon, arrow start codon) translating into a protein of 505 amino acids. The 5 'untranslated region is encoded by exonl,. while exon 11 encodes the 3 'untranslated region (black boxes) and is flanked by an poly(A)signal. Human FXRb contains two stop codons in exonl 1 and three frameshifts at exon/intron junctions (indicated by arrows), verified by sequencing of 3 independent genome loci and cDNAs. Untranslated regions are not verified for human locus, b, Splice variants of murine FXRb. Five splice variants of mFXRb were isolated by RT-PCR from adult liver and testis RNA. Isoforms differentially splice out parts of exon 3, 8 or 10 and thereby differ in the regions encoding the DBD (striped boxes) and the LBD (grey boxes). Numbers indicate exons.

Figure 2 shows the evolutionary analysis of FXRs a) Phylogenetic tree connecting FXRa and FXRb genes from different species. Numbers indicate the bootstrap support (out of 1,000) for the respective nodes. Notice also the longer branches in the cluster of mammalian FXRb se- quences compared to mammalian FXRa. * indicates putatively non-functional proteins in primates, b) Selection pressure on DBD and LBD of nuclear receptors. Table entries are the ratios of non-synonymous to synonymous substitution rates (Ka/Ks). Purifying selection causes a shift of these values towards zero. Comparisons were made between rat, mouse, and human sequences, except where indicated otherwise. Entries are ordered according to their Ka/Ks ratio in the LBD.

Figure 3 depicts the tissue distribution of murine FXRs. Transcription of FXRa and b was assayed by RT-PCR experiments amplifying a part of the LBD with 28 or 35 PCR cycles, respectively. Actin served as comparative control. Murine RNAs from indicated embryonic and adult tissues were used as template. M: DNA size marker.

Figure 4 represents mFXRb ligands. a) Lanosterol specifically induces cofactor interactions of mFXRb but not FXRa in HTRF at physiological concentrations. GSTmFXRbLBD and biotinylated mSRCl peptide were mixed in a reaction buffer and the effect of lanosterol and the FXRa agonist GW4064 on the interaction was assayed by HTRF (see Methods). Cofactor interaction is induced by lanosterol (EC50 value of ImM) whereas GW4064 only acts as a weak agonist. GSThFXRaLBD was mixed in a reaction buffer with biotinylated human his tagged Tif2 (aa 548-878) and the effect of lanosterol and GW4064 on interaction assayed by HTRF (see Methods). The known FXRa agonist GW4064 induces cofactor interaction (EC50 value of 70nM) whereas lanosterol does not show an effect. The ratio of 665nm acceptor signal versus 615 nm donor signal was multiplied by 1000 and the mean value of triplicates was plotted against log concentrations of lanosterol (in μM). Brackets indicate standard deviation values between triplicates, b) A diverse set of compounds induce cofactor interaction with mFXRb but not FXRa. A group of 100 compounds were evaluated for inducing SCR1 interaction with mFXRb and h FXRa in a HTRF assay (see Methods). Shown are EC₅o values and the relative efficacy of SRC 1 recruitment (efficacy of lanosterol set to 100%) calculated from dose response experiments for a selected group of compounds found to be active for FXRb. These compounds have no or very low (pregnenolone, cholestensteraloid, data not shown) efficacy in FXRa assays.

SEQ ID No: 1 to 6 depicts DNA-sequences of npFXRb and splicing variants thereof (cf. Table 1). SEQ ID No: 7 to 24 depict sequences of primers that were used for the following experiments

(cf. Table 2).

SEQ ID No: 25 depicts the amino-acid sequence of npFXRb.

EXAMPLES

Example 1: Bioinformatic Analysis

A database of all nuclear receptor protein sequences was constructed and used to search the daily human genome updates (Genbank), employing the BLASTX tool with default parameters. Two npFXRb containing BACs were identified, genbanknew: AC026039 and emblnew: AL390235.

Evolutionary Analysis. Phylogenetic trees were calculated on the basis of protein alignments using PHYLIP (Felsenstein, J. PHYLIP (phylogeny interference package). Distributed by the author. (1993)). More specifically, pairwise distances using the PAM250 substitution matrix were computed from 1,000 bootstrapped data sets. These distance matrices served as input to the neighbour algorithm, the output of which was in turn used as the input to the computation of the consensus tree. Finally, the Fitch-Margolisch algorithm was used to fit branch lengths to the consensus tree. Codon-specific DNA sequence alignments were generated by translating the cDNA sequences aligning the translations and backtranslating them based on the triplets in the original cDNA. These sequence manipulations were carried out using the EMBOSS bioinformatics software package (Rice, P., Bleasy, A. EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics 16, 276-277 (2000).). Substitution rates were calculated according to the Method by Nei and Gojobori (Nei, M., Gojobori, T. Simple methods for estimating the numbers of synonymous and non-synonymous nucleotide substitutions. Molecular Biology and Evolution 3, 418-426 (1986)). as implemented in the program DnaSP (Rozas, J., Rozas, R. DnaSP version 3: an integrated program for molecular population genetics and evolutionary analysis. Bioinformatics 15, 174-175 (1999)). , We used a stan-r dard chi-square test to assess the null hypothesis that the rate of synonymous substitutions is equal to the rate of non-synonymous substitutions.

DNA Cloning. Human npFXRb cDNAs were isolated by RT-PCR on adult human testis RNA . (Clontech) using primers 5'

CCAGACCAACCTATTCTTCCTCGAGAAATAAGGGAC 3' (SEQ ID No.: 7) and 5' TGGGGTCCTTTGTTTTCCAAGTGCTAAGTATTTCTG 3' (SEQ ID No.: 8) in 35 PCR cycles. Nested PCR was run with 30 cycles and primers 5' CCAGACCAACCTATTCTTCCTCGAGAAATAAGGGAC 3' (SEQ ID No.: 9) and 5' GTTCTCAGTTCAGTATGCTTCCATATGAGATGGGC 3' (SEQ ID No.: 10). npFXRb cDNAs from all other species were isolated using adult liver and testis RNA from the respective species and primers 5' TGGGGTCCTTTGTTTTCCA 3' (SEQ ID No.: 11) and 5' GTGAAATGGACATGTACATGCG 3^' (SEQ ID No.: 12) in 35 PCR cycles. Full length cDNAs from mouse and human npFXRb transcripts including untranslated regions were isolated by RACE using GeneRacer™ kit (Invitrogen). For nested RACE experiments the following primers were used: human 3' RACE: 5' GGCCAATGAGGATCAAACTGCACTACAGAAGGGA 3 ' (SEQ ID No.: 13) and 5' AAACTGAAGTGATATTTCTCCATGGGGCCCAAC 3'; (SEQ ID No.: 14) human 5' RACE: 5' TCCCTTCTGTAGTGCAGTTTGATCCTCATTGGCC 3' (SEQ ID No.: 15) and 5' ACGTATGTGTAGGACTGCTGTCTCTGAGAGTTGC 3 ' (SEQ ID No.: 16); mouse 5' RACE: 5' CTTCCACTTGGATGGCAGGGTACAG.GGCAGGC 3 ' (SEQ ID No.: 17) and 5' GCCAACATCCCCACCGCCTTACACTTCTTCAG 3' (SEQ ID No.: 18); mouse 3' RACE: 5' CCATCCAGAAGACCCACAGCATTTCGCCCACC 3' (SEQ ID No.: 19) and 5' GGGAGGCTTACTGAACTGAGAACTCTGAGTCACAGC 3' (SEQ ID No.: 20). (Accession numbers are currently being processed by GenBank). For the amplification of primate npFXRb sequences, same PCR primers were used on genomic DNA and RNA from the respective species as kindly provided by S. Paabo. To isolate the murine genome locus, the BAG library Easy-to-Screen DNA Pools: BAC Mouse ES (Release II) from Incyte Genomics was screened with above described murine PCR primers. BAC clones from plate 427 P8 and 476 H24 were purchased and the npFXRb locus sequenced using PCR products generated with above described primers. Flanking regions were sequenced by primer walking.

Expression analysis. For RT-PCR expression analysis of human npFXRb we used primers and PCR conditions as described above on a panel of human adult and embryonic RNAs (Clontech). RT-PCR expression analysis of murine npFXRb was performed using primers 5' TCATCCAGCACCAGATCTGGGAAAG 3' (SEQ ID No.: 21) and 5' GTCCTTTGTTTTCCACATGCGAAGG 3' (SEQ ID No.: 22), for mnpFXRa 5' GGGATGTTGGCTGAATGTTTGTTAACTG 3' (SEQ ID No.: 23) and 5' TCACTGCACATCCCAGATCTCACAG 5' (SEQ ID No.: 24) in 35 and 28 cycles PCR cycles, respectively, with a panel of RNA from adult (Clontech) and embryonic RNAs (Quantum Appligene). Plasmid construction and protein purification. A gateway cassette (Life technologies) was cloned into the pAGGHLT Polylinker (Pharmingen) and PCR fragments of the mnpFXRb and the hLXRa LBDs amplified from cDNA were recombinated into this construct to be used in Pharmingenes Baculovirus Expression vector system. For E. coli expression of a GST fusion of the LBD of hFXRa and a His tagged NR interaction domain of hTif2 respective PCR fragments amplified from human cDNA were recombinated into pDest 15 and pDestl7 (Life technologies). Transformed E. coli BL21 strains were cultured in LB medium with lOOμg/ml Ampicillin at 37°C to a OD600 of 0.4-0,8. 0,4 mM IPTG was added and growth continued at 30°c for 6-8 h. Cells were harvested for 10 min at 5000 x g and used for GST or His affinity purification according to the recommended procedures from Pharmacia and QIAGEN. Recombinant baculovirus was constructed using the Pharmingen Baculovirus Expression vector system as recommended. SF9 cells were infected by the virus and cells were harvested 3 days after infection. His affinity purification was performed as recommended by Pharmingene but cells were lysed on ice by 5 subsequent sonication pulses using a sonicator needle. For all purifications detergent was omitted from all buffers.

HTRF. His-hTif2BD was biotinylated using the Biotin protein labelling Kit from Roche. Bi- otinylation was performed in 20mM Tris/HCl pH 7.5, 150mM KCI instead of the recommended buffer. Proteins and compounds were mixed with 1 nM Europium-cryptate-antiGST antibody and lOOng Streptavidin-Allophycocyanin in black 384 well microtiterplates (NUNC Fluotrac200). Reaction volume was 25 μl. Plates were shaken for 2 minutes at 800 rpm_. and incubated for 60 minutes at room temperature. Measurement of fluorescence from both fluo- rophores occurred in a time window from 50-400μs after excitation on a Victor V HTRF Reader. Ratios, multiplied by a factor of 1000 of the emission intensity at 665nm and at 615nm were used for plotting. EC50 values were calculated automatically by Prism (Graph- Pad Software Inc.)

Table 1 : DNA-sequences and Protein sequence of npFXRb and splicing variants thereof

Seq ID No. Sequence 5' -> 3'

Seq ID No.l bCTCATTCCATGAGGAGGAGGGCAGACTGCAGGAACACTACCCAATT- GTTTTGTTCTGAAAGTGTTTAGCATTGCTGAAGGAAACAACTAGAAGC

(mmusfull TGGACCAGAGAATGGCAAATACCTATGTTGCTACGTCTGATGGGTACT length) ACCTCGCTGAACCCACACAGTATTATGATATTTTGCCAGAACAATTCC ATTATCAGCTGTGTGATACAGATTTCCAAGAACCACCCTATTGTCAAT ATTCTACCGCTCAGTTTCCTCCAGCGTTACAGTCCCCATCTTTACAAA GTCATTTCAACACACATGGCTTGGATCCACAGTACAGTGGAGGCAGTT

GGTGTGGACTCGACGCTCGAGAATCTGGTCAGTCCACTTATGTGGTTG

TTCAGGATGATGAAGATGAATTCCCTGGGGCACAAAGGTGCAGAGCAA

CTTGTTCTTTACGCTGGAAGGGTCAAGATGACATGCTCTGCATGGTCT

GCGGTGATAAGGCATCAGGATATCACTACAATGCAGTTACTTGTGAGG

GGTGCAAAGGCTTTTTCCGGCGTAgCATTACCAAGAATGCAGTGTAT-

TCTTGCAAGAACGGTGGTCACTGTGAAATGGACATGTACATGCGCAGA

AAATGCCAAGAGTGCAGACTGAAGAAGTGTaAGGCGGTGGGGATGTT-

GGCAGAATGTTTGCTCACAGAGATCCAGTGTAAGTCAAAGAGACTTCG

CAAGAACTTCAAGCACGGGCCTGCCCTGTACCCTGCCATCCAAGTGGA

AGATGAAGGAGCAGACACCAAACACG.TGTCATCCAGCACCAGATCTGG

GAAAGGGGTTCAGGACAACATGACTCTAACTCAAGAGGAACATCGGCT

TCTGAATACCATAGTGACTGCTCACCAAAAATCCATGATTCCCTTGGG

AGAAACAAGCAAACTTCTGCAGGAGGGTTCCAACCCCGAACTAAGTTT

TCTGAGACTCTCAGAGGTATCAGTCCTGCACATACAAGGGCTAATGAA

GTTTACCAAGGGACTCCCAGGATTTGAAAATTTAACCACTGAGGATCA

GGCTGCATTACAGAAGGCGTCAAAAACTGAAGTGATGTTCCTTCATGT

AGCCCAGCTTTATGGTGGGAAAGACTCAACCTCTGGAAGTACTATGAG

ACCAGCAAAGCCCTCAGCTGGGACACTAGAGGTGCATAATCCTAGCGC

TGATGAAAGTGTTCATTCTCcGGAAAACTTTcTCAAGGAAGGcTACC-

CTTCGGCTCCTCTaaCtGATATTACTAAAgAAtTTATTGCcTcaC-

TAtcTTACTTcTaCAGAAGAATGAGTGAACTTCATGTATCGGATACT-

GAATATGCTCTGCTTACGGCGACAACAGTGCTTTTCTCAGATCGTCCA

TGCCTTAAAAATAAGCAGCATATAGAAAACCTACAAGAACCAGTCCTG

CAACTTTTGTTTAAGTTTTCAAAAATGTACCATCCAGAAGACCCACAG

CATTTCGCCCACCTCATAGGGAGGCTTACTGAACTGAGAACTCTGAGT

CACAGCCACTCTGAAATCctTCGCATGTGGAAAACAAAGGACCCCAG-

GTTGGTGATGTTATTCTCTGAGAAATGGGATCTGCACTCATTTTCCTG

AAAATTTACAGTGGTGAGACTTGCCTTAAAATTTCAAAATCAAAATTG

GATGAATGATTTGAAAAAAATGTGATAACTCAAAATCATTGAGTTATC

ACACATCACCTTAA/-ATGGAGATCCAAAAGGCTCATCAGGGTTTGTGG

AACTTTCAACTCTTAGAAGATGAACACTCCAGGATTCATCTCAAGATT

GGATGCAAAGGAAAAATGGAGCCATGTCGTGGCATAGGCTCTGTACTT

GAGAGACAGAGACAAGAGGACCCCATGTTTAAGGCAGTCCTGGGCTAT

GTAGCGAGACCTCATCTCAGAAGAAAAAAGAGAAGAACTGATGGCCAT

CATTTTGTCTCCAATTCTCACCTAAAGAAACTAATTTTTTTGTTGTTG

TTGTTTAGCTGAAACACATAATGTGTA-AATGTGAACACAGGTAGGTTA

TGGTTAGGATAAGGCAGTTTGTATGCTTCACTGGTTAAGGACACATGG

ATGGTGTAGCAGCTAATGCTTAGAACCTCTTCTTCATGAATGATTAAC

GAGGAACAAGCCTTTTCAAATTGTATCTGCTTTATATTTTTAATAAGT

ATGTGAT ATTGT CAGAATAGTCAATATTGTACAGAAT TTTCAAT

TGGGAATTTCATGTGTATATATTAGATATTTCTGGCATATTTGCCACC

CACCCTGTGATGCCCACACTGCCAATGCCCTTCTCATCCCCCTAACAT

TTCCCTTCCTCTGCCTTGCCCTAAAACACCAGATTCATACGTGCAAGA

AAACATTTTTTAAACAAATTTTATTAGATATTTTCTTCATTTACAATT

CAAATGCTATCCCAAAAGTCCCTTATTCCCTCCCCTTATCCTGCTCTC

CAACCCACCCACTTCTGCTTCCTGGCCCTGTTATTCCCCTGTATTGGG

GTATATAATCTTTGCAAGACCAAGGGCCTCTCCTCCCAACGATGGTCC

ATTAGGCCATCTGCAAGAAAACATTTGATTCCAGTCTGAGTCTGTTTT

ATTTCCTTTGAGTATGACTATCTCGTCAATATCTGCAAATGACATAAT

TTCATTCCTCTTAAGGATGGAAAGGACTGGATTGTGTATGTATATCTG

TATATATTATTTTATTTATCCACCTGCTGATGACACCTAGTTTGATTC TGTATCTTGGCCACTGT.GAATCGTGCTATAATAAACCTATGTAGGTT- TTTAGATTTTAAAaaaaaAAAAAAAAAAAAAAAAAAA . .

Seq ID No.2 ATGGCAAATACCTATGTTGCTACGTCTGATGGGTACTACCTCGCTGAA

CCCACACAGTATTATGATATTTTGCCAGAACAATTCCATTATCAGCTG Mmusl TGTGATACAGATTTCCAAGAACCACCCTATTGTCAATATTCTACCGCT

CAGTTTCCTCCAGCGTTACAGTCCCCATCTTTACAAAGTCATTTCAAC

ACACATGGCTTGGATCCACAGTACAGTGGAGGCAGTTGGTGTGGACTC

GACGCTCGAGAATCTGGTCAGTCCACTTATGTGGTTGTTCACGATGAT

GAAGATGAATTCCCTGGGGCACAAAGGTGCAGAGCAACTTGTTCTTTA

CGCTGGAAGGGTCAAGATGACATGCTCTGCATGGTCTGCGGTGATAAG

GCATCAGGATATCACTACAATGCACTTACTTGTGAGGGGTGCAAAGG-

CTTTTTCCGGCGTAgCATTACCAAGAATGCAGTGTATTCTTGCAA-.

GAACGGTGGTCACTGTGAAATGGACATGTACATGCGCAGAAAATGCCA

AGAGTGCAGACTGAAGAAGTGTaAGGCGGTGGGGATGTTGGCAGAAT-

GTTTGCTCACAGAGATCCAGTGTAAGTCAAAGAGACTTCGCAAGAACT

TCAAGCACGGGCCTGCCCTGTACCCTGCCATCCAAGTGGAAGATGAAG

GAGCAGACACCAAACACGTGTCATCCAGCACCAGATCTGGGAAAGGGG

TTCAGGACAACATGACTCTAACTCAAGAGGAACATCGGCTTCTGAATA

CCATAGTGACTGCTCAGCAAAAATCCATGATTCCCTTGGGAGAAACAA

GCAAACTTCTGCAGGAGGGTTCCAACCCCGAACTAAGTTTTCTGAGAC

TCTCAGAGGTATCAGTCCTGCACATACAAGGGCTAATGAAGTTTACCA

AGGGACTCCCAGGATTTGAAAATTTAACCACTGAGG TCAGGCTGCAT

TACAGAAGGCGTCAAAAACTGAAGTGATGTTCCTTCATGTAGCCCAGC

TTTATGGTGGGAAAGACTCAACCTCTGGAAGTACTATGAGACCAGCAA

AGCCCTCAGCTGGGACACTAGAGGTGCATAATCCTAGCGCTGAT-

GAAAGTGTTCATTCTCcGGAAAACTTTcTCAAGGAAGGcTACCCT-

TCGGCTCCTCTaaCtGATATTACTAAAgAAtTTATTGCcTcaCTAtc-

TTACTTcTaCAGAAGAATGAGTGAACTTCATGTATCGGATACTGA-

ATATGCTCTGCTTACGGCGACAACAGTGCTTTTCTCAGATCGTCCATG

CCTT-AAAAATAAGCAGCATATAGAAAACCTACAAGAACCAGTCCTGCA

ACTTTTGTTTAAGTTTTCAAAAATGTACCATCCAGAAGACCCACAGCA

TTTCGCCCACCTCATAGGGAGGCTTACTGAACTGAGAACTCTGAGTCA

CAGCCACTCTGAAATCctTCGCATGTG^'GAAAACAAAGGACCCCAG-

GTTGGTGATGTTATTCTCTGAGAAATGGGATCTGCACTCATTTTCCTG-

A

Seq ID No.3 ATGGCAAATACCTATGTTGCTACGTCTGATGGGTACTACCTCGCTGAA CCCACACAGTATTATGATATTTTGCCAGAACAATTCCATTATCAGCTG Mmus2 TGTGATACAGATTTCCAAGAACCACCCTATTGTCAATATTCTACCGCT CAGTTTCCTCCAGCGTTACAGTCCCCATCTTTACAAAGTCATTTCAAC ACACATGGCTTGGATCCACAGTACAGTGGAGGCAGTTGGTGTGGACTC GACGCTCGAGAATCTGGTCAGTCCACTTATGTGGTTGTTCACGATGAT GAAGATGAATTCCCTGGGGCACAAAGGTGCAGAGCAACTTGTTCTTTA CGCTGGAAGGGTCAAGATGACATGCTCTGCATGGTCTGCGGTGATAAG GCATCAGGATATCACTACAATGCACTTACTTGTGAGGGGTGCAAAGGC TTTTTCCGGCGTAGCATTACCAAGAATGCAGTGTATTCTTGCAAGAAC GGTGGTCACTGTGAAATGGACATGTACATGCGCAGAAAATGCCAAGAG TGCAGACTGAAGAAGTGTAAGGCGGTGGGGATGTTGGCAGAATGTTTG CTCACAGAGATCCAGTGTAAGTCAAAGAGACTTCGCAAGAACTTCAAG CACGGGCCTGCCCTGTACCCTGCCATCCAAGTGGAAGATGAAGGAGCA GACACCAAACACGTGTCATCCAGCACCAGATCTGGGAAAGGGGTTCAG GACAACATGACTCTAACTCAAGAGGAACATCGGCTTCTGAATACCATA GTGACTGCTCACCAAAAATCCATGATTCCCTTGGGAGAAACAAGCAAA. CTTCTGCAGGAGGGTTCCAACCCCGAACTAAGTTTTCTGAGACTCTCA GAGGTATCAGTCCTGCACATACAAGGGCTAATGAAGTTTACCAAGGGA CTCCCAGGATTTGAAAATTTAACCACTGAGGATCAGGCTGCATTACAG AAGGCGTCAAAAACTGAAGTGATGTTCCTTCATGTAGCCCAGCTTTAT GGTACTATGAGACCAGCAAAGCCCTCAGCTGGGACACTAGAGGTGeAT AATCCTAGCGCTGATGAAAGTGTTCATTCTCCGGAAAACTTTCTCAAG GAAGGCTACCCTTCGGCTCCTCTAACTGATATTACTAAAGAATTTATT GCCTCACTATCTTACTTCTAC.AGAAGAATGAGTGAACTTCATGTATCG GATACTGAATATGCTCTGCTTACGGCGACAACAGTGCTTTTCTCAGAT CGTCCATGCCTTAAAAATAAGCAGCATATAGAAAACCTACAAGAACCA GTCCTGCAACTTTTGTTTAAGTTTTCAAAAATGTACCATCCAGAAGAC CCACAGCATTTCGCCCACCTCATAGGGAGGCTTACTGAACTGAGAACT CTGAGTCACAGCCACTCTGAAATCCTTCGCATGTGGAAAACAAAGGAC CCCAGGTTGGTGATGTTATTCTCTGAGAAATGGGATCTGCACTCATTT TCCTGA

Seq ID No.4 ATGGCAAATACCTATGTTGCTACGTCTGATGGGTACTACCTCGCTGAA

CCCACACAGTATTATGATATTTTGCCAGAACAATTCCATTATCAGCTG Mmus3 TGTGATACAGATTTCCAAGAACCACCCTATTGTCAATATTCTACCGCT

CAGTTTCCTCCAGCGTTACAGTCCCCATCTTTACAAAGTCATTTCAAC

ACACATGGCTTGGATCCACAGTACAGTGGAGGCAGTTGGTGTGGACTC

GACGCTCGAGAATCTGGTCAGTCCACTTATGTGGTTGTTCACGATGAT

GAAGATGAATTCCCTGGGGCACAAAGGTGCAGAGCAACTTGTTCTTTA

CGCTGGAAGGGTCAAGATGACATGCTCTGCATGGTCTGCGGTGATAAG

GCATCAGGATATCACTACAATGCACTTACTTGTGAGGGGTGCAAAGG-

CTTTTTCCGGCGTAgCATTACCAAGAATGCAGTGTATTCTTGCAA-

GAACGGTGGTCACTGTGAAATGGACATGTACATGCGCAGAAAATGCCA

AGAGTGCAGACTGAAGAAGTGTaAGGCGGTGGGGATGTTGGCAGAAT-

GTTTGCTCACAGAGATCCAGTGTAAGTCAAAGAGACTTC^'GCAAGAACT

TCAAGCACGGGCCTGCCCTGTACCCTGCCATCCAAGTGGAAGATGAAG

GAGCAGACACCAAACACGTGTCATCCAGCACCAGATCTGGGAAAGGGG

TTCAGGACAACATGACTCTAACTCAAGAGGAACATCGGCTTCTGAATA

CCATAGTGACTGCTCACCAAAAATCCATGATTCCCTTGGGAGAAACAA

GCAAACTTCTGCAGGAGGGTTCCAACCCCGAACTAAGTTTTCTGAGAC

TCTCAGAGGTATCAGTCCTGCACATACAAGGGCTAATGAAGTTTACCA

AGGGACTCCCAGGATTTGAAAATTTAACCACTGAGGATCAGGCTGCAT

TACAGAAGGCGTCAAAAACTGAAGTGATGTTCCTTCATGTAGCCCAGC

TTTATGGTGGGAAAGACTCAACCTCTGGAAGTACTATGAGACCAGCAA

AGCCCTCAGCTGGGACACTAGAGGTGCATAATCCTAGCGCTGAT-

GAAAGTGTTCATTCTCcGGAAAACTTTcTCAAGGAAGGcTACCCT-

TCGGCTCCTCTaaCtGAAGAATGAGTGAACTTCATGTATCGGATACT-

GAATATGCTCTGCTTACGGCGACAACAGTGCTTTTCTCAGATCGTCCA

TGCCTTAAAAATAAGCAGCATATAGAAAACCTACAAGAAC^'CAGTCCTG

CAACTTTTGTTTAAGTTTTCAAAAATGTACCATCCAGAAGACCCACAG

CATT.TCGCCCACCTCATAGGGAGGCTTACTGAACTGAGAACTCTGAGT

CACAGCCACTCTGAAATCctTCGCATGTGGAAAACAAAGGACCCCAG-

GTTGGTGATGTTATTCTCTGAGAAATGGGATCTGCAGTCATTTTCCTG

A

Seq ID No.5 ATGGCAAATACCTATGTTGCTACGTCTGATGGGTACTACCTCGCTGAA CCCACACAGTATTATGATATTTTGCCAGAACAATTCCATTATCAGCTG Mmus4 TGTGATACAGATTTCCAAGAACCACCCTATTGTCAATATTCTACCGCT CAGTTTCCTCCAGCGTTACAGTCCCCATCTTTACAAAGTCATTTCAAC ACACATGGCTTGGATCCACAGTACAGTGGAGGCAGTTGGTGTGGACTC GACGCTCGAGAATCTGGTCAGTCCACTTATGTGGTTGTTCACGAT-

GATGAAGATGAaTTCCCTGGGGCACAAAGGTGCAgAGCAACTTGTTC-

TTTACGCTGgAAGGGTCAAGATGACATGCTCTGCATGGTCTGCGGT-

GATAAGGCATCAGGATATCACTACAATGCACTTACTTGTGAGGGGTGC

AAAGGCTTTTTCCGGCGTAGCATTACCAAGAATGCAGTGTATTCTTGC

AAGAACGGTGGTCACTGTGAAATGGACATGTACATGCGCAGAAAATGC

CAAGAGTGCAGACTGAAGAAGTGTAAGGCGGTGGGGATGTTGGCAGAA

TGTTTGCTCACAGAGATCCAGTGTAAGTCAAAGAGACTTCGCAAGAAC

TTCAAGCACGGGCCTGCCCTGTACCCTGCCATCCAAGTGGAAGATGAA

GGAGCAGACACCAAACACGTGTCATCCAGCACCAGATCTGGGAAAGGG.

GTTCAGGACAACATGACTCTAACTCAAGAGGAACATCGGCTTCTGAAT

ACCATAGTGACTGCTCACCAAAAATCCATGATTCCCTTGGGAGAAACA

AGCAAACTTCTGCAGGAGGGTTCCAACCCCGAACTAAGTTTTCTGAGA

CTCTCAGAGGTATCAGTCCTGCACATACAAGGGCTAATGAAGTTTACC

AAGGGACTCCCAGGATTTGAAAATTTAACCACTGAGGATCAGGCTGCA

TTACAGAAGGCGTCAAAAACTGAAGTGATGTTCCTTCATGTAGCCCAG

CTTTATGGTACTATGAGACCAGCAAAGCCCTCAGCTGGGACACTAGAG

GTGCATAATCCTAGCGCTGATGAAAGTGTTCATTCTCCGGAAAACTTT

CTCAAGGAAGGCTACCCTTCGGCTCCTCTAACTGAAGAATGAGTGAAC

TTCATGTATCGGATACTGAATATGCTCTGCTTACGGCGACAACAGTGC

TTTTCTCAGATCGTCCATGCCTTAAAAATAAGCAGCATATAGAAAACC

TACAAGAACCAGTCCTGCAACTTTTGTTTAAGTTTTCAAAAATGTaC-

CATCCAGAAGACCCACAGCATTTCGCCCACCTCATAGGGAGGCTTACT

GAACTGAGAACTCTGAGTCACAGCCACTCTGAAAtCCTTCGCATGTG-^'

GAAAACAAAGGACCCCAGGTTGGTGATGTTATTCTCTGAGAAATGGGA

TCTGCACTCATTTTCCTGA ^{■ ■}

Seq ID No.6 ATGGCAAATACCTATGTTGCTACGTCTGATGGGTACTACCTCGCTGAA CCCACACAGTATTATGATATTTTGCCAGAACAATTCCATTATCAGCTG Mmus5 TGTGATACAGATTTCCAAGAACCACCCTATTGTCAATATTCTACCGCT CAGTTTCCTCCAGCGTTACAGTCCCCATCTTTACAAAGTCATTTCAAC ACACATGGCTTGGATCCACAGTACAGTGGAGGCAGTTGGTGTGGACTC GACGCTCGAGAATCTGGCTTTTTCCGGCGTAGCATTACCAAGAATGCA GTGTATTCTTGCAAGAACGGTGGTCACTGTGAAATGGACATGTACATG CGCAGAAAATGCCAAGAGTGCAGACTGAAGAAGTGTAAGGCGGTGGGG ATGTTGGCAGAATGTTTGCTCACAGAGATCCAGTGTAAGTCAAAGAGA CTTCGCAAGAACTTCAAGCACGGGCCTGCCCTGTACCCTGCCATCCAA GTGGAAGATGAAGGAGCAGACACCAAAC CGTGTCATCCAGCACCAGA TCTGGGAAAGGGGTTCAGGACAACATGACTCTAACTCAAGAGGAACAT CGGCTTCTGAATACCATAGTGACTGCTCACCAAAAATCCATGATTCCC TTGGGAGAAACAAGCAAACTTCTGCAGGAGGGTTCCAACCCCGAACTA AGTTTTCTGAGACTCTCAGAGGTATCAGTCCTGCACATACAAGGGCTA ATGAAGTTTACCAAGGGACTCCCAGGATTTGAAAATTTAACCACTGAG GATCAGGCTGCATTACAGAAGGCGTCAAAAACTGAAGTGATGTTCCTT CATGTAGCCCAGCTTTATGGTGGGAAAGACTCAACCTCTGGAAGTACT ATGAGACCAGCAAAGCCCTCAGCTGGGACACTAGAGGTGCATAATCCT AGCGCTGATGAAAGTGTTCATTCTCCGGAAAACTTTCTCAAGGAAGGC TACCCTTCGGCTCCTCTAACTGATATTACTAAAGAATTTATTGCCTCA CTATCTTACTTCTACAGAAGAATGAGTGAACTTCATGTATCGGATACT GAATATGCTCTGCTTACGGCGACAACAGTGCTTTTCTCAGATCGTCCA TGCCTTAAAAATAAGCAGCATATAGAAAACCTACAAGAACCAGTCCTG CAACTTTTGTTTAAGTTTTCAAA-AATGTACCATCCAGAAGACCCACAG CATTTCGCCCACCTCATAGGGAGGCTTACTGAACTGAGAACTCTGAGT. CACAGCCACTCTGAAATCCTTCGCATGTGGAAAACAAAGGACCCCAGG TTGGTGATGTTATTCTCTGAGAAATGGGATCTGCACTCATTTTCCTGA

Seq ID No.25 MANTYVATSDGYYLAEPTQYYDILPEQFHYQLCDTDFQEPPYCQ

YSTAQFPPALQSPSLQSHFNTHGLDPQYSGGSWCGLDARESGQS Mouse FXR TYVVVHDDEDEFPGAQRCRATCSLRWKGQDDMLCMNCGDKAS beta fullength GYHYNALTCEGCKGFFRRSITKNAVYSCKNGGHCEMDMYMRR

KCQECRLKKCKAVGMLAECLLTEIQCKSKRLRKNFKHGPALYP

AIQVEDEGADTKHVSSSTRSGKGVQDNMTLTQEEHRLLNTIVTA

HQKSMIPLGETSKLLQEGSNPELSFLRLSEVSVLHIQGLMKFTKG

LPGFENLTTEDQAALQKASKTEVMFLHVAQLYGGKDSTSGSTM

RPAKPSAGTLEVHNPSADESVHSPENFLKEGYPSAPLTDITKEFIA

SLSYFYRRMSELHVSDTEYALLTATTVLFSDRPCLKNKQHIENLQ

EPVLQLLFKFSKMYHPEDPQHFAHLIGRLTELRTLSHSHSEILRM.

WRTKDPRLVMLFSEKWDLHSFS

Table 2: Sequences of Primers that were used for the above experiments:

Claims

Claims:

1. A nucleic acid molecule coding for the non-primate nuclear receptor npFXRb or splice variants thereof, which is selected from the group comprising:

a) the nucleotide sequences set forth in SEQ ID NOs: 1 to 6 or the complements thereof; b) a nucleic acid which hybridizes to a nucleic acid of SEQ ID NOs: 1 to 6 under conditions of high stringency, and

2. A nucleic acid molecule according to claim 1, encoding a splice variant of the nuclear receptor npFXRb

3. A nucleic acid molecule comprising the nucleic acid molecule of any of claims 1 or 2 and a label attached thereto.

4. A vector, expression vector and/or genetic knock-out construct, comprising the nucleic acid molecule of claim 1 or 2.

5. A host cell transfected with the vector, expression vector and/or genetic knock-out construct of claim 4.

6. A non-primate host cell according to claim 5, lacking a functional npFXRb and/or splice variants thereof.

7. A non-primate transgenic animal, comprising a genetic knock-out construct according to claim 4.

8. A method of producing a polypeptide comprising the step of culturing the host cell of claim 5 in an appropriate culture medium to, thereby, produce the polypeptide.

9. An isolated polypeptide encoded by any portion of the nucleic acid of claim 1 or 2.

10. A polypeptide selected from the group comprising the amino acid sequence set forth in SEQ ID N0.25.

11. A method for identification for agents which are capable of modulating the cellular function of the non-primate nuclear receptor npFXRb or splice variants thereof, comprising the steps of: a) contacting one or more candidate agents with a polypeptide according to claim 9 or 10 and/or a host cell according to claim 5 or 6, b) removing unbound agent(s) c) detecting whether the agent(s) interact with said polypeptide of the nuclear receptor and/or said host cell.

12. A method for identification for agents according to claim 11, wherein the synthesis of lanosterol and cholesterol from lanosterol is detected and or' with developmental processes are detected.

13. An agent that modulates the cellular function of the non-primate nuclear receptor npFXRb or splice variants thereof, obtained through the method of claim 11.

14. Agent according to claim 13 that is lanosterol.

15. A method for the production of a pharmaceutical preparation, comprising a) the method according to claim 11 or 12, and b) admixing the agent with a pharmaceutically acceptable carrier.

16. A method for modulating the cellular function of the nuclear receptor, comprising the steps of: a) contacting a cell with a binding agent of a polypeptide according to claim 9 or 10, whereby the cellular function of npFXRb is modulated.

17. Use of a host cell according to claim 5 or 6. or a transgenic animal according to claim 7 for the identification of agents which are capable of modulating the cellular function of the non-primate nuclear receptor npFXRb or splice variants thereof.

18. Use of a transgenic animal, in particular a knock-out animal according to claim 7, for the analysis of agents, in particular according to claim 13 or 14, that modulate cholesterol, bile acids, and triglyceride synthesis.

19. Use according to claim 18, wherein the agent is further selected from FXRa modulating agents, LXRa or LXRb modulating agents, HNF4a, HNF4b, HNF4g modulating agents, PPARa, PPARb, PPARg modulating agents SCAP modulating agents, ACAT modulating agents, HMG-CoA-reductase modulating agents, and the like.

20. Use of an agent according to claim 13 or 14 for the modulation of the synthesis of lanosterol or cholesterol from lanosterol or bile acids or triglycerides in non-primate animals, modulation of developmental processes in non-primate animals,, and/or the modulation of the woolfat synthesis in e.g. sheep or lamas.

21. Use of an agent according to claim 13 or 14 for the fertility control of non-primate animals, including mice, rats, rabbits, frogs, birds, fish, non- vertebrates.