MXPA06009600A - Functional method for generating or screening for ligands which modulate steroid hormone receptors - Google Patents

Abstract

This application presents the discovery that the Vitamin D3 receptor (VDR), a member of the nuclear hormone ligand-activated receptor superfamily, interacts with a MNAR, a scaffolding protein. This interaction results in the formation of a ternary complex between VDR, MNAR, and the Src or PI3 kinase families of tyrosine kinases to mediate cell signaling, especially in osteoblast cells.

Description

FUNCTIONAL METHOD TO GENERATE OR SELECT LIGANDS THAT MODULATE RECEIVERS OF STEROID HORMONES FIELD OF THE INVENTION The present invention provides a method for functionally designing, or identifying by selection, selective ligands of the receptor. of vitamin D (VDR) that can be used for the treatment of osteoporosis and other disorders for which signaling has been implicated through the VDR. The invention relates to the discovery that the VDR interacts as MNAR, a scaffold protein previously demonstrated to be responsible for the interaction with other nuclear hormone receptors and because the ligand interaction between VDR and MNAR leads to the activation of the pathway of the Src / MAP kinase.

BACKGROUND OF THE INVENTION Nuclear hormone receptors Nuclear hormone receptors are a superfamily of ligand-inducible transcription factors, which, as a class, are involved in ligand-dependent transcriptional control of gene expression. The binding of a specific ligand, inducing conformational changes in the receptor molecule, affects the interaction of the receptor with other factors of REF: 175050 transcription and, in the end, the formation of the pre-initiation complex. This process regulates the rate or rate of gene transcription (D.J. Mangelsdorf, et al., Cell 1995; 83: 835-9). The superfamily of nuclear hormone receptors includes steroid hormone receptors, non-steroid hormone receptors and orphan receptors. The receptors for glucocorticoids (GR), mineral corticosteroids (MR), progestins (PR), androgens (AR), and estrogens (ER) are examples of classical steroid receptors. In addition to steroid hormone receptors, this superfamily of nuclear hormone receptors consists of receptors for non-steroidal hormones, such as vitamin D3, thyroid hormones, and retinoids. In addition, a range of sequences similar to the nuclear receptor has been identified, which code for the so-called "orphan" receptors. These orphan receptors are structurally related to and, therefore, classified as nuclear hormone receptors, although putative ligands have not yet been identified. (Cell 1995, 83: 851-857). The superfamilies of nuclear hormone receptors are structurally and functionally characterized by a modular structure in which they comprise six distinct structural and functional domains, A to F. More specifically, these receptors have a variable N-terminal region (A / B domain); followed by a highly conserved, centrally located DNA binding domain (hereafter referred to as DBD, domain C); a variable hinge region (domain D); a ligand binding domain, conserved, (hereinafter referred to as LBD, domain E); and a C-terminal variable region (F domain, Cell supra). The N-terminal region, which is highly variable in size and sequence, is poorly conserved among the different 'members of the superfamily. "This part of the receptor is involved in the modulation of transcription activation.The DBD consists of approximately 66 to 70 amino acids and is responsible for the binding activity DNA: This domain directs the receptor towards specific DNA sequences, called elements that respond to hormones, (hereinafter referred to as HRE) within the unit of control of the transcription of specific target genes on chromatin. Steroid receptors such as GR, MR, PR and AR recognize similar HRE DNA sequences, whereas ER recognizes a different sequence of HRE DNA. After DNA binding, it is thought that the steroid receptor interacts with the components of the basal transcriptional machinery and with sequence-specific transcription factors, which modulates the expression of specific target genes. The LBD is located in the C-terminal part of the receptor, and is primarily responsible for the ligand binding activity. In this way, LBD is essential for the recognition and binding of the hormone ligand and, in addition, it has a function of transcription activation, which determines the specificity and selectivity of the receptor's hormonal response. Although moderately conserved in structure, LBDs are known to vary considerably in homology between individual members of the nuclear hormone receptor superfamily. When a hormone ligand for a nuclear receptor enters the cell and is recognized by the LBD, it will bind to the specific receptor protein, thereby initiating an allosteric alteration of the receptor protein.

(Cell supra). As a result of this alteration, the ligand / receptor complex changes to a transcriptionally active state and, as such is able to bind through the presence of the DBD with high affinity to the corresponding HRE on the chromatin DNA. In this way, the ligand / receptor complex modulates the expression of the specific target genes. The diversity achieved by this receptor family results from its ability to respond to different ligands. In addition to the modulation of genomic activity, hormone receptor complexes can have important and varied non-genomic effects. This non-genomic activity is characterized by rapid and transient activation of some important signaling pathways that affect cellular functions, such as differentiation and proliferation. More generally, steroid hormone receptors are connected with embryonic development, homeostasis in adults and organ physiology. Various diseases and abnormalities are ascribed to a disturbance in the action of steroid hormones. Since steroid receptors exert their influence as modulators of hormone-activated transcription and as hormone-activated stimulators of non-genomic activity, further investigation into various procedures to modify, interact with or modulate these receptors is an area of immense significance . For example, mutations and defects in these receptors, as well as over-stimulation or blockage of these receptors, may provide a better insight into the underlying mechanism of the hormone signal transduction pathway, thereby leading to a increased efficacy in the treatment of a wide variety of diseases and abnormalities linked to the steroid receptor.

Vitamin D3 and VDR and cellular regulation The vitamin D3 receptor (VDR) belongs to the superfamily of nuclear hormone receptors. In the past few years, there has been a dramatic increase in evidence supporting the rapid signaling action of various vitamin V3 analogs (vit D3). Vitamin D3 has been shown to evolve transcellular movement of calcium through the cell membrane. The classical signaling pathway of vitamin D3 uses VDR, which is a transcription factor for target genes of vitamins D3. The effects of this pathway include the inhibition of cell development and cell invasion. The cytoplasmic signaling pathways are becoming increasingly recognized, which similarly can regulate the growth of differentiation but also apoptosis. Vitamin D3 inhibits the cell cycle at the Gl / S checkpoint by upregulating cyclin dependent p27 and p21 kinase inhibitors and by inhibiting cyclin Di. Indirect mechanisms include upregulation of TGF-β, and down regulation of the epidermal growth factor receptor. Vitamin D3 can induce apoptosis either indirectly through effects on the insulin-like growth receptor and tumor necrosis factor, more directly via the Bcl-2 family system, the ceramide pathway, the receptor death (for example, Fas) and protein kinase pathways activated by stress or tension (N-terminal kinase Jun and p38). Osteoporosis and other bone disorders. The development and homeostasis of bone is controlled to a large extent by two different cell types: osteoblasts and osteoclasts. The bone matrix is secreted by the osteoblasts, the cells that lie on the surface of the existing bone matrix and deposit fresh layers of bones on it. Mature osteoclasts are multinucleated cells of monocytic / macrophage origin that reabsorb the calcified bone matrix. Ordinarily, the activities of these two cell types are closely coordinated to maintain the structure and integrity of the bone in an organism. Nevertheless, the mechanisms that regulate the activities of these two cell types remain poorly understood and are largely unknown. A number of diseases and disorders are associated with abnormal bone growth or abnormal increase or decrease in bone mass. For example, osteopetrosis is a thickening of the bone matrix and has been associated with defects in the maturation of the osteoclasts, which renders them incapable of absorbing bone (see, for example, Kong et al., 1999, 397: 315-323).; Soriano et al., Cell 1991, 64: 693: 702; Iotsova et al., Nat. Med. 1997, 3: 1285-1289). In contrast, osteoporosis is a disease characterized by an increase in the activity of osteoclasts, resulting in bones that are extremely porous, easily fractured and slow to heal. Numerous other diseases and disorders that involve or are associated with abnormal bone growth and resorption are also known, including Paget's disease, osteogenesis imperfecta, fibrous displeasure. hypophosphatase, primary hyperparathyroidism, arthritis and periodontal disease, to name a few. In addition, osteolysis can be induced by many malignant tumors that reside in or are distant from bone, for example, skeletal metastases in cancers of the breast, lung, prostate, thyroid and kidney, humoral hypercalcemia during malignancy and multiple myelomas. Such diseases and disorders represent a major problem for public health in the United States and other countries. For example, it has been estimated that 10 million Americans, 80% of whom are women, are already affected with osteoporosis, while another 10 million individuals have low bone mass and are therefore at increased risk for the disease. There is, therefore, a need for methods and compositions that can be used to identify cells such as osteoblasts and / or osteoclasts (e.g., in samples of cells or tissues), and regulate or modulate the activities of such cells. There is also a need for methods and compositions for treating diseases and disorders associated with abnormal bone growth and resorption, including the diseases discussed above, for example, by modulating the activities of osteoblast and osteoclast cells. These and other needs in the art are addressed by the present invention. Vitamin D3 and bone remodeling. In osteoblasts, treatment with vitamin D3 leads to the activation of Src, phospholipase C and formation of inositol triphosphate. Vitamin D3 increases the expression of TGF-β and the TGF-β receptor, which play an important role in the coupling of bone formation with resorption, and with this the maintenance of bone mass. Vitamin D3 also interacts with the Smad signaling system, downstream of the activation of the TGF-β receptor. Vitamin D3 has an important impact on the local control of bone remodeling. It has antipro-life and prodifferential actions on the Osteoblasts, while playing a crucial role in bone resorption by stimulating the activity and formulation of osteoclasts. Contrary to osteoblasts, however, osteoclasts do not express VDR. Therefore, vitamin D3 regulates them indirectly, by controlling the expression of some cytokines and growth factors involved in the regulation of osteoclastogenesis and the function of osteoclasts. Vitamin D3 and skin. The vitamin D receptor has been detected in most skin cells, which means that keratinization, hair growth, melanogenesis, fibrogenesis, angiogenesis, and processes mediated by the immune system, are targeted potentials for vitamin D3. Vitamin D3 has been shown to regulate the function of melanocytes by suppressing tyrosinase activity (necessary for melanogenesis, or melanin production) in normal melanocytes (Abdel-Malek et al., J Cell Physiol., 1988; 136 (2) : 273-80). In addition, it has shown that physiological concentrations of vitamin D3 exhibit a growth inhibitory effect in primary melanocytes and conferred resistance to several inducers of programmed cell death, including tumor necrosis factor a and ultraviolet radiation (Sauer et al., Melanoma Res. 2003; 13 (4): 339-47). Vitamin D3 and cancer. The mechanisms of inhibition of tumor invasion and potential of metastasis that have been demonstrated, include the inhibition of serine proteins, metalloproteinases and angiogenesis. The lines of evidence for an effect of vitamin D3 in systemic cancer are the demonstration of the effects on cell growth, differentiation, apoptosis, invasion of malignant cells and metastasis; the epidemiological findings of an association of the appearance and production of cancers with derangements of vitamin D3 and its precursor, and the association of functional polymorphisms of VDR with the appearance of certain cancers. In addition, vitamin D3 analogues are being developed as cancer chemotherapy agents. There is accumulated evidence that the vitamin D3 / 1, 25 (OH) 2D3 / DVR axis is involved in malignant melanoma. Melanoma cells express VRD, and the antiproliferative and prolifferentiation effects of l, 25 (OH) 2D3 have been shown in cultured melanocytes, melanoma cells and melanoma genografts. Recently, an inhibitory effect on the dispersion of melanoma cells has been demonstrated. In addition, patients have been reported to have serum levels of 1, 25 (OH) 2D3, and VDR polymorphisms have been shown to be associated with the onset and production of malignant melanoma. As in other cancers, there is evidence of a protective effect of vitamin D3 in melanoma, but ultraviolet radiation, which is a major source of vitamin D3, is mutagenic.

Estrogen and regulation of ER and cellular Estrogen (E2) exerts numerous biological effects in different tissues, through an interaction with the ER. Analysis of the amino acid sequence, transient transfection studies and mutational dissections of ER indicate that ER have the classical modular structure described above. The terminal A / B domain of ER contains a transactivation function called transcriptional activation function 1 (TAF-1). The DBD contains two zinc fingers and is responsible for DNA recognition. The LBD and a second transactivation function, referred to as TAF-2, is located at the C-terminus of ER. After the link to the hormone, the ER undergoes an activation and a transformation step. The activated ER interacts with the specific elements of estrogen response (EREs) that are located in the promoter region of the genes regulated by estrogen and that influence its transcription of the target gene. In the last decade, numerous studies have provided a basic understanding of both ligand effects (agonist / antagonist) on ER and the relationship between ER structure and function. However, little is known about the mechanisms for the non-genomic activity of ER. ERß appears to be different from the more commonly known estrogen receptor, termed ERa. Collectively, ERa and ERß are referred to herein as ER. The DBD or the ERß is 90% identical to that of ERa. However, the complete homology between the ligand binding domain (LBD) of ERa and ERβ is less than 55%. As ERa, ERβ can stimulate transcription from an ERE in a "ligand-dependent manner." It has been established that estrogens induce rapid and transient increases in intracellular second messenger levels, including calcium and cyclic AMP (cAMP). , and that estrogen induces activation of mitogen-activated protein kinase (MAPK) and phospholipase C (Collins and Webb, 1999) .In fact, numerous studies have shown that estrogens induce rapid and transient activation of the pathway. Phosphorylation of the Src / Ras / MAP kinase Activation of this pathway triggers vital cellular functions that include cellular proliferation and differentiation.The time course of these acute events is paralleled by that promoted by the peptide hormones, supporting This way the hypothesis that these events do not involve the "classic" genomic action of estrogen.

Recent data suggest a direct connection between the estrogen receptor and the signaling cascade of the mitogen-activated protein kinase (MAP). MAP 'kinases are a family of serine-threonine kinases that are phosphorylated and activated to a variety of signals. These enzymes transduce extracellular signals from multiple membrane receptors to intracellular targets, including transcription factors, cytoskeletal proteins and enzymes. The family of MAP kinases includes the kinases related to the extracellular signal (ERKs), p38 and N-terminal kinase cJun that signal "through a pathway that involves sequential activation-of Ras, Raf and the mitogen-activated protein kinase (MEK).

(S.M. Thomas, J. S. Brugge, Annual Review of Cell &Developmental Biology 13, 513-609, 1997). In pulmonary endothelial cells, neuronal cells, osteoblasts and osteoclasts, it has been reported that 17β-estradiol (E2) rapidly activates the MAPK pathway, resulting in activities such as the induction of acute dilation of blood vessels, neuroprotection in primary cortical neurons after the excitotoxity of glutamate, and regulation of cell proliferation and differentiation in osteoclasts, leading to increased bone formation. In cell lines derived from breast cancer, E2 activates the signal transduction pathway of Src / Ras / Erk. The Src / Ras / Erk signaling pathway is a well-known target of growth factors. Importantly, activation of this pathway requires direct interaction of ER with Src. The activation of this pathway activates different cellular responses such as proliferation or differentiation.

Androgens and androgen receptors and cellular regulation. Androgen (AR) receptors are ligand-activated transcription factors that promote highly tissue-specific, highly selective effects through the regulation of divergent target genes.

Modulation of the non-genomic activity of the estrogen receptor (MNAR) The present inventors have recently identified a new scaffolding protein, designated as (non-genomic estrogen receptor activity modulator (MNAR).) MNAR interacts with the estrogen receptor (ER) and this interaction is increased by 17β-estradiol (E2). MNAR controls the interaction of ER with the members of the Src family of tyrosine-p60src (Src) and p56lck (Lck) kinases. This interaction induces the stimulation of the enzymatic activity of Src and the activation of the kinase pathway.

• MAP (Wong et al., Proc. Nati Acad Sci USA 2003; 99: 14783-14788). It has been shown that MNAR interacts with Src with the SH3 domain of SRc through a PXXP portion, and that ER interacts with Src via the SH2 domain of Src. It has also recently been shown that MNAR is a mediator of AR signaling similar to that for ER via a ternary complex of AR / MNAR / Src (Unni et al., Cancer Res. 2004; 64: 7156-68). These data suggest that MNAR can potentially play a more general role in the regulation of cellular processes. In addition, the structure-function analysis of MNAR indicates that MNAR is a scaffolding protein that allows the formation of multiple protein-protein contacts. The presence of several LXXLL portions in the MNAR molecule indicates that MNAR can accommodate the multiple NR bond. In addition to the interaction of ER using LXXLL, the interactions with PR, GR and AR were also confirmed. Furthermore, the presence of the proline-rich, extended portion and several classical PXXP portions suggest the potential interaction with proteins containing SH3 domains, which are present in multiple kinases and other signaling molecules. It has also recently been shown that MNAR interacts with the SH3 domains of Src and p85 - the regulatory subunit of the PI3 kinase. It has been well established that nuclear receptors can regulate cellular processes using the mechanism different from direct transcriptional regulation. ER, PR, AR and other nuclear receptors can activate multiple signaling cascades, which regulate the expression of critically important genes for cell proliferation, differentiation and survival. The previously presented data, as well as the analysis of the functional organization of -MNAR, suggest therefore that MNAR could potentially be, if not the main connection, at least one of the binding proteins that incorporates NR and other signaling cules that act in the cellular communication cascades. It is therefore reasonable to propose that MNAR can potentially represent an important objective for the development of functionally selective ligands of nuclear receptor ligands, which can regulate important cellular functions. It has previously been shown that VDR in the presence of vitamin D3 interacts with cSrc. It has also been previously shown that vitamin D3 increases the expression of osteocalcin and alkaline phosphatase in cells of the osteoblastic line. Accordingly, the present inventors have undertaken the evaluation of the effect of overexpression of MNAR on the expression of osteocalcin and alkaline phosphatase in osteosarcoma UMR 106 cells (ATCC CRL 1661 or ECACC 90111314) and ROS 17 / 2.8 cells. Considering that osteocalcin and alkaline phosphatase are critical markers for the differentiation of osteoblasts, any regulation by MNAR could implicate MNAR as an important regulator of bone development.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a novel interaction between the protein modulator of non-genomic activity of the nuclear receptors (MNAR) and the nuclear hormone receptor of vitamin D3 (VDR). Specifically, it has been discovered that VDR binds to the fifth "LXXLL" portion within the amino acid sequence of MNAR. Through this ligand-dependent interaction, the MNAR protein interacts with VDR and activates MAP kinase signaling via the formation of a ternary complex of VDR / MNAR / Src, leading to increased VDR transcriptional activity. In one embodiment, the present invention provides a method for identifying a ligand that modulates the interaction of MNAR with VDR, by adding a test compound to a reaction mixture of an MNAR polypeptide and a VDR polypeptide and evaluating the formation of a binding complex compared to the same reaction mixture in the absence of the test complex. It is contemplated that the reaction mixture is cell-free or cell-based. In a further embodiment, the method comprises detecting a complex between VDR and the test compound. The present invention also provides a method for identifying a ligand that modulates the activity of VDR in the presence of MNAR, by contacting a reaction mixture-containing an MNAR polypeptide, and a host cell comprising a functional VDR, and detecting VDR activity in the presence and absence of the test compound. In one embodiment, the activity is the phosphorylation of a MAP kinase. In a specific embodiment, the kinase is Erk 1 and / or Erk 2. In yet another embodiment, the activity is the expression of a gene induced by the activation of the VDR. In a specific modality, the gene is osteocalcin or alkaline phosphatase. The present invention also provides a method for modulating the ligand-dependent activity in a cell expressing a functional VDR, which comprises contacting an MNAR polypeptide for the cell in the presence of a VDR ligand. In one embodiment, the activity is the phosphorylation of a MAP kinase. In a specific embodiment, the kinase is Erk 1 and / or Erk 2. In another modality, the activity is the expression of a gene induced by the activation of the VDR. In a specific modality, the gene is osteocalcin or alkaline phosphatase.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. MNAR interacts with the ligand binding domain of VDR. The full-length MNAR was radiolabelled with 35S by in vitro transcription / translation and incubated with the binding domain fusion protein GST-VDR, in the presence or absence of 100 nM l, 25 (OH) 2Vit D3. The bound material was isolated using glutathione-sepharose and analyzed by SDS-PAGE and autoradiography. Figure 2. VDR interacts with MNAR LXXLL # 5. GST-VRD-LBD was incubated with the vehicle or with 1, 25 (OH) 2Vit. D3 at a concentration of 100 nM and then the immobilized peptides corresponding to different portions MNAR LXXLL. The VDR link was detected using anti-GST antiserum fused to HRP in the Wallac Victor 1420 multiple marker counter. The portions of MNAR LXXLL are numbered starting with the most N-terminal portion and designated LXXLL 1-9, respectively. Figure 3. MNAR increases activation of Erk stimulated with vitamin D3. Transfected UMR-106 cells - with the control or with the MNAR expression plasmids were treated with 1, 25 (OH) 2Vit. D3 10 nM for 0, 5, 10 or 20 minutes. The cells were harvested and the phosphorylation level of Erk 1 and 2 was determined using Western Blotting analysis. Figures 4A-4B. MNAR modulate the expression of the VDR-dependent gene. Figure 4A. The levels of osteocalcin RNA were determined using the analysis TaqMan from the RNA isolated from the UMR-106 cells transfected with the control or with the MNAR expression plasmid and- was treated with vehicle or with, 25 (OH) 2Vit. D3 100 nM per 24 hours. Figure 4B. Alkaline phosphatase RNA levels were determined using TaqMan analysis from RNA isolated from Ros 17 / 2.8 cells transfected with the control or with the MNAR expression plasmid and treated with the vehicle or 1, 25 (OH) 2Vit. D3 100 nM for 48 hours. Figure 5. This figure describes the schematic diagram of the MNAR organization. The N-terminal portion of MNAR is called the nuclear receptor interaction domain (NRID), due to the presence of multiple portions of LXXLL, while the C-terminal region is called the proline-rich domain and glutamic acid (PERD ), due to the presence of many proline and glutamic acid residues. Portions LXXLL and PXXP are designated 1-10 and 1.2, respectively, starting from the most N-terminal portion.

DETAILED DESCRIPTION OF THE INVENTION The present invention demonstrates that VDR MNAR and cSrc interact in a ligand-dependent manner. The direct interactions in DVR / MNAR and VDR / cSrc are mediated through the ligand binding domain of VDR, while the VDR / MNAR interaction requires the number 5 of the LXXLL portion of MNAR. In addition, the SH2 domain of cSrc is sufficient for the VDR / Src interaction, while the MNAR / Src interaction occurs via the SH3 domain of Src as previously demonstrated. The present invention demonstrates that MNAR interacts with VDR and forms a ternary complex with Src; and that the overexpression of MNAR strongly enhances the expression of the osteocalcin and alkaline phosphatase genes in osteosarcoma cells UMR 106 and ROS 17 / 2.8. As established previously, since osteocalcin and alkaline phosphatase are critical markers for the differentiation of osteoblasts, these data indicate that MNAR is an important regulator of bone development. Consequently, the generation or identification of functionally selective ligands that modulate the interaction of MNAR with VDR and, therefore, regulate the expression of osteocalcin and alkaline phosphatase, will be useful for the treatment of osteoporosis. These results provide additional evidence that MNAR is a scaffolding protein that incorporates nuclear receptors within Src-mediated cellular signaling and are consistent with the MNAR action model.

DEFINITIONS Vitamin D3 and its precursor, 1,25-dihydroxy-vitamin D3 (vitamin D3 and 1, 25 (OH) 2D3, respectively) is also known with cholecalciferol, (+) - 5, 7-cholestadien-3ß-ol or Racumin D (CAS No. 67-97-0) and is necessary for the use of calcium and phosphorus, by stimulating the absorption and for the assimilation of vitamin A. The human vitamin D3 receptor, or VDR, in a specific modality contains a sequence as described in the SwisProt database No P11473 and GenBank Accession No. J03258. The cDNA nucleotide sequence and the amino acid sequences for the human VDR are shown in SEQ ID Nos .: 1 and 2, respectively. A "functional VDR" is a VDR expressed in a host cell that is capable of signaling by second messenger, for example, phosphorylation of kinases in the MAP kinase pathway such as Erk 1 and Erk 2, and / or it is capable of regulating the transcription of ligand-dependent genes, for example, of osteocalcin or alkaline phosphatase, and is capable of promoting any other activity that has been demonstrated after coming into contact with a natural or synthetic ligand. A "VDR ligand" includes any natural ligand of the VDR, such as l, 25 (OH) 2D3 and any synthetic or analogous ligand. More than 3,000 synthetic Vit D3 analogs are known (Carlberg et al., Expert Opin. Ther.Patents 2003; 13 (6) -761-72). A "VDR ligand binding domain" or VDR-LBD comprises at least one C terminal region of the amino acid sequence in the SEQ. ID No .: 2, or another VDR ortholog. The VDR-LBD sequence begins at residue 121 and continues to approximately residue 427. Preferably, the LBD encompasses residues 110-427. The modulator of non-genomic estrogen receptor activity (MNAR) refers to the protein having an amino acid nucleotide sequence described in GenBank accession No. AF547989 (SEQ ID Nos .: 3 and 4, respectively). As shown in Figure 5, the LXXLL portions are in the N-terminal region, and are numbered 1-10. As used herein, a "ternary complex" refers to a complex that includes a nuclear hormone receptor, MNAR and Src or the pI3 kinase (or other signaling kinase). In one embodiment, the ternary complex is the VDR / MNAR / Src complex or the VDR / MNAR / PI3 kinase complex. More specifically, ~ the VDR / MNAR / Src complex is formed through an interaction of the ligand binding domain of VDR with MNAR via the LXXLL domain of MNAR, and with cSrc via the SH2 domain of cSrc; and the interaction of a PXXP portion of MNAR with the SH3 domain of cSrc. Additional ternary complexes include ER / MNAR / PI3 kinase and AR / MNAR / src. The term "bone or bone formation" is the process of bone synthesis and mineralization. The osteoblast cell modulates the process. The term "bone growth or development" is the process of skeletal expansion. This process occurs in one of two ways: (1) intramembranous bone formation that arises directly from the mesenchymal cells or the bone marrow; (2) longitudinal and endochondrial bone formation arising from bone from cartilage. The term "osteogenesis" is synonymous with the term bone formation defined above. The terms "disorder related to bone development", "disorder associated with bone development", "disorder of bone development", "disease of bone development", and other variants thereof, as they are generally used in the present, mean any disease or disorder related to the growth, development of repair, resorption, degradation or abnormal homeostasis of the bone tissue. Disorders related to bone development may therefore include diseases and disorders that are associated with abnormal increases, as well as with abnormal decreases in bone mass in. individuals Also, disorders related to bone development that are the subject of the present invention may include, but are not limited to, disorders that are associated with abnormal, (eg, increased or decreased) activity of osteoclast cells. Disorders related to bone development that are the subject of the present invention further include disorders that are associated with the abnormal (eg, increased or decreased) activity of osteoblast cells. Exemplary bone development related disorders that can be diagnosed or treated according to the methods and compositions of the present invention include osteopetrosis, osteoporosis, Paget's disease, osteogenesis imperfecta, fibrous dysplasia, hypophosphatase, primary hyperparathyroid arthritis, periodontal disease. and myeloma blood diseases to name a few. In addition, osteolysis can be induced by many malignant tumors that reside in or are distant from bone, for example, skeletal metastases in cancers of the breast, lung, prostate, thyroid and kidney, humoral hypercalcemia during malignancy, and multiple myelomas. Human osteocalcin, also known as gla-bone protein or BGP, has the nucleic acid (cDNA) and amino acid sequences as described in GenBank Accession No. NM_199173, and SEQ ID Nos .: 5 and 6, respectively. Alkaline phosphatase - is well known in the pertinent art. As an example, the nucleic acid and amino acid sequences of rat alkaline phosphatase, as found in rat osteosarcoma cell lines described herein, can be found in GenBank Accession No. J03572, and are described in SEQ ID Nos. : 7 and 8, respectively. The term "label" refers to an entity that is directly detectable or that can be linked to another molecule to allow detection. Direct markers include enzymes, fluorophores, chromophores, radioisotopes, dyes, colloidal gold, colloidal carbon, latex particles and chemiluminescent agents. Indirect markers include glutathione-S-transferase (which binds to glutathione and antibodies); FLAG, the myc marker, which binds to the antibodies; the His marker, which chelates the nickel; biotin, which binds to avidin; streptavidin (or any of the three that are linked to biotin); and the domains of the Ig constant region that bind to the antibodies in the Fe molecules. The markers can be covalently introduced, for example, using the conjugation chemistry - as part of a fusion construct (as exemplified further ahead) .

MOLECULAR BIOLOGY As used herein, the term "Isolated" means that the referred material is removed from the environment in which it is normally found. In this way, an isolated biological material can be free of cellular components, for example, components of the cells in which the material is found or produced. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. In another modality more. An isolated nucleic acid is preferably excised from the chromosome in which it can be found, and more preferably is no longer bound to the non-coding, non-regulatory regions or to other genes located upstream or downstream of the gene contained by the molecule isolated from nucleic acid when it is in the chromosome. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes and the like. In this way, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein can be associated with other proteins or other nucleic acids or both, with which it is associated in the cell, with cell membranes if this is a membrane-associated protein. An organelle, cell or isolated tissue is removed from the anatomical site in which it is found in an organism.

An isolated material can be, but it does not need to be purified. The term "purified" as used herein, refers to the material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, for example, contaminants including native materials from which the material is obtained. For example, a purified protein is preferably substantially free of other nucleic acid proteins with which it is associated in a cell; a purified nucleic acid molecule - is preferably substantially free of proteins or other nucleic acid molecules unrelated to which can be found within a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, the purified material is substantially free of contaminants is at least 50% pure; more preferably at least 90% pure, and still more preferably at least 99% pure. The purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. Methods for purification are well known in the art. For example, nucleic acids can be purified by precipitation, chromatography (including solid phase preparative chromatography, oligonucleotide hybridization and triple helix chromatography), ultracentrifugation and other means. The polypeptides and proteins can be purified by various methods including, without limitation, preparative disc gel electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation chromatography and salting, extraction, and countercurrent distribution. For the same purpose, this is preferably to produce a polypeptide in a recombinant system in which the protein contains an additional marker sequence that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a linking sequence. specifically to an antibody, such as FLAG or GST. The polypeptide can then be purified from a crude used host cell by chromatography on an appropriate solid phase matrix. Alternatively, antibodies raised against the protein or against peptides derived therefrom can be used as purification reagents. The cells can be purified by various techniques including centrifugation, separation in matrix (for example, separation with nylon wool), panoramic immunoselection techniques and others, depletion (for example depletion of the complement of contaminating cells), and cell sorting (for example, fluorescence-activated cell sorting [FACS] ]). Other purification methods are possible. A purified material may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90% of the cellular components with which it is originally associated. "Substantially pure" indicates the highest degree of purity that can be achieved using conventional purification techniques known in the art. In preferred embodiments, the terms "about" and "about" will generally mean an acceptable degree of error for the measured quantity, given the nature or precision of the measurements. Typical exemplary error rates are within 20 percent (%) preferably within 10% and more preferably within 5% of a given value or range of given values. Alternatively, and particularly in biological systems, the terms "about" and "about" may mean values that are within an order of magnitude, preferably within 5 times and preferably within 2 times a given value. The numerical quantities given herein are approximate unless otherwise stated, which means that the term "about" or "approximately" may be inferred when not expressly stated. The term "molecule" means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes. example, polypeptides and polynucleotides. As used herein, the term "ligand" refers to molecules, including chemical compounds, peptides, and peptide mimetics, which bind to MNAR and VDR, and thereby alter or affect the function or behavior of the cells that express VDR, or prevent or alter the effect that another biologically active protein might otherwise have on those cells. The term ligand and compound can be used interchangeably when referring to the design, selection and rational drug interaction assays. A "test substance" or "test compound" or "test ligand" (including peptides and peptidomimetics) is a substance that has been identified or designed to interact with MNAR, preferably via the LXXL portion of MNAR, whereby the MNAR interaction with the VDR is also allowed. A "leader compound" is a test compound that has been shown to bind to MNAR and modulate the activity of the second messenger (eg, transcriptional regulation) via the VDR over the interaction of MNAR and VDR. Non-human animals include, without limitation, laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, etc.; domestic animals such as dogs and cats; and farm animals such as sheep, goats, pigs, horses and cows, and especially animals processed transgenically with MNAR and / or human or murine VDRs. In accordance with the present invention, conventional molecular biology, microbiology and recombinant DNA techniques can be employed within the skill of the art. Such techniques are fully explained in the literature. See, for example, Sambrook, Fitsch and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Nucleic Acid Hybridization (B.D. Hamés and S.J. Higgins, eds., 1984); Animal Cell Cul ture (R. Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B.E. Perbal, A Practical Guide to Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocole in Molecular Biology, John Wiley & Sons, Inc. (1994). A "gene" is a sequence of nucleotides that encode a functional "gene product". In general, a gene product is a functional protein. However, a gene product can also be another type of molecule in a cell, such as an RNA (e.g., tRNA or rRNA). For the purposes of the present invention, a gene product also refers to an RNA sequence that can be found in a cell. For example, the measurement of the expression levels of the genes according to the invention may correspond to the measurement of mRNA levels. A gene can also comprise regulatory sequences (eg, non-coding) as well as coding sequences. Exemplary regulatory sequences include promoter sequences, which determine, for example, the conditions under which the gene is expressed. The transcribed region of the gene can also include the untranslated regions that include introns, a 5'-untranslated region (5'-UTR) and a 3'-untranslated region (3'-UTR). A "coding sequence" or a "coding sequence" and the expression product such as an RNA, polypeptide, protein or enzyme, is a nucleotide sequence which, when expressed, results in the production of that RNA, polypeptide, protein or enzyme; for example, the nucleotide sequence "encodes" that RNA, or it encodes the amino acid sequence for that polypeptide, protein or enzyme. A "promoter sequence" is a DNA regulatory region capable of binding to the RNA polymerase in one. cell and initiates the transcription of a coding sequence downstream (3 'direction). For purposes of defining the present invention, the promoter sequence is linked at its 3 'end by the transcription initiation site and extended upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at detectable levels above the antecedent. Within the promoter sequence will be found a transcription start site (conveniently found, for example, by mapping with the nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of the RNA polymerase . A coding sequence is "under the control of" or is "operatively associated with" the transcriptional and translational control sequences in a cell, when the RNA polymerase transcribes the coding sequence into RNA, which is -then spliced RNA-trans. (if it contains introns), if the sequence codes for a protein, it is translated into that protein. The term "express" and "expression" means allowing or causing the information of a gene or sequence of a DNA to become manifest, for example, by producing RNA (such as tRNA or mRNA) or a protein by activating cell functions involved in the transcription and translation of a corresponding gene or DNA sequence. A DNA is expressed by a cell to form an "expression product" such as an RNA (e.g., an mRNA or rRNA) or a protein. The product of expression itself, for example, the resulting RNA or protein, can also be said to be "expressed" by the cell. The term "transfection" means the introduction of a foreign nucleic acid into a cell. The term "transformation" means the introduction of a gene "foreign" (eg, extrinsic or extracellular), DNA or RNA sequence within a host cell, such that the host cell will express the introduced gene or sequence to produce a desired substance, in this invention typically an encoded RNA by the introduced gene or sequence, but also a protein or an enzyme encoded by the introduced gene or sequence. The introduced gene or sequence can also be called a "cloned" or "foreign" gene or sequence, it can include regulatory or control sequences (e.g., start, stop, promoter, signal, secretion or other sequences used. by the genetic machinery of a cell). The gene or sequence may include non-functional sequences or sequences with unknown function. A host cell that receives and expresses the DNA or RNA has been "transformed" and is a "transformant" or a "clone". The DNA or RNA introduced into a host cell can come from any source, including cells of the same genus or species as the host cells of a different genus or species. The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (eg, a foreign gene) can be introduced into a host cell to thereby transform the host and promote the expression (eg, transcription and translation) of the introduced sequence. The vectors can include plasmids, phages, viruses, etc., and are discussed in more detail below. A "cassette" refers to a sequence or segment of DNA coding of DNA that codes for an expression protein that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure the insertion of the cassette into a suitable reading structure. In general, foreign DNA is inserted into one or more restriction sites of the vector DNA, and then carried by the vector into a host cell, together with the transmissible vector DNA. A segment or DNA sequence having DNA inserted or added, such as an expression vector, can also be called a "DNA construct". A common type of vector is a "plasmid" which is generally a self-contained double-stranded DNA molecule, usually of bacterial origin, that can easily accept additional DNA (strange) and that can be easily introduced into a suitable host cell. A large number of vectors including plasmid and fungal vectors have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. The term "host cell" means any cell of any organism that is selected, modified, transformed, developed or used or manipulated in any way for the production of a substance by the cell. For example, a host cell can be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme. The host cells may also be used for screening assays or other assays that are described below. The host cells can be cultured in vitro or one or more cells in a non-human animal (eg, a transgenic animal or a transiently transfected animal). The term "expression system" means a host cell and the compatible vector under suitable conditions, for example, the expression of a protein encoded by the foreign DNA and carried by the vector introduced into the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells such as Sf9, Hi5 or S2 cells and Baculovirus vectors, Drosophila cells (Schneider cells) and expression • systems, and mammalian host cells and vectors. For example, MNAR or VDR can be expressed in cells PC12, COS-1 or C2C12.

Other suitable cells include CHO cells, HeLa cells, 293T cells (human kidney cells), mouse primary myoblasts and NIH 3T3 cells. In a preferred embodiment, the cells are osteosarcoma cells. The term "heterologous" refers to a combination of elements not of natural origin. For example, the present invention includes chimeric RNA molecules comprising a rRNA sequence and a heterologous RNA sequence that is not part of the rRNA sequence. In this context, the heterologous RNA sequence refers to an RNA sequence that is not naturally localized within the RNA ribosomal sequence. Alternatively, the heterologous RNA sequence may be naturally localized within the ribosomal RNA sequence, but is found at a site in the rRNA sequence where it does not appear naturally. As another example, heterologous DNA refers to DNA that is not naturally located in the cell, or at a chromosomal site in the cell. Preferably, the heterologous DNA includes a foreign gene for the cell. A regulatory element of expression, heterologous is a regulatory element operatively associated with a different gene than that with which it is operatively associated in nature. The terms "mutant" and "mutation" mean any detectable change in the genetic material, for example, DNA, or any process, mechanism or result of such change. This includes gene mutations, in which the structure (eg, the DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (eg, RNA, protein or enzyme) expressed by a modified gene or DNA sequence. The term "variant" can also be used to indicate a modified or altered gene, DNA sequence, RNA, enzyme, cell, etc. For example, the present invention relates to altered or "chimeric" RNA molecules comprising a rRNA sequence that is altered by the insertion of a heterologous RNA sequence that is not naturally part of this sequence or is not localized to natural way in the position of that rRNA sequence. Such chimeric rRNA sequences as well as DNA and genes encoding them are also referred to herein as "mutant" sequences. In addition, the variants include modified proteins or peptide fragments with altered chemical properties, which demonstrate enhanced binding to a target, eg, MNAR or VDR, which include certain commonly found amino acids, which are not genetically encoded, which can be used and which include, but are not limited to, [beta-alanine (B-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so on; α-aminoisobutyric acid (Aib); α-aminohixanoic acid (Aha); α-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); Ornithine (Orn); citrulline (Cit); t-butylalanine (T-BuA); t-butylglycine (t-BuG); N-methyl isoleucine (Melle) '; phenylglycine (phg); cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe (4-Cl)); 2- fluorophenylalanine (Phe (2-F)); 3-fluorophenylalanine (Phe (3-F)); 4-fluorophenylalanine (Phe (4-F)), penicillamine (Pen); 1,2,3-tetrahydroisoquinoline-3-carboxylic acid (Tic); [beta] -2-thienylalanine (Thi); methionine sulfoxide (MOS), homoarginine (hArg); N-acetyl-lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2, 3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe (pNH2)); N-methyl-valine (MeVal); homocysteine (hCys) and homoserin (hSer). The terms "array" and "microarray" are used interchangeably and refer generally to any array ordered (eg, on a surface or substrate) of different molecules, referred to herein as "probes". Each probe different from the arrays specifically recognizes and / or binds to a particular molecule, which is denominated in the present as its "objective". Microarrays are therefore useful for simultaneously detecting the presence or absence of a plurality of different target molecules, for example, in a sample. In preferred embodiments, the arrays used in the present invention are "steerable arrays" where each different probe is associated with a "particular address". For example, in preferred embodiments where the probes are mobilized on a surface or a substrate, each probe other than the targeting array can be immobilized at a particular known location on the surface or substrate. The presence or absence of the target molecule of that probe in a sample can therefore easily be determined simply by determining whether or not an objective has been linked to that particular site on the surface or the substrate. A nucleic acid molecule is "hybridizable" to another nucleic acid molecule (e.g., cDNA, genomic DNA or RNA) when a single-stranded form of the DNA molecule can be annealed to another nucleic acid molecule under appropriate conditions of temperature and ionic strength in solution (see for example, Sambrook et al., supra).

The conditions of temperature and ionic strength determine the "severity or requirement" of the hybridization. "Hybridization" requires that two nucleic acids contain complementary sequences, although depending on the severity of the hybridization, poor couplings between bases are possible.

The appropriate requirement to hybridize nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for the hybrids of the nucleic acids that have those sequences. For hybrids of more than 100 nucleotides in length, the equations for calculating Tm have been derived (see Sambrook et al., Supra, 9.50-9.51). In a specific embodiment, the term "standard hybridization conditions" refers to a Tm of about 55aC and uses conditions as described above. In a preferred embodiment, the Tm is 60SC; in a more preferred embodiment, the Tm is 652C. In a specific embodiment, the term "high requirement" refers to the conditions of hybridization and / or washing at 682C in SSC 0.2x to 42C in 50% formamide, SSC 4x, or under conditions that provide equivalent hybridization levels to those observed under either of these two conditions. Low stringency or stringency hybridization conditions corresponding to a melting temperature of about 55aC can be used (eg, 5x SSC, 0.1% SDS, 0.25% milk and no formamide, or alternatively, 30% formamide, SSC 5x and 0.5% SDS). The conditions of hybridization of moderate demand correspond to a Tm. higher, for example, 40% formamide with 5x or 6x SSC. The conditions of high-requirement hybridization correspond to the highest Tm, for example, 50% formamide, 5x or 6 x SSC. A solution lx SSC is understood to be a solution containing 0.15 M sodium chloride and 0.015 M sodium citrate.

APPLICATIONS AND USES The present invention relates to the discovery that MNAR interacts with the VDR, thereby mediating the binding of the VDR ligand. NMAR interacts with VDR specifically by means of the fifth LXXLL portion in the amino acid sequence of MNAR (SEQ ID No .: 2). Accordingly, ligands that bind to this portion and thereby agonize or antagonize at the link of VDR to MNAR, are contemplated for use to modulate signaling through the VDR, for example, through cSrc or PI3 kinase. . Using the methods described herein, it is also possible to identify guiding compounds or guiding peptides or compounds or peptides designed rationally by test that bind to or otherwise interact with MNAR and VDR. For example, vitamin D3 can be used to antagonize the binding of a test compound to a reaction comprising MNAR-VDR in order to determine whether, in the absence of vitamin D3, the test compound can replace and mediate the interaction of MNAR-VDR and / or its activity. To a minimum, the methods of the present invention depend on the portion LXXLL number 5 in MNAR (see figure 1), amino acids 110-427 of VDR, which encompass the VDR LBD and bind to MNAR.

INTERACTION AND ACTIVITY TESTS Interaction tests. The MNAR function agonists or antagonists could act either by modulating an interaction between MNAR and VDR or another nuclear hormone receptor, or MNAR and cSrc or the PI3 kinase or other kinase, or by modulating a MNAR activity or a VDR or other steroid hormone receptor. Any of the well-known two hybrid assays or other conventional assays can be employed to study the protein-protein interactions and the disintegration thereof by the test compounds. The in vitro systems can be easily designed to identify guiding ligands capable of specifically binding to MNAR according to the present invention. In general, such screening assays involve the preparation of a reaction mixture, comprising a wild-type MNAR protein and test compound, under conditions and for a sufficient time to allow the two compounds to interact (eg bind) with which A complex is formed that can be detected. The links can be conducted in any of a variety of different ways, including the use of microarrays. Protein binding assays and gel shift assays are useful methods for detecting "binding." Exemplary assays include the evaluation of the labeled VDR or the binding of cSrc to the immobilized MNAR and the labeled MNAR or the MNAR peptide that binds to the immobilized nuclear receptor (eg, VDR) or the modulator, eg, cSrc .. Many appropriate assays are suitable for high-throughput, large-scale use, suitable for large-volume drug selection. will be determined by the particular nature of the MNAR interactions The assays can employ a simple MNAR, fragments of MNAR, MNAR fusion products, partial complexes of MNAR, and the complete basal transcription complex comprising an MNAR nucleic acid The detection of MNAR-VDR-ciñasa complexes can be achieved using specific binding assays such as immunoassays, and drug interactions. biotin / avidin link (including streptavidin and neutravidin). Commercial antibodies to VDR and kinases such as cSrc, various markers such as myc and FLAG markers, and second messenger kinases that can be used to practice the invention, are available from various sources, including R &D Systems, Minneapolis, MN and Santa Cruz Biotechnology, Santa Cruz, CA. Antibodies to MNAR have been described and published in PCT Application WO2004 / 031223, incorporated by reference herein in its entirety. Such immunoassays include radioimmunoassay, Enzyme Linked Immunosorbent Assay (ELISA), sandwich or sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzymatic labels or radioisotopes, for example), Western blots, precipitation reactions, agglutination assays (eg, gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays and immunoelectrophoresis assays. In one embodiment, the binding of the antibody is detected by detecting a marker on the primary antibody. In yet another embodiment, the primary antibody is detected upon detection of the binding of a secondary antibody or secondary reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art to detect binding in an immunoassay, and are within the scope of the present invention. - Other assays, including but not limited to, two-hybrid assays and surface plasmon resonance (SPR) assays, could be used to identify such interactions. The two hybrid yeast assays as described in Young and Ozenberger in U.S. Patent No. 5,989,808, which is incorporated by reference herein. SPR is described in Wegner et al., Anal. Chem. 2002; 74: 5161-5168; Cooper et al., Anal Bioanal Chem. 2003; 377 (5): 834-42; and Gambari, Curr Med "Chem Anti-Canc Agents, 200; 1 (3): 277-91.) Microarrays: The terms" arrangement "and "microarray" are used interchangeably and refer generally to any ordered arrangement (e.g., on a surface or substrate) or different molecules, referred to herein as "probes". Each probe different from an array specifically recognizes and / or binds to a particular molecule, which is referred to herein as its "target". Microarrays are therefore useful for simultaneously detecting the presence or absence of a plurality of different target molecules, for example in a sample. The presence or absence of that target molecule of the probe in a sample can therefore be easily determined simply by analyzing whether an objective has or has not been linked to that particular site, on the surface or the substrate. 'Conventional microarrays generally comprise a non-porous solid substrate such as a glass slide or a computer chip. In a typical microarray application, the substrate is contacted with a sample containing the biomaterials to be analyzed. The substrate is then contacted with the probe molecules such as the labeled nucleic acids or polypeptides or other molecules. The labeled molecules are linked to the molecules in the sample. The unbound probe molecules are removed, for example, by washing, and the microarray is then read by a suitable signal detection device, for example, by fluorescence emission. For example, one embodiment comprises anchoring a protein, e.g., MNAR, or a test ligand, e.g., VDR or cSrc on a solid phase and detecting the complexes of the protein and the test ligand that are on the phase solid at the end of the reaction, and then removing (eg, by washing), the unbound ligands. For example, in a preferred embodiment of such a method, a protein can be anchored on a solid surface and a labeled compound or polypeptide is contacted with the surface. After incubating the test ligand for a sufficient time and under sufficient conditions so that a complex can be formed between the protein and the test compound or the test polypeptide, the unbound molecules of the test ligand are removed from the surface ( for example by washing) and the labeled molecules that remain are detected. In one embodiment, a mutant or variant protein can be anchored to a solid surface or support. Another labeled protein (the test compound), which can bind to the protein anchored to the solid surface, can be treated with a proteolytic enzyme, and its fragments can be allowed to interact with the protein bound to the solid surface. After washing,. the labeled, short peptide fragments of the treated protein may remain associated with the anchored protein. These peptides can be isolated if the region of the full-length protein from which they are derived can be identified by the amino acid sequence. In another alternative embodiment, the molecules of one or more different test compounds are linked to the solid phase and the molecules of a protein (eg, a marker MNAR polypeptide) can be contacted therewith. In such an embodiment, the molecules of different test compounds are preferably linked to the solid phase, at a particular site on the solid phase, so that the test compounds that bind to a mutant protein can be linked to determine the location of the protein. bound proteins on the solid phase or the solid surface. Again, mutant proteins and variants can be used as test compounds. In addition, the selection can be made using vitamin D3 as an antagonist to a well containing an MNAR and VDR polypeptide, to which well a test compound in the presence and absence of vitamin D3 is also added. After binding and forming a compound / MNAR / VDR interaction, it must be inhibited by the addition of unlabeled vitamin D3. The automated multiple well formats are the best developed high performance systems. 96-well, automated plate-based screening systems are widely used. The current trend in plate-based selection systems is to reduce the volume of the reaction wells further, thereby increasing the density of the wells per plate (96 wells to 384 wells, and 1536 wells per plate). The reduction in reaction volumes results in increased throughput, dramatically decreased bioreactive costs, and a decrease in the number of plates that need to be handled by automation. For a description of the protein arrays that can be used for high throughput screening, see U.S. Patent Nos. 6,475,809, 6,406,921 and 6,197,559, incorporated by reference herein. Activity tests. Once one or more guide compounds that bind specifically to an MNAR protein have been designated or identified and characterized, they can be used in tests to determine whether they modulate signaling through the VDR. The test examples are described below and in the examples. The activity assays are generally designed to measure the activity of a target protein in the presence or absence of a test agent. Activity assays, including but not limited to, mammalian transfection assays in which the transcriptional activity of VDR is observed, could be used to identify the leader ligands. For example, the ligands could be selected for their ability to modulate the increase of VDR transcriptional activity by MNAR. Alternatively, the ligands could be selected for their ability to affect signaling of the second messenger, such as Src, phospholipase C, the kinases of Erk 1 and 2. Such compounds can modulate the interactions of MNAR with VDR or can modulate a known activity or unknown of MNAR or VDR, including a transcriptional activity, for example, of osteocalcin or alkaline phosphatase, or the activation or enzymatic activity of kinases, such as, for example, phosphorylation. A method used to select a ligand that modulates the MNAR activity with VDR, comprises the steps of (a) contacting a test compound with an MNAR polypeptide comprising the LXXLL portion capable of binding to VDR; and (b) determining whether said test compound binds specifically to the polypeptide. In addition, the method may comprise the steps of (a) adding a test compound to a cell comprising the MNAR polypeptide and the VDR receptor; and (b) compare the activity of MNAR before and after the addition of the compound.

Additional methods to this method involve the addition of a test compound to a control sample comprising a mutant cell that lacks MNAR activity or significantly reduced MNAR activity. There are numerous methods to identify compounds that affect the non-genomic activity or genomic activity of nuclear receptors. In one embodiment, this comprises the steps of (a) adding a test compound to a cell comprising an MNAR and VDR complex; and (b) compare genomic versus non-genomic activity before and after. A selective genomic activity can be measured by conventional means. Preferably, the increase or positive effect of the non-genomic activity, as measured, is a double increase in, for example, transcription, after the addition of the test compound to a cell in the presence of MNAR and VDR, when compares the genomic activity with the test compound in the absence of MNAR and where no change in non-genomic activity is observed, after the addition of the test compound. In these methods, preferably, at least one ligand of a nuclear receptor is present in the cell, or a kinase activated by such a ligand is present, or both are present. Alternatively, when selecting the test compounds for non-genomic activity, there must be at least about a two-fold increase, for example, in transcription, after the addition of the test compound to a cell, in the presence of MNAR. -VDR when the non-genomic activity is compared to the test compound in the absence of MNAR, and where no change in genomic activity is observed after the addition of said test compound. The effect can be measured by determining an increase or decrease in the transcriptional activity of VDR in the presence of MNAR. In each of these methods a control is used to evaluate the non-genomic activity (or genomic activity) whose control comprises administering a compound to a cell in the presence of MNAR and the VDR, and then repeating the experiment in the presence of MNAR and comparing the level of non-genomic activity, for example, kinase activity. Preferred methods employ a cell that overexpresses MNAR or VDR and / or cSrc or PI3 kinase, or two or all three of them. The genomic activity of a compound is detected by having the VDR operatively associated with a reporter gene. One way to detect second messenger activity is through kinase assays. This can be accomplished using cell-free, cell-based assays or based on animal models. Such assays can be used as secondary selections for the activity of selected candidate compounds in a primary selection. Kinase assays detect the phosphorylation status of the kinases, typically by incorporating 32 P into the cell, isolating cellular proteins, and immunotransforming with an antibody specific for phosphorus. Such assays are well known in the art. Assays to detect changes, e.g., increases or decreases in the transcription of a gene within, e.g., mRNA, are well known in the art. Such assays include the reverse transcriptase polymerase chain reaction (RT-PCR), including quantitative RT-PCR, Northern hybridization, transfection assays of the gene of interest linked to a reporter gene, etc. .

Optimization Once one or more drug guides for a goal have been generated or identified, it is often still necessary to optimize a guide in order to improve its pharmacological efficacy. In this step, called as guide optimization, synthetic chemists chemically modify the guide in order to increase or decrease its link to the target, modify its susceptibility to degradative pathways or modify its pharmacokinetics. The optimization of rational guidance employ theoretical methods to determine a group of probable guide candidates for a given objective, before experimentally selecting in guide-objective bond. Rational guide optimization offers the possibility of reducing drug discovery time and drug costs by reducing the number of potential guide-target selections.

Rational drug design and selection for VDR agonists / antagonists, mediated by MNAR Drug discovery occurs in two steps: 1) identification of the objective and 2) identification and optimization of the guide. In the first step of identifying the target, a large molecule, which can be, but is not limited to, a cell surface receptor or an intracellular protein, named as the target can be identified with a particular biological pathway or structure. particular interest. Once a potential target has been identified, it should be selected against a large number of small molecules, to determine whether or not the target is appropriate for the binding and interaction of the small molecule. An MNAR-VDR interaction model can be used to create VDR ligands, mediated by MNAR, which, for example, do not affect VDR-mediated transcription, but regulate VDR-mediated cell signaling, including, for example, activation of the MAP kinase pathway and the C-gamma phospholipase pathway (PLC?). Structural identification. The identification and selection of the modulators is further facilitated by the determination of the structural characteristics of the protein, for example, MNAR or the interaction of MNAR with VDR, using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for the determination of the structure. These techniques provide for the rational design or identification of modulators, including agonists and antagonists and partial agonists and antagonists.

To design small molecules or peptidomimetics, it is beneficial to obtain a three-dimensional structure for the pharmacophore of one or more peptide compounds. The term "pharmacophore" refers to the collection of functional groups on a compound or peptidomimetic that are accommodated in the three-dimensional space in a manner complementary to the target protein. For example, MNAR, and "that are responsible for the biological activity as a result of the binding of the ligand to the target protein." The useful three-dimensional pharmacophore models are derived better from either the crystallographic or nuclear magnetic resonance structures of the related targets or structure-activity relations, quantitative, three-dimensional, derived from a previously discovered series of active compounds.The X-ray crystallography techniques are well known and are within the routine experience in the matter.For example, see Cantor &Schimmel, Biophysical Chemistry 1980 (Vols. 'I-III) WH Freeman and Company (particularly chapters 1-13 in Vol. I, and Chapter 13 in Vol. II.) See also, Macromolecular Crystallography, Parts AB (Carter &Sweet, Eds. ) In: Methods Enzymol, 1997, Vols 276-277, Jan Drenth, Principies of Protein X-Ray Crys allografphy (New York, Springer-Verlag, 1994).

The crystal structure of the VDR linked to its natural ligand is known (Rochel et al., Mol Cell 2000; 5 (1): 173-9). Similarly, the structurally and functionally important amino acids of the VDR agonist conformation are known, and include the amino acid residues H229, D232, E269, F279 and Y295 (Vaisanen et al., Mol Pharmacol.; 62 (4): 788-94). Consequently, given the LXXLL portion involved in linking MNAR to vitamin D3, coupled with the critical regions of VDR that must bind to a ligand for activation, rationally designed ligands can be designed to mimic the selective aspects of these activities . Alternatively, the three-dimensional structures of MNAR-VDR interactions can be generally determined using nuclear magnetic resonance (NMR) techniques that are well known in the art. The acquisition of NMR data is preferably carried out in aqueous systems that closely mimic physiological conditions, to ensure that a relevant structure is obtained. In summary, NMR techniques use the magnetic properties of certain atomic nuclei (such as 1H, 13C, 15N and 31P), which have a magnetic moment or spin, to probe the chemical environment of such nuclei.

The NMR data can be used to determine the distances between atoms in the molecule, which can be used to derive a three-dimensional model or molecule.

POTENTIAL LIGANDS According to the present invention, the ligands include peptide ligands (which further include "peptidomimetics that are linear or cyclic) or chemical compounds, which selectively mimic the biological activity of the biologically active protein, or selectively block or selectively agonize the activity of the biologically active protein, such as the interaction of VDR with MNAR - Compounds: The structural domains that mediate the interactions between the molecules, for example, VDR and MNAR, and which also confer biological activity on the cells that expressing VDR receptors (or VDR-like receptors), can be used to rationally design modulators of compounds.These structural domains, and other functional domains, that can modulate the activity of these structural domains, can all be modified through of a variety of media, including but not limited to site-directed mutagenesis, in order to increase or reduce biological activity. The structure and topology of these domains can all be used as a basis for the rational design of pharmaceutical products (conventional small molecule drugs or new high molecular weight biological compounds based on carbohydrates, lipids, DNA / RNA or proteins). ) to modulate (increase or decrease) the activity of VDR, or the VDR-MNAR complex, and / or the activity of the VDR / MNAR / other protein complexes. For example, the use of structural prediction calculations, possibly in conjunction with spectroscopic data such as nuclear magnetic resonance, circular dichroism, and other physico-chemical structural data, or crystallographic data, or both, can generate molecular models for structure of MNAR and / or VDR. These models, in turn, are important for the rational design of drugs. For selection, classes of compounds that can be identified include, but are not limited to, small molecules (e.g., organic or inorganic molecules that are less than 2 kD in molecular weight, which are more preferably less than about 1. kD in molecular weight, as well as macromolecules (eg, molecules larger than about 2 kD in molecular weight) The compounds identified by these screening assays also preferably include peptides and polypeptides, eg, soluble peptides, peptide members. of fusion of the combinatorial libraries (such as those described by Lam et al., Nature 1991, 354: 82-84, and by Houghton et al., Nature 19'91, 354: 84-86), members of libraries derived by combinatorial chemistry, such as the molecular libraries of the amino acids of D and / or L configuration, phosphopeptides, such as the members of the phosphopeptide libraries as, random or partially degenerate (see for example, Songyang et al., Cell 1993, 72: 767-778). In addition, libraries of small high purity organic ligands and peptide agonists having well-documented pharmacological activities are available from Sigma-Aldrich (LOPAC LIBRARYm and LlGAND-SETSm.) Also available from Sigma Aldrich is an Aldrich library of rare chemicals , which is a diverse library of more than 100,000 small molecule compounds, including plant extracts and microbial cultures, Other libraries of compounds are available from Tripos (LeadQuest®), TimTech (including libraries targeted for kinase modulators). Provided libraries of compounds include the following: 3-Dimensional Pharmaceuticals, Inc., Advanced ChemTech, Abinitio PharmaSciences, Albany Molecular, Aramed, Inc. (formerly Bearsden Bio, Inc.), ASINEX, AVANT Imunotherapeutics, AXYS Pharmaceuticals, Bachem, Bentley. Pharmaceuticals, Bicoll Group, Biofor Inc., BioProspect Australia Limited, Biosepra Inc. .; Cadus Pharmaceutical Corp .; Cambridge Research Biochemicals; Cetek Corporation; Charybdis Technologies, Inc .; ChemBridge Corporation; ChemDiv, Inc .; ChemGenics Pharmaceuticals Inc .; ChemOvation Ltd .; ChemStar, Ltd .; Chrysalon; ComGenex, Inc .; Compugen Inc .; Cytokinetics; Dextra Laboratories Ltd.; Discovery Partners International Inc .; Discovery Technologies Ltd .; Diversity Corporation; Dovetail Technologies, Inc .; Drug Discovery Ltd .; ECM Pharma; - Galilaeus Oy; Janssen Pharmaceutica; Jerini Bio Tools; J-Star Research; KOSAN Biosciences, Inc .; KP Pharmaceutical Technology, Inc .; Lexicon Genetics Inc .; Libris Discovery; MicroBotánica, Inc .; MicroChemistry Ltd .; MicroSource Discovery Systems, Inc .; Midwest Bio-tech Inc .; Molecular Design & Discovery; MorphoSys AG; Nanosyn, Inc .; Ontogen Corporation; Organix, Inc .; Pharmacopeia, Inc .; Pherin Pharmaceuticals; Phytera, Inc .; PTRL East, Inc .; REPLICor Inc .; RSP Amino Acid Analogues, Inc .; Sanofi-Synthelab Pharmaceuticals; Sequitur, Inc .; Signature BioScience Inc .; Spectrum Info Ltd .; Heel Cheminformatics Inc .; Telik, Inc .; Tera Biotechnology Corporation; Tocris Cookson; Torrey Pines Institute for Molecular Studies; Trega Biosciences, Inc .; and WorldMolecules / MMD. In addition, the Institute of Chemistry and Cell Biology (ICCB), maintained by the Harvard Medical School, provides the following chemical libraries, including libraries of natural products, for selection: Chem Bridge DiverSet E (16,320 compounds); Bionet 1 (4,800 compounds) -; CEREP (4,800 compounds); Maybridge 1 (8,800 compounds); Maybridge 2 (704 compounds); - Peakdale 1 (2,816 compounds); Peakdale 2 (352 compounds); ChemDiv Combilab and International (28,864 compounds); Mixed Commercial Píate 1 (352 compounds); Mixed Commercial Píate 2 (320 compounds); Mixed Commercial Píate 3 (251 compounds); Mixed Commercial Píate 4 (331 compounds); ChemBridge Microformat (50,000 compounds); Commercial Diversity Setl (5,056 compounds); NC collections: Structural Diversity Set, version 2 (1,900 compounds); Mechanistie Diversity Set - (879 compounds); Open Collection 1 (90,000 compounds); Open Collection 2 (10,240 compounds); Known Bioactive Collections: NINDS Custom Collection (1,040 compounds); ICCB Bioactives 1 (489 compounds); SpecPlus Collection (960 compounds); ICCB Discretes Collections. The following ICCB compounds were collected individually from the chemists of ICCB, Harvard and other collaborating institutions: ICCBl (190 compounds); ICCB2 (352 compounds); ICCB3 (352 compounds); ICCB4 (352 compounds).

Excerpts from natural products: NCI Marine Extracts (352 wells); Organic Fractions - Fungal Extracts and NCI plants (1,408 wells); Extracts of Philippine Plants 1 (200 wells); Synthesis-oriented Diversity Collections (DOS) of ICCB-ICG; DDSl (DOS Diversity Group) (9600 wells). In a preferred embodiment, the libraries of compounds or designed peptides are based on the objective. There are numerous techniques available to create libraries of more focused compounds, rather than large and diverse ones. Chemical Computing Group, Inc. (Montreal). has developed the software (computer hardware) with a new procedure for the design of high performance drugs. The company's method uses experimental data from high-throughput (HTS) selection 'to create a Probabilistic Quantitative Relationship Structure-Activity (QSAR) model, which is subsequently used to select building blocks in a virtual combinatorial library This is based on statistical estimation instead of standard regression analysis. In addition, ArQule, Inc. (Woburn, MA) has also integrated technologies to perform automated high-throughput production of chemical compounds, and to distribute these compounds of known structure and high purity in sufficient quantities for guide optimization. Its Automated Molecular Assembly Plant (lsMA'PMR) performs high performance chemical synthesis for each phase of compound discovery.

Similarly, compounds are frequently provided in online databases or in cederroms for selective "collection" of compounds. See for example, Ablnitio PharmaSciences; ActiMol; Aral Biosynthetics; ASDI Biosciences; Biotechnology Corporation of America; Chembridge; ChemDiv; Florida Center - Heterocyclic Compounds; Microsource / MSDI; NorthStar; Peakdale; Texas Retaining Group; Zelinsky Institute; Advanced Chem / Tech; Ambinter; AnalytiCon Discovery; Aurora Fine - Chemicals; Biofocus; Bionet / Key; Comgenex; Key Organics; LaboTest; Polyphor; SPECS and Biospecs; and Bharavi Laboratories. Peptides Peptide libraries useful for selection are available from the following sources: American Peptide Co. , Inc .; BIOMOL Research Laboratories Inc .; Cell Science Inc .; GenoMechanix, LLC; Phoenix Pharmaceuticals Inc .; United States Biological; Advanced ChemTech Inc .; AerBio Ltd .; Amphotech Ltd .; AnaSpec Inc .; ANAWA Trading SA; Biomar Diagnostic Systems GmbH; BioSource International Inc.; Dalton Chemical Laboratories Inc.; Enzyme Systems Products Inc .; Peptides Internacional Inc. '; Princeton BioMolecules Corp .; Protein Technologies Inc .; Sigma-RBI; Synpep Corp .; and Xaia Custom Peptides. Another procedure uses the recombinant bacteriophage to produce large libraries. Using the "phage method" (Scott and Smith, Science 1990, 249: 386-390; Cwirla, et al., Proc. Nati. Acad. Sci. USA 1990, 87: 6378-6382; Devlin et al., Science 1990, 49: 404-406), very large libraries can be constructed (106-108 chemical entities). A second procedure uses mainly chemical methods of which the Geysen method is exemplary (Geysen et al., Molecular Immunology 1986, 23: 709-715; Geysen et al. J. Im unol. Meth. 1987, 102: 259-244; and the method of Fodor et al. (Science 1991, - 251: 767-773).

Furka et al. (14th International Congress of Biochemistry 1988, Volume # 5, Excerpt FR: 013, Furka, Int. J. Peptide Protein Res. 1991, 37: 487-493), Houghton. (Patent of the United States No. 4,631,211) and Rutter et al. (Patent of the United States No. 5,010,175) describes methods for producing a mixture of peptides that can be tested as agonists or antagonists. Phage display kits are available, for example, from Ph.D.101. Peptides can be rationally designed and generated by identifying specific linear and constricted discrete portions of biologically active proteins involved in protein-protein interactions, eg, MNAR and VDR. By identifying such specific and discrete portions, biologically active peptides can be constructed, which mimic the biological activity of the biologically active protein, or which block the activity of the biologically active protein, or which selectively activate the biologically active protein. In this way, biologically active peptides can be constructed, which act as ligands that act on mammalian cells by binding to the receptor sites of those cells, to alter or affect their function or behavior, or to prevent the binding of the biologically active, natural protein to the cellular receptor, thereby preventing the biologically active protein from affecting the cell, or selectively modulating the receptor. The . Peptides can be synthesized by those of ordinary skill in the art, using well-known techniques and readily available starting materials. According to the invention, references to synthesis or construction of peptides, is considered herein to refer to the production of peptides similar in sequence or structure to the corresponding regions of MNAR and VDR identified to be involved in protein-protein interactions. . These peptides can be produced using any method known in the art, including but not limited to, chemical synthesis as well as biological synthesis in vitro or in vivo in a eukaryotic or prokaryotic expression system. The peptides may consist solely of the corresponding regions or may comprise the corresponding sequences and the addition sequences. The peptides of the invention may be biologically active as they are produced, or may require modification in order to assume a three-dimensional conformation that is biologically active. In general, the peptides are active as they are produced. However, some modifications may be necessary for the activity, and some modifications may be desirable to improve or alter the activity. Modifications that can be made, using standard techniques according to the invention include but are not limited to, cyclization, disulfide bond formation, glycosylation, phosphorylation or addition or subtraction of amino acid residues that include amino acid residues that serve to produce a useful three-dimensional conformation via a chemical bond that is not generally found in natural peptides and / or mimetics including but not limited to those described in Freidinger et al., 1980, Science 210: 656; Hinds et al., 1988, J. Chem. Soc. Chem. Comm. 1447; Kemp et al., 1984, J. Org. Chem. 49: 2296; Kemp et al, 1985, J. Org. Chem. 50: 5834; Kemp et al., 1988, Tetrahedron Lett. 29: 5077; Jones et al., 1988, Tetrahedron Lett 29: 3853. In addition, modifications can be made, using standard techniques, according to the invention to create dimers or oligomers of the curl or multiple curl structures. Peptidomimetics Peptidomimetics are compounds in which at least a portion of the MNAR is modified, such that the three-dimensional structure of the peptidomimetic remains substantially the same as that of the relevant interaction regions of the mature MNAR protein, and the compound retains the ability to bind to and modulate VDR In one embodiment, the peptidomimetic is designed to bind to the VDR in a manner independent of the ligand. In yet another embodiment, the peptidomimetic, as a single molecule, mimics the MNAR binding region (to VDR), and a region of the natural or synthetic ligand of VDR, and thus modulates VDR in a manner similar to the MNAR interaction. -linking-VDR. The peptidomimetics can be peptide analogues which are, per se, cyclic peptides containing one or more substitutions, or other modifications within the MNAR sequence. Alternatively, at least a portion of the sequence can be replaced with a non-peptidic structure such that the three-dimensional structure of the MNAR is substantially conserved. In other words, one, two or three amino acid residues within, for example, the LXXLL sequence can be replaced with a non-peptide structure for the improved link.

In addition, other peptide portions of MNAR can, but need not, be replaced with a non-peptidic structure. Peptidomimetics (peptide and non-peptidyl analogs) may have improved properties (eg, decreased proteolysis, increased retention, or increased bioavailability). The peptidomimetics generally have improved oral availability, which makes them especially suitable for the treatment of conditions such as cancer or osteoporosis. It should be noted that peptidomimetics may or may not have similar two-dimensional chemical structures, but they share three-dimensional structural features and common geometry. Each peptidomimetic may also have one or more additional linking elements. The present invention provides methods for identifying peptidomimetics. A variety of peptide modifications are known in the art and can be used to generate peptidomimetic compounds. See, for example, International Patent Publication No. WO 01/53331. Such modifications can also be used in the present invention to generate peptidomimetic compounds, as well as the specific modifications described below. All peptidomimetics will ideally have a three-dimensional structure that is substantially similar to a three-dimensional structure for example of the LXXLL portion of MNAR. In general, two dimensional structures are said to be substantially structurally similar to each other, if their pharmacophore atomic coordinates have a mean square root deviation - (RMSD) less than or equal to 1 angstrom, as calculated using the module of molecular similarity within the QUANTA program (QUANTA, available from Molecular Simulations Inc., San Diego, California). All peptidomimetics have at least one low energy three dimensional structure that is substantially similar to at least one low energy three dimensional structure of MNAR.

LIVING SELECTION In one embodiment of the invention, drugs that inhibit the activity or formation of protein-protein complexes are identified, tested, or optimized to prevent, for example, osteoporosis, transgenic mice or mice deleted in some gene such as the osteoblast ablation mouse (Mizumo et al., Biochem Biophys Res Commun., 1998 29; 247 (3): 610-5) or a soluble osteoclast differentiation factor, of overexpression in mice (Mizumo et al., Bone Miner Metab., 2002; 20 (6): 337-44). The test system of animal suppressed in some gene, or transgenic, are used to test the agents or drugs that reduce or inhibit osteoporosis or other bone disorders by modulating the expression or activity of the target proteins of drugs such as MNAR, specifically, the drugs that modulate the interactions between MNAR and VDR and / or the PI3 or cSrc kinase. In yet another aspect of the present invention, transgenic mice of MNAR or deleted in the MNAR gene can be used to further elucidate the role of MNAR in bone disorders, and any other disorders associated with steroid hormone receptors known to interact with MNAR, or to elucidate other proteins with which MNAR can interact and modulate its activity. Transgenic animals for use in the present invention can be prepared by any method, including but not limited to, modification of embryonic pluripotent stem (ES) and heteronuclear injection into blast cells, and such cells are known in the art ( see, for example, Coffman, Semin. Nephrol., 18: 404, 1997;? sther et al., Lab. Invest., 74: 953, 1996; Heddle, Environ Mol Mutagen 32: 110 4, 1998; Werner et al., Arzneimittelforschung. 48: 870 80, 1998; U.S. Patent Nos. 4,736,866 (Leder and Steward); 4,870,009 (Evans et al.); 5,718,883 (Harán and June); 5,614,396 (Bradley et al.) And 5,650,503 (Archibald et al. ).

A "deleted mammal in a gene" is a mammal (mouse) that contains within its genome a specific gene that has been inactivated by the method of directing the gene (see, for example, U.S. Patent Nos. 5,777,195 and 5,616,491) . A deleted mammal in a gene can be either a heterozygous deleted in a gene (eg, a defective allele and a wild-type allele) or a mutant or homozygous. A "substituted in a gene" mammal is a mammal in which an endogenous gene is substituted with a heterologous gene (Roamer et al., New Biol. 1991; 3: 331).

Preferably, the heterologous gene is "aggregated in a gene" to a locus of interest, either the subject of the evaluation (in which case the gene may be a reporter gene, see Ellegant et al., Proc. Nati, Acad. Sci. USA; 95: 11897, 1998) of the expression or function of a homologous gene, thereby linking the expression of the heterologous transcription gene from the appropriate promoter. This can be achieved by homologous recombination by transposon (Westphal and Leder, Curr Biol 1997; 7: 530), using mutant recombination sites (Araki et al., Nucleic Acids Res, 25: 868; 1997) or PCR (Zhang and Henderson , Biotechniques 1998; 25: 584). For example, transgenic animals "aggregated into a gene" can be created 'in which the MNAR gene is stably inserted into the transgenic animal genome; or where the animals are genetically engineered to constitutively express the endogenous MNAR, for example, by gene activation technology.

Treatment of Bone Disorders, Skin Disorders and Cancer As used herein, the term "therapeutically effective amount" is understood to refer to an amount or a compound that produces a medicinal effect observed as the reduction in the rate of loss. Bone in an individual when a therapeutically effective amount of a compound is administered to an individual who is susceptible to or suffering from a disease characterized by bone loss. Therapeutically effective amounts are typically determined by the effect they have compared to the effect observed when administered to a similarly situated individual, a composition that does not include the active ingredient. For example, VDR ligands that are found to selectively stimulate MPA kinase activity may possess significant bone-saving action, without affecting calcium homeostasis, which can be used for the treatment of osteoporosis. Alternatively, VDR ligands that selectively regulate VDR-mediated cell signaling can be used to treat patients with skin cancers. This could be a significant improvement to the treatment - using vitamin D3, since these drugs would not have the hypercalcemic effects of vitamin D3. In the end, the selective nature of rationally designed ligands could reduce or abrogate the plurality of side effects often associated with vitamin D3 treatment, such as hypercalcemia, which frequently results in nausea, headache, bone pain, high blood pressure, and kidney damage.

EXAMPLES The invention is also described by means of particular examples. However, the use of such examples is illustrative only and in no way limits the scope and meaning of the invention and any exemplified terms. Likewise, the invention is not limited to the particular preferred embodiments described herein. Rather many modifications and variations of the invention will be apparent to those skilled in the art after reading this specification and can be made without departing from its spirit and scope. The invention is therefore limited only by the terms of the appended claims, together with the full scope of the equivalents for which the claims are authorized. The following experiments indicate that VDR interacts independently of the ligand, with 'MNAR and that this interaction is measured by portion number 5 of LXXLL of MNAR. VDR in the presence of vitamin D3 interacts with cSrc (via the SH2 domain of cSrc) and overexpression of MNAR increases vitamin D3-induced activation of the MAP kinase pathway through the MNAR-cSrc interaction (via the domain SH3 from cSrc through a PXXP portion) and the phosphorylation of Erk kinases 1 and 2. The interaction of VDR1 / MNAR / PI3 kinase is also expected to be based on the results observed with the formation of the ternary complex involving ER / MNAR / PI3 kinase, described below. It was found that similarly to cSrc, MNAR interacts with the PI3 kinase via the SH3 domain of the p85 subunit of the PI3 kinase. These results provide additional evidence that MNAR is a scaffolding protein that incorporates nuclear receptor interaction within cell signaling mediated by kinase and are consistent with the MNAR action model that was previously proposed.

EXAMPLE 1: MNAR ASSOCIATION WITH VDR Reactive Methods. 1, 25 (OH) 2Vit. D3 was obtained from Sigma.

The biotinylated peptides corresponding to different LXXLL portions of MNAR were synthesized and purified by peptide chemistry group in Wyeth Research. Glutathione-sepharose spheres were obtained from Sigma. The anti-phosphotyrosine antibody, the SuperSignal Elisa Pico peroxidase substrate and the microplate coated with Reacti-Bindm NeutrAvidin ™ were from Pierce. GST abatement interaction analysis. A fusion of GST to the ligand binding domain of VDR, amino acids 110-427, designated GST-VDR-LBD, was expressed in BL-21 cells and linked to glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ). Wild-type MNAR was transcribed / translated and radiolabeled with 35S using the Promega TNT Quick (Madison, WI) coupled transcription / translation system and incubated for 1 hour at room temperature with the GST-VDR-LBD fusion protein bound to glutathione- Sepharose 4B in the absence or presence of 1, 25 (OH) 2Vit. D3 100 nM in linker buffer (50 mM Tris, pH 8.0, 150 mM sodium chloride, 1 mM DTT, 1 mM PMSF, 1 mM EDTA, 0.05% NP-40, IX protease cocktail). The spheres were then washed 4 times with the binding buffer and the bound proteins were diluted by the addition of the SDS buffer and analyzed by SDS-PAGE and autoradiography. Analysis of VDR-MNAR interaction based on ELISA.

A non-isotropic, rapid ELISA method was used for the characterization of VDR-MNAR interactions. The biotinylated peptides corresponding to different LXXLL portions of MNAR (designated 1-9, sequentially from the most N-terminal portion) were synthesized and immobilized on a microplate coated with Reacti-Bind * 111 NeutrAvidin1"1 (Pierce Biotechnology, Rockford, IL) The microplate was washed twice with the binding buffer (50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1 mM DTT, 1 mM EDTA, 0.01% NP-40 and 0.01% BSA) The peptides were diluted in 100 μl of binding buffer to a final concentration of 50 μM, and incubated with the microplate coated with Reacti-Bind1 NeutrAvidin ™ for 1 hour at room temperature, washed 4 times with the binding buffer. VDR-LBD, preincubated with vehicle ol, 25 (OH) 2D3 1 μM was allowed to interact with the immobilized peptide corresponding to one of each of the LXXLL portions of MNAR, for 2 hours at room temperature. the link absorber e incubated with anti-GST antibodies fused to HRP (Sigma, St. Louis, MO) for 1 hour in binding buffer, and washed 4 times more. The chemiluminescent substrate of SuperSignal® peak ELISA (Pierce Biotechnology, Rockford, IL) was used to detect the antigen / antibody complex, the signal was read using a Wallac Victor2 1420 Multilabel counter (PerkinElmer Lifesciences, Boston, MA).

RESULTS MNAR interacts with the ligand binding domain of the VDR. GST abatement demonstrates that MNAR and VDR interact. The data presented in Figure 1 indicate that MNAR interacts directly with VDR-LBD in a ligand-dependent manner. VDR interacts with the # 5 portion of LXXLL from MNAR. MNAR contains 10 putative LXXLL portions that could potentially mediate the interaction of MNAR with nuclear hormone receptors. The assays of the ELISA result using the anti-GST antibody fused to HRP (Figure 2) demonstrate that the VDR ligand binding domain dependently on the ligand, interacted with the peptide corresponding to the # 5 portion of LXXLL of MNAR.

EXAMPLE 2: AGONIST ACTIVATION OF VDR ACTIVITY BY MNAR Methods Analysis of Western Blotting. UMR-106 cells were transfected with the control and the MNAR expression vector, using the Lipofectamine 2000 reagent following the procedures suggested by the manufacturer. Cells were cultured for an additional 48 hours after transfection in medium supplemented with FBS purified with 2% char. After. 48 hours, the cells were treated with 10 nM l, 25 (OH) 2D3 for the indicated time. The cells were rinsed and harvested in cold PBS, then centrifuged and the supernatant was removed. The cells were lysed with 2 volumes of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, beta-glycerophosphate 1 mM, 1 mM Na2V04, 1 μg / μl leupeptin). Cell debris was removed by centrifugation and an equal amount of proteins was run on SDS-PAGE. The separated proteins were transferred to the microcellulose membrane and the levels of MNAR, ERK and p-ERK were determined by Western Blotting analysis. Quantitative PCR analysis (TaqMan). The UMR-106 and ROS 17 / 2.8 cells were transfected with the control with the MNAR expression vectors using the lipofectamine 2000 reagent following the procedures suggested by the manufacturers. After transfection, the cells were cultured in medium supplemented with FBS purified with 2% mineral carbon. 16 hours after transfection the cells were treated with 1, 25 (OH) 2Vit. D3 '100 nM for 24 to 48 hours. The cells were lysed and the total RNA was isolated using the RNeasy kit (Quiagen). Quantitative PCR (TaqMan) was performed to determine osteocalcin levels in UMR-106 cells and alkaline phosphatase levels in ROS 17 / 2.8 cells.

RESULTS MNAR increases the stimulation of Erk's activity by visum D3. The UMR-106 cells were transfected with the control or with the MNAR expression vectors and treated with 10 nM 1, 25 (OH) 2Vit. D3 for 0, 5, 10 or 20 minutes. The phosphorylation levels of Erk 1 and 2 in. Extracts of UMR-106 cells were evaluated using Western Blotting analysis with anti-Erk 1 antibody and phosphorylated 2 (Figure 3). Treatment of UMR-106 cells with 1, 25 (OH) 2Vit.D3 for 10 or 20 minutes resulted in increased levels of phosphorylated Erk 1 and 2, in the absence of MNAR. However, overexpression of MNAR led to a dramatic increase in Erk phosphorylation. These results indicate that MNAR controls the activation of the MPA kinase pathway induced by l, 25 (OH) 2Vit.D3. MNAR modulates gene expression dependent on vi-tamine D. Quantitative PCR was performed with RNA isolated from UMR 106 and Ros 17/2.8 cells treated with 1, 25 (OH) 2Vit. D3, to determine if MNAR can affect the gene expression induced by 1, 25 (OH) 2Vit .D3. The UMR 106 cells were transfected with the control or the MNAR expression plasmids, the RNA was isolated and analyzed using the TaqMan analysis (Figure 4A). Treatment with 1, 25 (0H) 2Vit. D3 did not affect the level of osteocalcin expression. However, the overexpression of MNAR resulted in a strong potentiation in the basal osteocalcin levels and stimulated with 1.25 (0H) 2Vit. D3. It was also determined whether MNAR affects the expression of alkaline phosphatase (AP) - an important marker for the differentiation of osteoblasts. The RNA from the Ros 17 / 2.8 cells treated with vehicle or with 1,25 (OH) 2Vit. D3, transfected with the control or the MNAR expression plasmids, was isolated and used for the TaqMan analysis (Figure 4B) . Overexpression of MNAR strongly increased the induction of AP expression level by 1,25 (OH) 2Vit .D3. These results demonstrate that MNAR can regulate the expression of the 1,25 (OH) 2Vit. D3 dependent gene, and the differentiation of osteoblast cells and suggests that MNAR plays an important role in bone development mediated by vitamin D3.

EXAMPLE 3: MODULATION OF PI3 KINASE IN ER BY MNAR Methods It was recently shown that MNAR affects the signaling mediated by the Pl3 / Akt kinase in ER via the formation of a ternary complex of ER / MNAR / PI3 kinase. MNAR was expected to have similar effects on the Pl3 / Akt kinases that signal through the VDR. Transfections and preparations of cellular used. - The MCF-7 cells were transfected with the control, the MNAR expression vector, or non-specific siRNA or MNAR-specific siRNA using the lipofectamine 2000 reagent following the procedures suggested by the manufacturers. The cells were cultured for an additional 48 hours after transfection in media supplemented with FBS purified with 2% charcoal. After 48 hours, the cells were treated with 10 nM 17β-estradiol for the indicated time. The cells were rinsed and harvested in cold PBS, then centrifuged and the supernatant was removed, the cells were lysed with two volumes of lysis buffer (20 mM Tris HCl, pH 7.5, 150 mM sodium chloride, 1 mM Na2EDTA, EGTA 1 mM, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na2V04, 1 μg / μl leupeptin). Cell debris was removed by centrifugation. For DNA interference, the non-specific siRNA or. The MNAR-specific siRNA was developed by Dharmacon Inc. Immunoprecipitation and kinase assays. Er was imunoprecipitated from the cellular ones (1 mg / ml protein) using the anti-ERa monoclonal antibody (D12, Santa Cruz) for 60 minutes 4aC, then added with 20μl of 50% suspension of G-Sepharose protein and incubated for an additional 60 minutes. The samples were centrifuged and the pellets were washed with 1 ml of lysis buffer 4 times, and analyzed by immunoblotting. p85 was immuno-precipitated from cell lysates (1 mg / ml protein) using the anti-p85 polyclonal antibody (Upstate Biotech). For the PI3-K reaction, p85 was immunoprecipitated from the cell lysates as described above. The kinase activity in the immunoprecipitate was detected using the E3-kinase Pl3-kinase ELISA kit, according to the manufacturer's instructions. Briefly, the reaction was run in a volume of 50 μl by the addition of 5 μl of the 10X reaction buffer and 10 μl of the PI (4.5) P2 substrate solution (10 μM) to the p85 immunoprecipitate. After the PI3K reactions were completed, the reaction products were first mixed and incubated with an antibody that detects PI (3,4,5) P3, then added to the microplate coated with PI (3,4,5) P3 for the competitive link. A secondary detection reagent linked to peroxidase and colorimetric staining was used to detect the antibody that detects PI (3,4,5) P3, which binds to the plate. The colorimetric signal was inversely proportional to the amount of PI (3,4,5) P3 produced by the activity of PI3-K. Akt kinase activity was detected in the immunoprecipitate with the anti-Akt antibody using the Cell Signaling kit (catalog # 9840). Briefly, 1 mg of the fusion protein of GSK-3 was added to the alkynoprecipitate of Akt as a substrate. The kinase reaction was run in a reaction buffer supplied by the manufacturer. The level of p-GSK-3 produced in the kinase reaction was analyzed by Western Blotting analysis. Analysis of Western Blotting. An equal amount of protein from the cell lysates was run on SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes and the Akt and p-Akt levels were determined by Western Blotting analysis. The antibody for Akt was from Cell Signaling (anti-Akt catalog 9272, anti-p-Akt catalog # 9271).

RESULTS Era, MNAR and p85 interact in MCF-7 cells. It has previously been shown that ER interacts with the p85 subunit of PI3 kinase (Migliaccio et al., J. Steroid Biochem.Mol. Biol. 2002; 83 (1-5 ): 31-5). It was first evaluated whether the endogenous ER, MNAR and the PI3-K-p85 regulatory subunit, interact in MCF-7 cells. To address this question MCF7 cells at rest were not stimulated, or stimulated with 10 mM estradiol for 20 minutes. The cells were lysed with the lysis buffer and the cell lysates were used to immunoprecipitate with either anti-ERa or anti-p85a antibodies. Each immunoprecipitate was analyzed by immunoblotting with anti-p85a, anti-ERa or anti-MNAR antibodies. The results show that the "treatment" with estradiol triggered the immunoprecipitation of MNAR, p85 and ERa.An association was not detected in the control immunoprecipitates.These data indicate that endogenous MNAR-ERa and p85 interact in MCF7 cells.Specifically, MNAR interacts with p85 via the SH3 domain of p85: Role of MNAR in the oestradiol-dependent activation of pl3-K To investigate the effect of MNAR on the activation induced by estradiol of PI3-K, MCF-7 cells were already transfected with the plasmid expressing MNAR or the MNAR-specific siRNA to down-regulate MNAR expression 48 hours after transfections, the cells were untreated or treated with 10 nM estradiol for 20 minutes, harvested and the activity level was evaluated. PI3-K in cell extracts To evaluate the activity of PI3 kinase, the production of Ptdlns-3P by immunoprecipitates of p85 was measured. The presence of MCF-7 cells with estradiol leads to the stimulation of PI3 kinase activity. Overexpression of MNAR increased the activation of estrogen-dependent PI3-K. In contrast, the depletion of the cells from MNAR using the MNAR specific siRNA, resulted in a decreased level of activation of PI3-K by estradiol. In addition, the Pl3-k inhibitor, LY294002, as well as the ER antagonist, ICI 182 780, blocked the effect of estradiol on PI3-K activity. These data indicate that the interaction between MNAR-ER and p85 lead to the activation of PI3 kinase. MNAR modulates the activation of Akt dependent on estradiol. It has been well established that activation of PI3 kinase leads to activation of Akt, and that Akt is the primary mediator of signaling initiated by PI3-K.

By modulating the activity of the targets downstream (3 '), Akt promotes cell survival and cell cycle progression. To assess whether MNAR affects Akt phosphorylation and activation in response to estradiol, MCF-7 cells were transfected with either the MNAR-expressing plasmid or MNAR-specific siRNA. 48 hours after the transfections, the cells were untreated or treated with 10 mM estradiol for 20 minutes. The cell extracts were then prepared and analyzed for the phosphorylated Akt level using the Western Blotting analysis. It was found that the treatment of MCF-7 cells with estradiol increased the level of phospho-Akt. In addition, the strong attenuation of Akt phosphorylation was observed in cells transfected with MNAR-specific siRNA, but not in cells transfected with non-specific siRNA. These results are consistent with the strong increase in the level of phospho-Akt detected in cells transfected with the plasmid that expresses MNAR, comparing cells transfected with the empty vector. These data indicate that MNAR regulates the level of Akt phosphorylation in response to estradiol. To determine whether increased phosphorylation of Akt leads to its activation, MCF-7 cells were transfected with either the empty vector or a plasmid for overexpression of MNAR. 48 hours after the. transfection, the cells were untreated or treated with estradiol for 20 minutes, then harvested and the Akt was immunoprecipitated with the anti-Akt antibody. The kinase reaction was performed with Akt precipitated using a polypeptide substrate derived from GSK-3. It is well known that activated Akt phosphorylates GSK-3. The phosphorylation level of GSK-3 was evaluated using Western Blotting analysis. The results of this experiment demonstrate that overexpression of MNAR stimulates Akt activation induced by estradiol. Taken together, these data demonstrate that MNAR plays a critical role in the activation of the Pl3 / Akt kinase pathway in response to estradiol. The same can be expected from the signaling of the Pl3 / Akt kinase via VDR, via the interaction and formation of the ternary complex of VDR / MNAR / p85. The present invention is not limited in scope by the specific embodiments described herein. Rather, various modifications of the invention in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. It is intended that such modifications fall within the scope of the appended claims. It should also be understood that all values are approximate, and are provided for description. Patents, patent applications, publications, product descriptions and protocols are cited throughout this application, the descriptions of which are incorporated by reference herein in their entirety, for all purposes. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (33)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for identifying a ligand that modulates the interaction of MNAR with the vitamin D3 receptor, characterized in that it comprises: (a) contacting a test compound with a reaction mixture comprising: (i) an MNAR polypeptide containing the fifth portion LXXLL; and (ii) a vitamin D3 receptor polypeptide containing the ligand binding domain, wherein the conditions of the reaction mixture allow the binding of the MNAR polypeptide to the vitamin D3 receptor polypeptide, to form a binding complex; (b) detecting the levels of binding complex formation in the reaction mixture, in the presence of the test compound; and (c) comparing the level of the binding complex formed in the presence of the test compound, at the level of the binding complex formed in the absence of said test compound, wherein an increase in the level of the binding complex formed in the presence of the compound test, indicates that the test compound can be a guide compound.

The method according to claim 1, characterized in that the MNAR polypeptide comprises the amino acid sequence described in SEQ ID No .: 4.

The method according to claim 1, characterized in that the reaction mixture is based on in cells.

4. The method according to claim 1, characterized in that the reaction mixture is cell-free.

5. The method according to claim 1, characterized in that the MNAR polypeptide comprises a minimum of the number 5 portion of LXXLL.

6. The method of compliance with the claim 1, characterized in that it further comprises detection of the binding of the test compound to the vitamin D3 receptor polypeptide.

7. A method for identifying a ligand that modulates the activity of a vitamin D3 receptor after the interaction of MNAR with the vitamin D3 receptor, characterized in that it comprises: (a) contacting a test compound with a mixture of a reaction comprising (i) an MNAR polypeptide; and (ii) a host cell comprising a functional vitamin D3 receptor polypeptide, wherein the conditions of the reaction mixture allow the binding of the MNAR polypeptide to the vitamin D3 receptor to form a binding complex; (b) detecting the activity of the vitamin D3 receptor in the reaction mixture in the presence of the test compound; and (c) comparing the level of the activity in the presence of the test compound, at the activity level in the absence of said test compound, wherein an increase in the level of activity in the presence of the test compound indicates that the compound of test can be a ligand that modulates VDR activity.

8. The method according to claim 7, characterized in that the MNAR polypeptide comprises. the amino acid sequence described in SEQ ID No .: 4.

The method according to claim 7, characterized in that the MNAR polypeptide comprises the number 5 of LXXLL.

The method according to claim 7, characterized in that it further comprises detecting the binding of the test compound to the polypeptide 'vitamin D3 receptor.

11. The method according to claim 7, characterized in that the host cell is an osteosarcoma cell.

The method according to claim 11, characterized in that the osteosarcoma cell is a UMR 106 cell or a ROS 17 / 2.8 cell.

13. The method according to claim 7, characterized in that the activity detected is the phosphorylation of the "Erk 1 or Erk -2 kinase 1.

The method according to the claim 7, characterized in that the activity detected is the expression of a. gene induced by the activation of the vitamin D3 receptor.

15. The method according to claim 14, characterized in that the gene is osteocalcin or alkaline phosphatase.

The method according to claim 15, characterized in that the gene is the osteocalcin having a nucleotide sequence described in SEQ ID NO: 5, or a nucleotide sequence that hybridizes to the nucleotide sequence described in SEQ ID No. 5.

The method according to claim 15, characterized in that the gene is alkaline phosphatase having a nucleotide sequence described in SEQ ID No .: 7, or having a nucleotide sequence that hybridizes to the nucleotide sequence described in SEQ ID No .: 7.

18. A method for modulating the VDR ligand-dependent activity in a cell, characterized in that it comprises contacting a MNAR polypeptide with a mixture of A reaction comprising: (i) a host cell that includes a functional vitamin D3 receptor polypeptide; and (ii) a ligand of vitamin D3, wherein the activity of the vitamin D3 receptor in the reaction mixture in the presence of the MNAR polypeptide is different compared to the activity of the vitamin D3 receptor in the absence of the MNAR polypeptide .

19. The method according to the claim 18, characterized in that the host cell is an osteosarcoma cell.

20. The method of compliance with the claim 19, characterized in that the osteosarcoma cell is a UMR 106 cell or a ROS 17 / 2.8 cell.

21. The method according to claim 20, characterized in that the activity detected is the phosphorylation of the Erk 1 or Erk 2 kinase.

22. The method according to claim 18, characterized in that the activity detected is the increase in the expression of a gene induced by the activation of the vitamin D3 receptor.

23. The method according to claim 22, characterized in that the gene is osteocalcin or alkaline phosphatase.

24. The method according to claim 23, characterized in that the gene is osteocalcin having a nucleotide sequence described in SEQ ID NO: 5, or a nucleotide sequence that hybridizes to the nucleotide sequence described in SEQ ID No. : 5.

The method according to claim 23, characterized in that the gene is alkaline phosphatase having a nucleotide sequence described in SEQ ID No .: 7, or having a nucleotide sequence that hybridizes to the nucleotide sequence described in SEQ ID No .: 7.

26. A peptide, characterized in that it comprises the number 5 of LXXLL of MNAR, and a detectable marker, wherein the peptide is a fragment of MNAR.

27. The peptide according to claim 26, characterized in that the peptide is LPGLLTSLL.

28. The peptide according to claim 26, characterized in that the label is biotin.

29. A composition, characterized in that it comprises the components according to claim 1.

30. A method for identifying a ligand that modulates signaling through the VDR receptor, characterized in that it comprises: (a) contacting a test compound with a reaction mixture comprising: (i) an MNAR polypeptide containing the fifth portion of LXXLL; and (ii) a vitamin D3 receptor polypeptide that contains the ligand binding domain, (iii). a functional cSrc or PI3 kinase; wherein the conditions of the reaction mixture allow the binding of the MNAR polypeptide to the vitamin D3 receptor polypeptide and the cSrc or PI3 kinase to form a ternary complex; (b) detecting the formation levels of the ternary complex in the reaction mixture in the presence of the test compound; and (c) comparing the level of the ternary complex formed in the presence of the test compound at the level of the binding complex formed in the absence of the test compound, wherein an increase in the level of the ternary complex formed in the presence of the test compound, indicates that the test compound can be a guide compound.

31. A method for identifying a ligand that modulates the activity of a vitamin D3 receptor after the interaction of MNAR with the vitamin D3 receptor, characterized in that it comprises: (a) contacting a test compound with a reaction mixture comprising: (i) an MNAR polypeptide; and (ii) a host cell comprising a functional polypeptide of the vitamin D3 receptor and PI3 kinase or functional cSrc, wherein the conditions of the reaction mixture allow the binding of the MNAR polypeptide to the receptor "of vitamin D3 and the kinase. cSrc or PI3 to form a ternary complex, (b) detect the activity of the vitamin receptor D3 in the reaction mixture in the presence of the test complex; and (c) comparing the level of the activity in the presence of the test compound at the activity level in the absence of the test compound., wherein an increase in the activity level in the presence of the test compound indicates that the test compound can be a ligand that modulates VDR activity.

32. The method according to claim 31, characterized in that the kinase is the PI3 kinase.

33. The method according to claim 32, characterized in that the activity detected is the phosphorylation of the Akt kinase.