Reagents and methods for modulating dkk-mediated interactions

Abstract

The present invention provides reagents, compounds, compositions, and methods relating to novel interactions of the extracellular domain of LRP5, HBM (a variant of LRP5), and/or LRP6 with Dkk, including Dkk-1. The various nucleic acids, polypeptides, antibodies, assay methods, diagnostic methods, and methods of treatment of the present invention are related to and impact on Dkk, LRP5, LRP6, HBM, and Wnt signaling. Dkk, LRP5, LRP6, HBM, and Wnt are implicated in bone and lipid cellular signaling. Thus, the present invention provides reagents and methods for modulating lipid levels and/or bone mass and is useful in the treatment and diagnosis of abnormal lipid levels and bone mass disorders, such as osteoporosis.

We claim:  
     
         1 . A method of regulating LRP5, LRP6, or HBM activity in a subject comprising administering a composition which modulates a Dkk activity in an amount effective to regulate LRP5, LRP6, or HBM activity.  
     
     
         2 . The method of any of claims  1 ,  24 ,  28 ,  33 ,  36 ,  37 ,  48 ,  64 ,  65 ,  93 ,  98 ,  101 ,  105 ,  107 ,  111 , or  112 , wherein the Dkk is Dkk-1.  
     
     
         3 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein the Dkk is Dkk-1 and the Dkk activity is inhibited.  
     
     
         4 . The method of claims  1  or  24 , wherein the Dkk activity modulates bone mass and/or lipid levels.  
     
     
         5 . The method of  claim 4 , wherein bone mass is increased and/or lipid levels are decreased.  
     
     
         6 . The method of  claim 5 , wherein the increase in bone mass is determined via one or more of a decrease in fracture rate, an increase in bone strength, an increase in bone density, an increase in bone mineral density, an increase in trabecular connectivity, an increase in trabecular density, an increase in cortical density, an increase in bone diameter, and an increase in inorganic bone content.  
     
     
         7 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition comprises one or more compounds selected from the group consisting of Dkk interacting proteins, or a Dkk-binding fragment thereof.  
     
     
         8 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition comprises an antisense, a siRNA, or shRNA molecule which recognizes and binds to a nucleic acid encoding one or more Dkk interacting proteins.  
     
     
         9 . The method of any of claims  1 ,  24 ,  28 , or  33 , and wherein said composition comprises a Dkk peptide aptamer.  
     
     
         10 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition comprises a mimetic of a Dkk peptide aptamer.  
     
     
         11 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition inhibits Dkk binding to LRP5, LRP6, or HBM.  
     
     
         12 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition enhances binding of Dkk to LRP5, LRP6, or HBM.  
     
     
         13 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition comprises a Dkk interacting protein peptide aptamer.  
     
     
         14 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition comprises a mimetic of a Dkk interacting protein peptide aptamer.  
     
     
         15 . The method of any of claims  1 ,  24 ,  28  or  33 , wherein said composition inhibits Dkk interacting protein or Dkk-binding fragment thereof binding to Dkk.  
     
     
         16 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition enhances binding of Dkk interacting protein or Dkk-binding fragment thereof to Dkk.  
     
     
         17 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said subject is a vertebrate or an invertebrate organism.  
     
     
         18 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said subject is a mammal.  
     
     
         19 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said subject is a canine, a feline, an ovine, a primate, an equine, a porcine, a caprine, a camelid, an avian, a bovine, or a rodent.  
     
     
         20 . The method of  claim 19 , wherein said primate is a human.  
     
     
         21 . The method of any of claims  1 ,  24 ,  28 , or  33 , wherein said composition comprises an LRP5 peptide aptamer.  
     
     
         22 . The method of  claim 21 , wherein said peptide aptamer is OST262 (SEQ ID NO:208).  
     
     
         23 . The method of any of claims  1 ,  24 ,  28  or  33 , wherein the composition comprises an LRP5 antibody or an immunologically active fragment thereof.  
     
     
         24 . A method of regulating Dkk-Wnt pathway activity in a subject comprising administering a composition which modulates Dkk activity in an amount effective to regulate Dkk-Wnt pathway activity.  
     
     
         25 . The method of claims  24 ,  101 , or  107 , wherein the Wnt is one or more of Wnt1-Wnt19.  
     
     
         26 . The method of  claim 25 , wherein the Wnt is Wnt1, Wnt3, Wnt3a, or Wnt10b.  
     
     
         27 . The method of  claim 24  wherein said composition which modulates Dkk activity or modulates Dkk interaction with LRP5/LRP6/HBM is administered in an amount effective to modulate Wnt signaling.  
     
     
         28 . A method of modulating bone mass in a subject comprising administering to the subject a composition which modulates Dkk activity or Dkk interaction with LRP5, LRP6, or HBM in an amount effective to modulate bone mass in the subject.  
     
     
         29 . The method of  claim 28 , wherein bone mass is increased.  
     
     
         30 . The method of the previous claim, wherein the increase in bone mass is determined via one or more of a decrease in fracture rate, an increase in bone strength, an increase in bone density, an increase in bone mineral density, an increase in trabecular connectivity, an increase in trabecular density, an increase in cortical density, an increase in bone diameter, and an increase in inorganic bone content.  
     
     
         31 . The method of claims  28  or  36 , wherein said subject has a bone mass disorder selected from the group consisting of a bone development disorder, a bone fracture, age-related loss of bone, chrondrodystrophy, drug-induced bone disorder, high bone turnover, hypercalcemia, hyperostosis, osteogenesis imperfecta, osteomalacia, osteomyelitis, osteoporosis, Paget's disease, osteoarthritis, and rickets.  
     
     
         32 . The method of  claim 28 , wherein the composition which modulates Dkk activity or Dkk interaction with LRP5, LRP6, or HBM is administered in an amount effective to modulate the amount of trabecular and/or cortical tissue.  
     
     
         33 . A method of modulating lipid levels in a subject comprising administering to the subject a composition which modulates Dkk activity or Dkk interaction with LRP5, LRP6, or HBM in an amount effective to modulate lipid levels in the subject.  
     
     
         34 . The method of  claim 33 , wherein lipid levels are decreased.  
     
     
         35 . The method of  claim 33  or 36, wherein the subject has a lipid-modulated disorder and wherein the lipid-modulated disorder is selected from the group consisting of a cardiac condition, atherosclerosis, familial lipoprotein lipase deficiency, familial apoprotein CII deficiency, familial type 3 hyperlipoproteinemia, familial hypercholesterolemia, familial hypertriglyceridemia, multiple lipoprotein-type hyperlipidemia, elevated lipid levels due to dialysis and/or diabetes, and elevated lipid levels of unknown etiology.  
     
     
         36 . A method of diagnosing low or high bone mass and/or high or low lipid levels in a subject comprising examining expression of Dkk, LRP5, LRP6, HBM, or and HBM-like variant in the subject and determining whether Dkk, LRP5, LRP6, HBM, or an HBM-like variant is over- or under-expressed to determine whether subject has (a) high or low bone mass and/or (b) has high or low lipid levels.  
     
     
         37 . A method of screening for a compound which modulates the interaction of Dkk with LRP5, LRP6, HBM, or a Dkk-binding fragment of LRP5, LRP6, or HBM comprising: 
 (a) exposing Dkk and a LRP5, LRP6, and/or HBM binding fragment thereof to a compound; and    (b) determining whether said compound modulates Dkk interaction with the LRP5/LRP6/HBM binding fragment.    
     
     
         38 . The method of  claim 37 , wherein said modulation is determined by whether said compound binds to Dkk or the LRP5, LRP6, or HBM binding fragment thereof.  
     
     
         39 . The method of  claim 37 , wherein Dkk or a LRP-binding fragment thereof is attached to a substrate.  
     
     
         40 . The method of  claim 37 , wherein said compound comprises one or more compounds selected from the group consisting of Dkk interacting proteins, or a Dkk-binding fragment thereof.  
     
     
         41 . The method of  claim 37  or  48 , wherein said compound comprises a Dkk peptide aptamer.  
     
     
         42 . The method of  claim 37  or  48 , wherein said compound comprises a mimetic of a Dkk peptide aptamer.  
     
     
         43 . The method of  claim 37  or  48 , wherein said compound comprises a Dkk interacting protein peptide aptamer.  
     
     
         44 . The method of  claim 37  or  48 , wherein the compound comprises an LRP5 peptide aptamer.  
     
     
         45 . The method of  claim 44 , wherein the peptide aptamer is OST262 (SEQ ID NO:208).  
     
     
         46 . The method of  claim 37  or  48 , wherein the compound comprises an LRP5 antibody.  
     
     
         47 . The method of  claim 37  or  48 , wherein said compound is a mimetic of a Dkk interacting protein peptide aptamer.  
     
     
         48 . A method of screening for a compound which modulates the interaction of Dkk with a Dkk interacting protein comprising: 
 (a) exposing a Dkk interacting protein or a Dkk-binding fragment thereof to a compound; and    (b) determining whether said compound bound to a Dkk interacting protein or the Dkk-binding fragment thereof; and    (c) further determining whether said compound modulates the interaction of Dkk interacting protein and Dkk.    
     
     
         49 . The method of  claim 48 , wherein the Dkk interacting protein or a Dkk-binding fragment thereof is attached to a substrate.  
     
     
         50 . A composition comprising a LRP5, LRP6, or HBM activity-modulating compound and a pharmaceutically acceptable carrier therefor.  
     
     
         51 . The composition of  claim 50 , wherein said LRP5, LRP6, or HBM activity-modulating compound comprises a compound which binds to Dkk thereby modulating the interaction of Dkk with LRP5, LRP6, or HBM.  
     
     
         52 . The composition of  claim 50 , wherein said LRP5, LRP6, or HBM modulating compound comprises one or more Dkk interacting proteins and Dkk-binding fragments thereof.  
     
     
         53 . The composition of  claim 50 , wherein said LRP5, or LRP6, or HBM modulating compound is a monoclonal antibody or an immunologically active fragment thereof which binds to a Dkk interacting protein, or a Dkk-binding fragment thereof.  
     
     
         54 . The composition of  claim 53 , wherein the monoclonal antibody is human, chimeric, humanized, primatized®, or bispecific.  
     
     
         55 . The composition of  claim 50 , wherein said LRP5, LRP6, or HBM modulating compound comprises an antisense, a siRNA, or shRNA molecule which recognizes and binds to a nucleic acid encoding one or more Dkk interacting proteins.  
     
     
         56 . The composition of  claim 50 , wherein said LRP5, LRP6, or HBM modulating compound comprises a Dkk peptide aptamer.  
     
     
         57 . The composition of  claim 50 , wherein said LRP5, LRP6, or HBM modulating compound comprises a mimetic of a Dkk peptide aptamer.  
     
     
         58 . The composition of  claim 50 , wherein said LRP5, LRP6, or HBM modulating compound comprises a Dkk interacting protein peptide aptamer.  
     
     
         59 . The composition of  claim 50 , wherein said LRP5, LRP6, or HBM modulating compound comprises a mimetic of a Dkk interacting protein peptide aptamer.  
     
     
         60 . The composition of  claim 50 , wherein the compound comprises an LRP5 peptide aptamer.  
     
     
         61 . The composition of  claim 60 , wherein the peptide aptamer is OST262.  
     
     
         62 . The composition of  claim 50 , wherein the compound comprises an LRP5 antibody.  
     
     
         63 . A pharmaceutical composition comprising a compound which modulates Dkk activity and a pharmaceutically acceptable carrier therefor.  
     
     
         64 . A method for identifying compounds which modulate Dkk and LRP5/LRP6/HBM interactions comprising: 
 (a) creating an LRP5, LRP6, or HBM fluorescent fusion protein using a first fluorescent tag; and    (b) creating a Dkk fusion protein comprising a second fluorescent tag;    (c) adding a test compound; and    (d) assessing changes in the ratio of fluorescent tag emissions using Fluorescence Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET) to determine whether the compound modulates Dkk and LRP5/LRP6/HBM interactions.    
     
     
         65 . A method of identifying binding partners for a Dkk protein comprising the steps of: 
 (a) exposing the Dkk protein(s) or a LRP5/LRP6 binding fragment thereof to a potential binding partner; and    (b) determining if the potential binding partner binds to a Dkk protein or the LRP5/LRP6 binding fragment thereof.    
     
     
         66 . A nucleic acid encoding a Dkk interacting protein peptide aptamer comprising a nucleic acid encoding a scaffold protein in-frame with the activation domain of Gal4 or LexA that is in-frame with a nucleic acid that encodes a Dkk interacting protein amino acid sequence.  
     
     
         67 . A vector comprising the nucleic acid of  claim 66 .  
     
     
         68 . The nucleic acid of  claim 66 , wherein the scaffold protein is trxA.  
     
     
         69 . A method of detecting a modulatory activity of a compound on the binding interaction of a first peptide and a second peptide of a peptide binding pair that bind through extracellular interaction in their natural environment, comprising: 
 (i) culturing at least one eukaryotic cell comprising: 
 a) a nucleotide sequence encoding a first heterologous fusion protein comprising the first peptide or a segment thereof joined to a transcriptional activation protein DNA binding domain;  
 b) a nucleotide sequence encoding a second heterologous fusion protein comprising the second peptide or a segment thereof joined to a transcriptional activation protein transcriptional activation domain;  
 wherein binding of the first peptide or segment thereof and the second peptide or segment thereof reconstitutes a transcriptional activation protein; and  
 c) a reporter element activated under positive transcriptional control of the reconstituted transcriptional activation protein, wherein expression of the reporter element produces a selected phenotype;  
   (ii) incubating the eukaryotic cell in the presence of a compound under conditions suitable to detect the selected phenotype; and    (iii) detecting the ability of the compound to affect the binding interaction of the peptide binding pair by determining whether the compound affects the expression of the reporter element which produces the selected phenotype;    wherein (1) said first peptide is a Dkk peptide and the second peptide is a peptide selected from LRP5, HBM, LRP6 and the Dkk-binding portion of LRP5/LRP6/HBM or (2) said first peptide is a Dkk interacting protein or the Dkk-binding fragment thereof and said second peptide is a Dkk peptide.    
     
     
         70 . The method of  claim 69 , wherein the eukaryotic cell is a yeast cell.  
     
     
         71 . The method of  claim 70 , wherein the yeast cell is Saccharomyces.  
     
     
         72 . The method of  claim 71 , wherein the Saccharomyces cell is  Saccharomyces cerevisiae.    
     
     
         73 . The method of  claim 69 , wherein the Dkk is Dkk-1 and wherein the compound comprises one or more Dkk interacting proteins or a Dkk-binding fragment thereof.  
     
     
         74 . The method of  claim 73 , wherein the compound is directly added to assay.  
     
     
         75 . The method of  claim 73 , wherein the compound is recombinantly expressed by said eukaryotic cell in addition to said first and second peptides.  
     
     
         76 . The method of  claim 69 , wherein the compound comprises a Dkk peptide aptamer.  
     
     
         77 . The method of  claim 69 , wherein the compound comprises a mimetic of a Dkk peptide aptamer.  
     
     
         78 . The method of  claim 69 , wherein the compound comprises a Dkk interacting protein peptide aptamer.  
     
     
         79 . The method of  claim 69 , wherein the compound comprises a mimetic of a Dkk interacting protein peptide aptamer.  
     
     
         80 . The method of  claim 69 , wherein the eukaryotic cell further comprises at least one endogenous nucleotide sequence selected from the group consisting of a nucleotide sequence encoding the DNA binding domain of a transcriptional activation protein, a nucleotide sequence encoding the transcriptional activation domain of a transcriptional activation protein, and a nucleotide sequence encoding the reporter element, wherein at least one of the endogenous nucleotide sequences is inactivated by mutation or deletion.  
     
     
         81 . The method of  claim 69 , wherein the peptide binding pair comprises a ligand and a receptor to which the ligand binds.  
     
     
         82 . The method of  claim 69 , wherein the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, QalF, VP16, or a mammalian nuclear receptor.  
     
     
         83 . The method of  claim 69 , wherein at least one of the heterologous fusion proteins is expressed from an autonomously-replicating plasmid.  
     
     
         84 . The method of  claim 69 , wherein the DNA binding domain is a heterologous DNA-binding domain of a transcriptional activation protein.  
     
     
         85 . The method of  claim 84 , wherein the DNA binding protein is selected from the group consisting of a mammalian steroid receptor and bacterial LexA protein.  
     
     
         86 . The method of  claim 69 , wherein the reporter element is selected from the group consisting of lacZ, a polynucleotide encoding luciferase, a polynucleotide encoding green fluorescent protein (GFP), and a polynucleotide encoding chloramphenicol acetyltransferase.  
     
     
         87 . The method of  claim 86 , wherein the reporter element is LacZ.  
     
     
         88 . The method of  claim 69 , wherein the test sample comprises an LRP5 peptide aptamer.  
     
     
         89 . The method of  claim 88 , wherein the peptide aptamer is OST262 (SEQ ID NO:208).  
     
     
         90 . The method of  claim 69 , wherein the test sample comprises an LRP5 antibody.  
     
     
         91 . A transgenic animal wherein Dkk-1 is knocked out in a tissue-specific fashion.  
     
     
         92 . The transgenic animal of  claim 91 , wherein the tissue specificity is bone tissue, cancer tissue, or liver tissue.  
     
     
         93 . A method for identifying potential compounds which modulate Dkk activity comprising: 
 a) measuring the effect on binding of one or more Dkk interacting proteins, or a Dkk-binding fragment thereof, with Dkk or a fragment thereof in the presence and absence of a compound; and    b) identifying as a potential Dkk modulatory compound a compound which modulates the binding between one or more Dkk interacting proteins or Dkk-binding fragment thereof and Dkk or fragment thereof.    
     
     
         94 . A peptide aptamer of FIG. 3 (SEQ ID NOs:171-188) or FIG. 4 (SEQ ID NOs:189-192).  
     
     
         95 . An antibody or antibody fragment which recognizes and binds to one or more peptides of amino acid sequences GNKYQTIDNYQPYPC (SEQ ID NO:118), LDGYSRRTTLSSKMYHTKGQEG (SEQ ID NO:119), RIQKDHHQASNSSRLHTCQRH (SEQ ID NO:120), RGEIEETITESFGND (SEQ ID NO:121), EIFQRCYCGEGLSCRIQKD (SEQ ID NO:122), MYWTDWVETPRIE (SEQ ID NO:123), MYWTDWGETPRIE (SEQ ID NO:124), KRTGGKRKEILSA (SEQ ID NO:125), ERVEKTTGDKRTRIQGR (SEQ ID NO:126), KQQCDSFPDCIDGSDE (SEQ ID NO:127), or a Dkk-1 amino acid sequence selected from the group consisting Asn34-His266 (SEQ ID NO:110), Asn34-Cys245 (SEQ ID NO:111), Asn34-Lys182 (SEQ ID NO:112), Cys97-His266 (SEQ ID NO:113), Val139-His266 (SEQ ID NO:114), Gly183-His266 (SEQ ID NO:115), Cys97-Cys245 (SEQ ID NO:116), or Val139-Cys245 (SEQ ID NO:117).  
     
     
         96 . The antibody or antibody fragment of  claim 95 , wherein the antibody is a monoclonal antibody.  
     
     
         97 . The antibody or antibody fragment of  claim 95 , wherein the antibody is a polyclonal antibody  
     
     
         98 . A method of identifying Dkk interacting proteins which modulate the interaction of Dkk with the Wnt signaling pathway comprising: 
 (a) injecting Dkk and potential Dkk interacting protein mRNA into a Xenopus blastomere; and    (b) assessing axis duplication or analyzing marker gene expression; and    (c) identifying compositions which elicit changes in axis duplication or marker gene expression as Dkk interacting proteins which modulate the interaction of Dkk with the Wnt signaling pathway.    
     
     
         99 . The method of  claim 98 , wherein the mRNA of HBM, LRP5/6, any Wnt, Wnt antagonist, Wnt pathway modulator, or combination of these is co-injected into the Xenopus blastomere.  
     
     
         100 . The method of  claim 98 , wherein the marker gene analyzed is Siamois, Xnr3, slug, Xbra, HNK-1, endodermin, Xlhbox8, BMP2, BMP4, XLRP6, EF-1, or ODC.  
     
     
         101 . A method for identifying Dkk interacting proteins which modulate the interaction of Dkk with the Wnt signaling pathway comprising: 
 (a) transfecting cells with constructs containing Dkk and potential Dkk interacting proteins; and    (b) assessing changes in expression of a reporter gene linked to a Wnt-responsive promoter; and    (c) identifying as a Dkk interacting protein any protein which alters reporter gene expression compared with cells transfected with a Dkk construct alone.    
     
     
         102 . The method of  claim 101 , wherein the cells are HOB-03-CE6, HEK293, or U2OS cells.  
     
     
         103 . The method of  claim 101 , wherein the Wnt-responsive promoter is TCF or LEF.  
     
     
         104 . The method of  claim 101 , wherein the cells are co-transfected with CMV -galactosidase.  
     
     
         105 . A method for identifying compounds which modulate Dkk and LRP5/LRP6/HBM interactions comprising: 
 (a) immobilizing LRP5/LRP6/HBM to a solid surface; and    (b) treating the solid surface with a secreted Dkk protein or a secreted epitope-tagged Dkk and a test compound; and    (c) determining whether the compound regulates binding between Dkk and LRP5/LRP6/HBM using antibodies to Dkk or the epitope tag or by directly measuring activity of an epitope tag.    
     
     
         106 . The method of  claim 105 , wherein the epitope tag is alkaline phosphatase, histidine, or a V5 tag.  
     
     
         107 . A method for identifying compounds which modulate the interaction of Dkk with the Wnt signaling pathway comprising: 
 (a) transfecting cells with constructs containing Dkk and Wnt proteins;    (b) assessing changes in expression of a reporter element linked to a Wnt- responsive promoter; and    (c) identifying as a Dkk/Wnt interaction modulating compound any compound which alters reporter gene expression compared with cells transfected with a Dkk construct alone.    
     
     
         108 . The method according to  claim 107 , wherein Wnt3a and Wnt1 constructs are co-transfected into the cells.  
     
     
         109 . The method according to  claim 107 , wherein the cells are U2-OS, HOB-03-CE6, or HEK293 cells.  
     
     
         110 . The method according to  claim 107 , wherein the reporter element used is TCF-luciferase, tk-Renilla, or a combination thereof.  
     
     
         111 . A method of testing compounds that modulate Dkk-mediated activity in a mammal comprising 
 (a) providing a group of transgenic animals having (1) a regulatable one or more Dkk genes, (2) a knock-out of Dkk genes, or (3) a knock-in of one or more Dkk genes;    (b) providing a second group of control animals respectively for the group of transgenic animals in step (a); and    (c) exposing the transgenic animal group and control animal group to a potential Dkk-modulating compound which modulates bone mass or lipid levels; and    (d) comparing the transgenic animals and the control group of animals and determining the effect of the compound on bone mass or lipid levels in the transgenic animals as compared to the control animals.    
     
     
         112 . A method of screening for compounds or compositions which modulate the interaction of Dkk and a Dkk interacting protein comprising: 
 (a) exposing a Dkk interacting proteins or a Dkk-binding fragment thereof to a compound; and    (b) determining whether said compound binds to a Dkk interacting proteins or the Dkk-binding fragment thereof.    
     
     
         113 . The method of  claim 112 , wherein said modulation is determined by whether said compound binds to the Dkk interacting protein or the Dkk-binding fragment thereof.  
     
     
         114 . An antibody or antibody fragment which recognizes and binds to a sequence depicted in FIG. 3 (SEQ ID NOs:171-188) or FIG. 4 (SEQ ID NOs: 189-192).