Molecular targets and compounds, and methods to identify the same, useful in the treatment of joint degenerative and inflammatory diseases
The application discloses methods for identifying and using compounds that inhibit extra-cellular matrix (ECM) degradation and inflammation, using a polypeptide sequence including SEQ ID NO: 17-127 (hereinafter “TARGETS”) and fragments thereof, expression inhibitory agents such as antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), comprising a nucleic acid sequence complementary to, or engineered from, a naturally occurring polynucleotide sequence encoding a polypeptide of SEQ ID NO: 17-127, useful in pharmaceutical compositions comprising said agent, for the treatment, or prevention, of chronic joint degenerative and/or inflammatory diseases such as rheumatoid arthritis.
1. A method for identifying a compound that inhibits extra-cellular matrix degradation comprising
contacting a compound with a polypeptide comprising the amino acid sequence of SEQ ID NO:18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 or 64 in an in vitro cell-free preparation;
and measuring the binding affinity of said compound in said in vitro preparation;
selecting a candidate compound for confirmation as an inhibitor of extra-cellular matrix degradation, which candidate compound is selected based on its binding affinity for one or more of the polypeptide of the aforesaid SEQ ID Nos.
2. The method according to claim 1 , wherein said compound is a peptide in a phage display library or an antibody fragment library.
3. A method for identifying a compound that inhibits extra-cellular matrix degradation in a mammalian cell that expresses an extra-cellular matrix degrading protein, and that expresses a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, said method comprising
(a) contacting a compound with a polypeptide comprising the amino acid sequence of SEQ ID NO:18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 or 64, in an in vitro cell-free preparation;
(b) determining the binding affinity of said compound in said in vitro preparation;
(c) contacting said compound having binding affinity to, and capable of forming a complex with, said polypeptide of SEQ ID NO:18 with a mammalian cell, in which said polypeptide comprising the amino acid sequence of SEQ ID NO:18 is expressed, and which expresses an extra-cellular matrix degrading protein; and
(d) measuring levels of one or more extra-cellular matrix degrading proteins expressed by said mammalian cell; and
(e) determining if said levels of said one or more extra-cellular matrix degrading proteins are decreased compared to levels of said extra-cellular matrix degrading proteins expressed in said mammalian cell that is not contacted with said compound.
4. The method according to claim 3 , wherein the extra-cellular matrix degrading protein is selected from the group consisting of matrix metallo protease and Cathepsin.
5. The method according to claim 3 wherein said compound having binding affinity to, and capable of forming a complex with, said polypeptide of SEQ ID NO:18 exhibits a binding affinity of at least 10 micromolar.
6. The method according to claim 5 , wherein said mammalian cell is a synovial fibroblast.
7. The method according to claim 6 , wherein each of said mammalian synovial fibroblast that is contacted with said compound and that is not contacted with said compound is contacted with a cytokine agonist or with cytokine-producing cells.
8. A method for identifying a compound that inhibits extra-cellular matrix degradation in a mammalian cell that expresses an extra-cellular matrix degrading protein, and that expresses a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, said method comprising
(a) contacting a compound with a polypeptide comprising the amino acid sequence of SEQ ID NO:18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 or 64, in an in vitro cell-free preparation;
(b) determining the binding affinity of said compound in said in vitro preparation;
(c) contacting said compound having binding affinity to, and capable of forming a complex with, said polypeptide of SEQ ID NO:18 with a mammalian cell, in which said polypeptide comprising the amino acid sequence of SEQ ID NO:18 is expressed, and which expresses an extra-cellular matrix degrading protein; and
(d) measuring levels of ion channel activity in said mammalian cell; and
(e) determining if said levels of ion channel activity in said mammalian cell contacted with said compound are decreased as compared to levels of said ion channel activity in said mammalian cell that is not contacted with said compound;
selecting a candidate compound, based on its level of ion channel activity, for confirmation as an inhibitor of extra-cellular matrix degradation.
9. The method according to claim 8 further comprising the steps of
(f) measuring levels of one or more extra-cellular matrix degrading protein expressed by said mammalian cell; and
(g) determining if said levels of said one or more extra-cellular matrix degrading protein expressed by said mammalian cell are decreased as compared to levels of said extra-cellular matrix degrading protein expressed in said mammalian cell that is not contacted with said compound.
10. The method according to claim 9 , wherein the extra-cellular matrix degrading protein is selected from the group consisting of matrix metallo protease and Cathepsin.
11. The method according to claim 9 , wherein said compound having binding affinity to, and capable of forming a complex with, said polypeptide of SEQ ID NO:18 exhibits a binding affinity of at least 10 micromolar.
12. The method according to claim 9 , wherein said mammalian cell is a synovial fibroblast.
13. The method according to claim 12 , wherein each of said mammalian synovial fibroblast that is contacted with said compound and that is not contacted with said compound is contacted with a cytokine agonist or with cytokine-producing cells.
14. The method according to claim 3 , wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:18.
15. The method according to claim 3 , wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 or 64.
16. The method according to claim 8 , wherein said polypeptide comprises the amino acid sequence of SEQ ID NO:18.
17. The method according to claim 8 , wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 or 64.