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Compositions and methods for bone formation and remodelingRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Chewing Gum TypeCompositions and methods for bone formation and remodeling description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060198791, Compositions and methods for bone formation and remodeling. Brief Patent Description - Full Patent Description - Patent Application Claims
Detailed Description of the Invention REFERENCE TO RELATED PATENT APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/504,860, filed on September 22, 2003, entitled "Compositions and Methods for Stimulation of Bone Formation." [0002] This application is related to the patent application entitled "Compositions and Methods for the Stimulation or Enhancement of Bone Formation and the Self-Renewal of Cells", by Dan Wu, et al. filed on May 19, 2004, and its entire contents is hereby incorporated by reference, in its entirety. FIELD OF THE INVENTION [0003] The present invention relates to the field of therapeutic methods, compositions and uses thereof, in the treatment of bone fractures, bone disease, bone injury, bone abnormality, tumors, growths or viral infections. More particularly, the methods and compositions of the invention are directed to the stimulation, enhancement and inhibition of bone formation or bone remodeling. [0004] All patents, patent applications, patent publications, scientific articles, and the like, cited or identified in this application are hereby incorporated by reference in their entirety in order to describe more fully the state of the art to which the present invention pertains. BACKGROUND OF THE INVENTION [0005] Osteoporosis is a major public health problem, and it is especially prevalent in aging populations (1, 15, 21). The majority of fractures that occur in people over the age of 65 are due to osteoporosis (15, 40). Peak bone mass is a determining factor in establishing the risk of osteoporotic fracture (Heaney et al., 2000), and studies indicate that genetic factors contribute significantly to the variance in peak bone mass. One of the genes that regulate bone mass has recently been identified via positional cloning. Loss of function mutations in low density lipoprotein receptor-related protein 5 (LRP5), a co-receptor for the canonical Wnt signaling pathway (27), were found to be associated with Osteoporosis-Pseudoglioma Syndrome (OPPG), an autosomal recessive disorder which shows a reduction of bone density in humans (9). In addition, two independent kindreds that manifest familial High Bone Mass (HBM) phenotypes were found to harbor a Gly171 to Val substitution mutation (G171V) in LRP5 (5, 22). More recently, additional HBM mutations were reported in the same structural domain of the G171V mutation (36). Moreover, mice in which the LRP5 genes were inactivated by gene targeting showed phenotypes similar to those of OPPG patients (16), and transgenic expression of LRP5G171V in mice resulted in HBM (2). Furthermore, mouse primary osteoblasts showed reduced responsiveness to Wnt in the absence of LRP5 (16), and Wnt (9) or activated .beta.-catenin (4) stimulated the canonical Wnt signaling activity and induced the production of the osteoblast marker alkaline phosphatase (AP) in osteoblast-like cells. Together, these pieces of evidence indicate that the canonical Wnt signaling pathway plays an important role in the regulation of bone development. [0006] Until recently, the canonical Wnt signaling pathway was believed to start when Wnt bound to frizzled Fz proteins. The seven transmembrane domain-containing Fz proteins suppress the Glycogen synthase kinase 3 (GSK3)-dependent phosphorylation of .beta.-catenin through ill-defined mechanisms involving Dishevelled proteins. This suppression leads to the stabilization of .beta.-catenin. .beta.-catenin can then interact with transcription regulators, including lymphoid enhancing factor-1 (LEF-1) and T cell factors (TCF), to activate gene transcription (7, 10, 38). Recently, genetic and biochemical studies have provided solid evidence to indicate that co-receptors are required for canonical Wnt signaling in addition to Fz proteins (27, 28). The fly ortholog of LRP5/6 (LRP5 or LRP6), Arrow, was found to be required for the signaling of Wg, the fly ortholog of Wnt-1 (37). LRP5 and LRP6 are close homologues which basically function the same way, yet exhibit, different expression patterns. In addition, LRP6 was found to bind to Wnt1 and regulate Wnt-induced developmental processes in Xenopus embryos (34). Moreover, mice lacking LRP6 exhibited developmental defects that are similar to those caused by deficiencies in various Wnt proteins (30). Furthermore, LRP5, LRP6 and Arrow were found to be involved in transducing the canonical Wnt signals by binding Axin and leading to Axin degradation and .beta.-catenin stabilization (25, 35). The LRP5/6-mediated signaling process does not appear to depend on Dishevelled proteins (18, 31). Recently, a chaperon protein, Mesd, was identified as required for LRP5/6 transport to the cell surface (6, 11). [0007] Xenopus Dickkopf (Dkk)-1 was initially discovered as a Wnt antagonist that plays an important role in head formation (8). Thus far, four members of Dkk have been identified in mammals (17, 26). These include Dkk1, Dkk2, Dkk3 and Dkk4. Dkk1 and Dkk2 inhibit canonical Wnt signaling by simultaneously binding to LRP5 or LRP6 and a single transmembrane protein Kremen (3, 23, 24, 32). It has been previously reported that the LRP5 HBM G171V mutation appeared to attenuate Dkk1-mediated antagonism to the canonical Wnt signaling (5). The present invention describes the mechanism for this attenuation. SUMMARY OF THE INVENTION [0008] The present invention describes a model which explains the functional interactions of cavities on domains of receptors or co-receptors involved in bone formation or bone remodeling with Dkk, Wnt, Mesd, or other proteins which function in similar ways. These receptors include, but are not limited to, the LRP5 receptor, the LRP6 receptor, and the frizzled receptor. The LRP5 receptor is comprised of four YWTD repeat domains. Each domain contains multiple YWTD repeats of amino acids. The LRP5 receptor also has an LDL receptor repeat. Both LRP5 and LRP6 are close homologues and function in basically the same way although they possess different expression patterns. [0009] The invention provides methods for identifying non-native or exogenous compounds which bind to or interact with these cavities to cause the stimulation, inhibition or regulation of Wnt signaling, and thus bone formation, tumorigenesis and any other biological and pathological process regulated by Wnt signaling. A non-native compound comprises a compound that is not naturally or normally found in a cell or organism, as opposed to a native compound which is not introduced from an outside source. The compounds were identified from a National Cancer Institute (NCI) database through various screening methods and assays. These compounds could also be modified to create derivates or analogues not found in the NCI database or in nature which also function effectively. Compounds were identified which disrupted Dkk and LRP5/6 interactions, Wnt and LRP5/6 interactions and Mesd and LRP5/6 interactions. BRIEF DESCRIPTION OF THE DRAWINGS [0010] FIG. 1 shows a schematic representation of wildtype LRP5 and its deletion mutants. [0011] FIG. 2 illustrates that the G171V mutation disrupts LRP5 trafficking. HEK cells were transfected with expression plasmids as indicated in the figure. One day later, the cells were lysed and immunoprecipitation was carried out using an anti-Flag antibody. Mesd was Flag-tagged whereas all LRP5 molecules were HA-tagged. The G171V mutation disrupted the interactions of both LRP5 with Mesd (FIG. 2A, lanes 1 and 3), and R12 with Mesd (FIG. 2B, lanes 1 and 2), while the E721 mutation did not affect the interaction (FIG. 2A, Lanes 2 and 3). The lower panels of FIG. 2A and FIG. 2B show equal amounts of Wt and mutant LRP5 input for the immunoprecipitation. [HEK cells were transfected with the Mesd plasmid and the expression plasmids indicated in the figure.] R12TGV, R12T, R1-4 and R1-4GV (GV) are AP fusion proteins, which are LRP5 mutants lacking transmembrane domains that may be secreted in the supernatants of the cell cultures. One day later, conditioned medium (CM) was collected and centrifuged at a high speed. The supernatants were immunoprecipitated by an anti-HA antibody (FIG. 2C) or used for an AP assay (FIG. 2D). Cells were also lysed in the SDS-PAGE sample buffer and analyzed by Western blotting (lower panels of FIG. 2C and FIG. 2D). The data shows that the G171V mutation inhibited the secretion of R12 and R1-4. FIG. 2E confirms that the G171V mutation interferes with cell surface transport of LRP5 through the use of a binding assay which detects LRP5 on the cell surface. The levels of cell surface LRP5 molecules were detected by Western analysis using streptavidin-horse radish peroxidase (SA-HRP) after the cell surfaces were biotinylated and LRP5 molecules were precipitated with anti-HA antibody (FIG. 2E, upper panel). The levels of LRP5 in the immunocomplexes are shown in the lower panel of FIG. 2E. [0012] FIG. 3 shows that the HBM G171V mutation of LRP5 is less susceptible to Dkk1-mediated inhibition of coexpressed Wnt activity. The left panel of FIG. 3A shows that when HEK cells were transfected with plasmids as indicated together with LEF-1 luciferase reporter plasmids in the presence or absence of Wnt1, the HBM G171V mutation did not lead to an increase in LEF-1-dependent transcriptional activity compared to the wildtype (Wt) LRP5 (LRP5.sub.Wt). The right panel of FIG. 3A shows expression levels of LRP5, LRP5.sub.G171V, LRP6, and LRP6.sub.G158V as determined by antibodies specific to the HA tag carried by LRP5 proteins or anti-LRP6 antibodies. FIG. 3B shows that when HEK cells were transfected with LEF-1 luciferase reporter plasmids, Wnt-1, Dkk1 and Kremen in the presence of Wt or G171V LRP5 as indicated in the figure. LEF-1 reporters-indicated Wnt activity is significantly higher in HEK cells expressing LRP5.sub.G171V than those expressing LRP5.sub.Wt when Dkk is present. The protein expression levels of Dkk1, Kremen and LRP5 were verified by Western blotting, as shown in FIG. 3C. [0013] FIG. 4 illustrates that cells expressing LRP5G171 show less Dkk1 binding sites than those expressing LRP5.sub.Wt (FIG. 4A). FIG. 4B showsequal amounts of Wt and mutant LRP5 expression after transfection. [0014] FIG. 5 shows that the second domain of LRP5 is required for Wnt activity. HEK cells were transfected with LEF activity reporter plasmids and expression plasmids. One day later, LEF reporter activity was measured, as previously described. The results in FIG. 5 show that LRP5.sub.R494Q and LRP5.sub.G479V (LRP5 with point mutations in the second domain) may abolish Wnt signaling compared to LRP5.sub.Wt. [0015] FIG. 6 illustrates that the third domain of LRP5 is required for Dkk-mediated antagonism. FIG. 6A shows that the third YWTD repeat domain is required for Dkk-mediated inhibition. HEK cells were transfected with LEF activity reporter plasmids, Kremen1 plasmids and expression plasmids. LRP5R12 or LRP5R124, but not LRP5R34, could still potentiate Wnt-stimulated LEF-1 activity, suggesting that either LRP5R12 or LRP5R124 retains the Wnt coreceptor function. However, Dkk1 could not inhibit Wnt signaling when LRP5R12 or LRP5R124 was present despite the coexpression of Kremen. This suggests that the third YWTD repeat domain is required for Dkk1-mediated inhibition. The expression level of LRP5.sub.Wt and its mutant molecules are shown in FIG. 6B. FIG. 6C illustrates that LRP5R34 contains Dkk1 binding sites and that E721 in R34 is required for Dkk1 binding. FIG. 6D is a schematic representation of the mutations. [0016] FIG. 7 shows that amino acid residues in the third YWTD repeat domain, consisting of interaction surfaces, are required for Dkk-mediated inhibition of Wnt. In FIG. 7A, the space filled model of the third YWTD repeat domain was deduced based on the structure of the LDL receptor YWTD repeat domain (13). Based on the three-dimensional structure, 19 LRP5 mutants containing Ala substitution mutations on the surface of the third YWTD repeat domain were generated. The ability of these mutant LRP5 proteins to resist Dkk1-mediated inhibition was determined. Nine of the mutants (more than 5%) showed altered sensitivity to Dkk1-mediated inhibition, and they all contained mutations that were localized on the same surface. In FIG. 7B, HEK cells were transfected with LEF activity reporter plasmids, Kremin1 plasmid, and expression plasmids. The expression of Wt and mutant LRP5 molecules are shown in the lower panel. Among 19 mutations, the E721 mutation showed the strongest effect on Dkk1-mediated inhibition of Wnt, followed by W781, and then Y719. LRP5.sub.G171v also showed an effect on the Dkk1-mediated inhibition of Wnt. [0017] FIG. 8 shows the two dimensional structures of three compounds obtained from the National Cancer Institute (NCI). NCI106164 (FIG. 8A) shows a 68% inhibitory effect on Dkk1 binding while NCI39914 (FIG. 8B) and NCI660224 (FIG. 8C) increase Dkk1 binding by 654% and 276%, respectively. [0018] FIG. 9 illustrates the two-dimensional structure of anthra-9,10-quinone (FIG.9A), a common substructure in NCI39914 and NCI660224. FIG. 9B shows the two-dimensional structure of NCI 657566. FIG. 9C shows the template that was used for the two-dimensional similarity search. 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