Molecular targets and compounds, and methods to identify the same, useful in the treatment of bone and joint degenerative diseases

Abstract

The present invention relates to methods for identifying agents capable of inhibiting the expression or activity of proteins involved in the processes modulating osteoclastogenesis, which inhibition is useful in the prevention and/or treatment of bone and joint degenerative diseases and diseases involving aberrant activity or differentiation of osteoclasts. In particular, the present invention provides methods for identifying agents for use in the prevention and/or treatment of rheumatoid arthritis.

1 . A method for identifying a compound that inhibits bone resorption, comprising:
 (a) contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41-69 and 80, and fragments thereof; and   (b) measuring a compound-polypeptide property related to bone resorption.   
     
     
         2 . The method according to  claim 1 , wherein said polypeptide is in an in vitro cell-free preparation. 
     
     
         3 . The method according to  claim 1 , wherein said polypeptide is present in a mammalian cell. 
     
     
         4 . The method of  claim 2 , wherein said property is a binding affinity of said compound to said polypeptide. 
     
     
         5 . The method of  claim 4 , which additionally comprises the steps of:
 c) contacting a population of mammalian cells expressing said polypeptide with the compound that exhibits a binding affinity of at least 10 micromolar; and   d) identifying a compound that inhibits bone resorption.   
     
     
         6 . The method of  claim 1 , wherein said property is upregulation of a biological pathway producing a biochemical marker indicative of the inhibition of bone resorption. 
     
     
         7 . The method of  claim 6  wherein said indicator is osteoprotegerin. 
     
     
         8 . The method of  claim 1 , wherein said property is the activity of said polypeptide. 
     
     
         9 . The method of  claim 1 , wherein said property is the expression of said polypeptide. 
     
     
         10 . The method according to  claim 8  or  9 , which additionally comprises the steps of:
 c) contacting a population of mammalian cells expressing said polypeptide with the compound that significantly inhibits the expression or activity of the polypeptide; and 
 d) identifying the compound that inhibits bone resorption. 
 
     
     
         11 . The method according to  claim 1 , which additionally comprises the step of comparing the compound to be tested to a control. 
     
     
         12 . The method according to  claim 11 , wherein said control is where the polypeptide has not been contacted with said compound. 
     
     
         13 . The method according to  claim 5  or  10 , which additionally comprises the step of comparing the compound to a control, wherein said control is a population of mammalian cells that does not express said polypeptide. 
     
     
         14 . The method according to  claim 1 , wherein said compound is selected from the group consisting of compounds of a commercially available screening library and compounds having binding affinity for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41-69 and 80. 
     
     
         15 . The method according to  claim 1 , wherein said compound is a peptide in a phage display library or an antibody fragment library. 
     
     
         16 . An agent effective in inhibiting bone resorption, selected from the group consisting of an antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence of about 17 to about 30 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-29 and 40. 
     
     
         17 . The agent according to  claim 16 , wherein a vector in a mammalian cell expresses said agent. 
     
     
         18 . The agent according to  claim 16 , which is effective in inducing osteoprotegerin (OPG) expression in the OPG assay. 
     
     
         19 . The agent according to  claim 17 , wherein said vector is an adenoviral, retroviral, adeno-associated viral, lentiviral, a herpes simplex viral or a sendaiviral vector. 
     
     
         20 . The agent according to  claim 16 , wherein said antisense polynucleotide and said siRNA comprise an antisense strand of 17-25 nucleotides complementary to a sense strand, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-29 and 40. 
     
     
         21 . The agent according to  claim 20 , wherein said siRNA further comprises said sense strand. 
     
     
         22 . The agent according to  claim 21 , wherein said sense strand is selected from the group consisting of SEQ ID NO: 81-97 and 107. 
     
     
         23 . The agent according to  claim 20 , wherein said siRNA further comprises a loop region connecting said sense and said antisense strand. 
     
     
         24 . The agent according to  claim 23 , wherein said loop region comprises a nucleic acid sequence selected from the group consisting of UUGCUAUA or GUUUGCUAUAAC (SEQ ID NO: 108). 
     
     
         25 . The agent according to  claim 16 , wherein said agent is an antisense polynucleotide, ribozyme, or siRNA comprising a nucleic acid sequence complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 81-97 and 107. 
     
     
         26 . A bone resorption inhibiting pharmaceutical composition comprising a therapeutically effective amount of an agent according to  claim 16  in admixture with a pharmaceutically acceptable carrier. 
     
     
         27 . A method for treatment and/or prevention of a disease involving an imbalance in bone metabolism in a subject suffering from or susceptible to the disease comprising administering to the subject the pharmaceutical composition of  claim 26 . 
     
     
         28 . The method according to  claim 27  wherein the disease is a joint degenerative disease. 
     
     
         29 . The method according to  claim 28 , wherein the disease is rheumatoid arthritis. 
     
     
         30 . A method for treatment and/or prevention of a disease involving abnormal bone resorption in a subject comprising administering to the subject the agent of  claim 16 . 
     
     
         31 . The method according to  claim 30 , wherein the disease is selected from the group consisting of joint degenerative and inflammation diseases. 
     
     
         32 . The method according to  claim 30 , wherein the disease is rheumatoid arthritis. 
     
     
         33 . A method for treatment or prevention of a condition characterized by abnormal osteoprotegrin (OPG) expression and/or activity in a subject comprising administering to the subject the agent of  claim 16 . 
     
     
         34 . The method of any of  claim 27 ,  30  or  33  wherein the treatment and/or prevention additionally comprises administering said pharmaceutical composition or said OPG inducing agent in combination with a disease-modifying anti-rheumatic drug (DMARD) or an anti-inflammatory compound. 
     
     
         35 . The method according to  claim 34 , wherein said DMARD is selected from the group consisting of Infliximab, Etanercept, Adalimumab, Rituximab, CTLA4-Ig methotrexate, leflunomide and sulfasalazine. 
     
     
         36 . The method according to  claim 34 , wherein said anti-inflammatory agent is selected from the group consisting of corticosteroids or non-steroidal anti-inflammatory agents. 
     
     
         37 . A method for diagnosing a pathological condition involving abnormal bone resorption or a susceptibility to the condition in a subject, comprising determining a first amount of polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41-67 and 80 present in a biological sample obtained from said subject, and comparing said first amount with the ranges of amounts of the polypeptide determined in a population of healthy subjects, wherein an increase of the amount of polypeptide in said biological sample compared to the range of amounts determined for healthy subjects is indicative of the presence of the pathological condition.