Molecular targets and compounds, and methods to identify the same, useful in the treatment of fibrotic diseases
The present invention relates to methods and assays for identifying agents useful in the treatment of fibrotic conditions. The invention provides polypeptide and nucleic acid TARGETs, siRNA sequences based on these TARGETs and antibodies against the TARGETs. The invention is further related to pharmaceutical composition comprising siRNA sequences based on the TARGETs and antibodies against the TARGETs for use in the treatment of fibrotic conditions. The invention further provides in vitro methods for reduction or inhibition of macrophage differentiation into alternatively-activated macrophages (M2).
1. A method for identifying a compound that inhibits macrophage differentiation into M2 macrophages, said method comprising:
a) contacting a test compound with a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 26;
b) measuring a binding affinity of the test compound to said polypeptide;
c) contacting the test compound that demonstrates binding affinity to said polypeptide with a population of macrophage cells;
d) measuring a property related to differentiation of macrophages into M2 macrophages; and
e) identifying a compound capable of inhibiting macrophage differentiation into M2 macrophages.
2. The method according to claim 1 , which additionally comprises the step of comparing the compound to be tested to a control.
3. The method of claim 1 , wherein said polypeptide is coupled to a detectable label.
4. The method according to claim 1 , wherein said polypeptide in steps (a) and (b) is present in an in vitro cell-free preparation.
5. The method according to claim 1 , wherein said polypeptide in steps (a) and (b) is present in a cell.
6. The method according to claim 5 , wherein the cell naturally expresses said polypeptide.
7. The method according to claim 5 , wherein the cell has been engineered so as to express said polypeptide.
8. The method according to claim 5 , wherein said cell is a mammalian cell.
9. The method of claim 8 , wherein said cell is a macrophage cell.
10. The method of claim 1 , wherein said property is the inhibition of release or expression of a marker of M2 macrophages.
11. The method of claim 10 wherein said property is an expression or release of a marker selected from the group consisting of CCL18, CCL13, TGFβ, CCL22, CCL17, soluble fibronectin, folate receptor β, CD206, and CD163.
12. The method according to claim 1 wherein said cells have been triggered by a factor which induces macrophage differentiation into M2 macrophages (M2 inducing factor).
13. The method according to claim 1 , wherein said cells have been triggered by one or more M2 inducing factors selected from the group consisting of IL4, IL10, IL13, immune complexes and lipopolysaccharides.
14. The method of claim 1 wherein the method additionally comprises:
measuring a property related to the differentiation of macrophages into classically-activated (M1) macrophages and identifying a compound that does not inhibit said differentiation.
15. The method of claim 14 wherein said property is the level and/or expression of a marker of the M1 macrophage phenotype, and a compound is identified which does not increase the levels of said marker.
16. The method of claim 15 wherein said marker is TNFα.
17. A method for identifying a compound that inhibits macrophage differentiation into M2 macrophages, said method comprising:
a) contacting a test compound with a population of macrophage cells expressing a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 26;
b) measuring expression, activity, and/or amount of said polypeptide in said cells;
c) measuring a property related to differentiation of macrophages into M2 macrophages if the test compound reduces expression, activity, and/or amount of said polypeptide; and
d) identifying a compound capable of inhibiting macrophage differentiation into M2 macrophages.
18. The method according to claim 17 , which additionally comprises the step of comparing the compound to be tested to a control.
19. The method according to claim 17 , wherein said polypeptide is coupled to a detectable label.
20. The method according to claim 17 , wherein the cell naturally expresses said polypeptide.
21. The method according to claim 17 , wherein the cell has been engineered so as to express said polypeptide.
22. The method of claim 17 , wherein said property is the inhibition of release or expression of a marker of M2 macrophages.
23. The method of claim 22 wherein said property is an expression or release of a marker selected from the group consisting of CCL18, CCL13, TGFβ, CCL22, CCL17, soluble fibronectin, folate receptor β, CD206, and CD163.
24. The method according to claim 17 wherein said cells have been triggered by a factor which induces macrophage differentiation into M2 macrophages (M2 inducing factor).
25. The method according to claim 17 , wherein said cells have been triggered by one or more M2 inducing factors selected from the group consisting of IL4, IL10, IL13, immune complexes and lipopolysaccharides.
26. A method for identifying a compound that inhibits macrophage differentiation into M2 macrophages, said method comprising:
a) contacting a test compound with a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 26;
b) measuring an activity of said polypeptide;
c) contacting the test compound that inhibits the activity of said polypeptide with a population of macrophage cells;
d) measuring a property related to differentiation of macrophages into M2 macrophages if the test compound reduces the activity of said polypeptide; and
e) identifying a compound capable of inhibiting macrophage differentiation into M2 macrophages.
27. The method according to claim 26 , which additionally comprises the step of comparing the compound to be tested to a control.
28. The method according to claim 26 , wherein said polypeptide is coupled to a detectable label.
29. The method according to claim 26 , wherein said polypeptide in steps (a) and (b) is present in a cell.
30. The method of claim 26 , wherein said property is the inhibition of release or expression of a marker of M2 macrophages.
31. The method of claim 30 wherein said property is an expression or release of a marker selected from the group consisting of CCL18, CCL13, TGFβ, CCL22, CCL17, soluble fibronectin, folate receptor β, CD206, and CD163.
32. The method according to claim 26 wherein said cells have been triggered by a factor which induces macrophage differentiation into M2 macrophages (M2 inducing factor).
33. The method according to claim 26 , wherein said cells have been triggered by one or more M2 inducing factors selected from the group consisting of IL4, IL10, IL13, immune complexes and lipopolysaccharides.