Sclerostin and the inhibition of wnt signaling and bone formation

Abstract

The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass (HBM). In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293 cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by over-expression of Wnt coreceptor LRP5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transducing canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at the late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b. Given the plethora of evidence indicating that canonical Wnt signaling stimulates osteogenesis, we believe that the HBM phenotype associated with the loss of sclerostin may at least in part be attributed to an increase in canonical Wnt signaling resulting from the reduction in sclerostin-mediated Wnt antagonism.

1 - 97 . (canceled) 
     
     
         98 . A method for identifying a drug candidate or compound which promotes or inhibits a protein-protein interaction comprising:
 a. determining the virtual structure of a receptor protein;   b. identifying a particular binding cavity on said receptor protein;   c. identifying a specific site within said binding cavity; and   d. screening for a compound that fits into said specific site.   
     
     
         99 . The method of  claim 98  further comprising the step of identifying the compound with the highest binding affinity using the Cscore™ program. 
     
     
         100 . The method of  claim 98  wherein said step of identifying a particular binding cavity comprises conducting experiments based on the biological functions of the compounds. 
     
     
         101 . The method of  claim 100  wherein said experiments comprise mutational analyses. 
     
     
         102 . The method of  claim 98  wherein said receptor protein is non-soluble or membrane bound. 
     
     
         103 . A method for identifying a drug candidate or compound which promotes or inhibits a protein-protein interaction comprising:
 a. determining the virtual structure of a receptor protein;   b. identifying a particular binding cavity on said receptor protein;   c. identifying a specific site within said binding cavity;   d. screening for a compound that fits into said specific site using the UNITY™ program;   e. docking said compound into the cavity using the Flexx™ program; and   f. obtaining the compound with the highest binding affinity using the Cscore™ program.   
     
     
         104 . The method of  claim 103  wherein said step of identifying a particular binding cavity comprises conducting experiments based on the biological functions of the compounds. 
     
     
         105 . The method of  claim 104  wherein said experiments comprise mutational analyses. 
     
     
         106 . The method of  claim 103  wherein said receptor protein is non-soluble or membrane-bound. 
     
     
         107 . A method for identifying a drug candidate compound which binds to a receptor protein involved in bone formation or bone remodeling comprising:
 a. determining the virtual or computational structure of a non-soluble receptor protein comprising the use of amino acid sequencing, X-ray crystallography, NMR, analogs or derivatives of said receptor protein, or any combination thereof;   b. identifying a particular binding cavity on said receptor protein through the use of experiments based on biological function comprising mutational analysis, chemical modification of amino acids, co-crystallography, NMR, or any combination thereof;   c. identifying a specific binding site within said binding cavity based on mutations or chemical modifications;   d. screening for a compound that fits into said specific site using the UNITY™ program; and   e. identifying the compound with the highest binding affinity or the lowest energy using the Cscore™ program to find the compound with the best fit.   
     
     
         108 . A method for identifying a drug candidate compound which binds to a receptor protein involved in bone formation or bone remodeling comprising:
 a. determining the virtual or computational structure of a signal peptide comprising the use of amino acid sequencing, X-ray crystallography, NMR, analogs or derivatives of said signal peptide, or any combination thereof;   b. identifying a particular binding cavity on said receptor protein through the use of experiments based on biological function comprising mutational analysis, chemical modification, co-crystallography, NMR or any combination thereof;   c. identifying a specific binding site within said binding cavity based on the results obtained from said experiments;   d. screening for a compound that fits into said binding cavity or said specific site in said binding cavity using the UNITY™ program;   e. docking said compound into said binding cavity or said specific site in said binding cavity using the Flexx™ program; and   f. identifying the compound with the highest binding affinity or the lowest energy using the Cscore™ program.   
     
     
         109 . A method for identifying a drug candidate or compound which binds to a protein involved in a signal transduction system comprising:
 a. providing or determining the virtual or computational structure of said protein comprising the use of amino acid sequencing, X-ray crystallography, NMR, analogs or derivatives of said receptor protein, or any combination thereof;   b. identifying a particular binding cavity, site or domain on said protein using various experiments comprising mutational analysis, chemical modification of amino acids, co-crystallography, NMR, or any combination thereof;   c. identifying a specific binding site within said binding cavity, site or domain based on the results of said experiments; and   d. screening a library of drug candidate compounds to identify the drug candidate or compound having the best fit with said specific binding site or said binding cavity, site or domain.   
     
     
         110 . A method for identifying a drug candidate or compound which binds to a nonsoluble or membrane-bound protein involved in a signal transduction system comprising:
 a. providing or determining the virtual or computational structure of said protein comprising the use of amino acid sequencing, X-ray crystallography, NMR, analogs or derivatives of said receptor protein, or any combination thereof;   b. identifying a particular binding cavity, site or domain or said protein using various experiments comprising mutational analysis, chemical modification of amino acids, co-crystallography, NMR, or any combination thereof;   c. identifying a specific binding site within said binding cavity, site or domain based on the results of said experiments; and   d. screening a library of drug candidate compounds to identify the drug candidate or compound having the best fit with said specific binding site or said binding cavity, site or domain.   
     
     
         111 . A method for identifying a drug candidate or compound which binds to a protein involved in a Wnt signaling pathway comprising:
 a. providing or determining the virtual or computational structure of said protein comprising the use of amino acid sequencing, X-ray crystallography, NMR, analogs or derivatives of said receptor protein, or any combination thereof;   b. identifying a particular binding cavity, site or domain or said protein using various experiments comprising mutational analysis, chemical modification of amino acids, co-crystallography, NMR, or any combination thereof;   c. identifying a specific binding site within said binding cavity, site or domain based on the results of said experiments; and   d. screening a library of drug candidate compounds to identify the drug candidate or compound having the best fit with said specific binding site or said binding cavity, site or domain.   
     
     
         112 . A method for identifying a drug candidate or compound which binds to a protein involved in a signal transduction system for promoting or inhibiting bone growth comprising:
 a. providing or determining the virtual or computational structure of said protein comprising the use of amino acid sequencing, X-ray crystallography, NMR, analogs or derivatives of said receptor protein, or any combination thereof;   b. identifying a particular binding cavity, site or domain or said protein using various experiments comprising mutational analysis, chemical modification of amino acids, co-crystallography, NMR, or any combination thereof;   c. identifying a specific binding site within said binding cavity, site or domain based on the results of said experiments; and   d. screening a library of drug candidate compounds to identify the drug candidate or compound having the best fit with said specific binding site or said binding cavity, site or domain.   
     
     
         113 . A method for the regulation of bone formation or bone remodeling comprising administering at least one non-native compound, at least one fragment of a non-native compound, or any combination thereof, that decreases or eliminates the affinity of sclerostin to a receptor or co-receptor involved in bone formation or bone remodeling.