Histone deacetylase whole cell enzyme assay

• US 20060063210A1
• Filed: 09/21/2005
• Published: 03/23/2006
• Est. Priority Date: 09/22/2004
• Status: Abandoned Application

• Current Assignee: MethylGene, Inc.
• Original Assignee: MethylGene, Inc.
• Inventors: Li, Zuomei, Bonfils, Claire, Besterman, Jeffrey M.
• Application Number:
  US11/231,528
• Publication Number:
  US 20060063210A1
• Time in Patent Office: Days
• US Class Current: 435/7.900
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Abstract

The invention relates to enzymatic assays for protein deacetylases. More particularly, the invention relates to such assays utilizing whole cells. The invention provides assays which allow assessment of the level of a protein deacetylase activity in whole cells taken directly from the body of the mammal.

Claims

1. A method for assessing total protein deacetylase activity of a protein deacetylase family in whole cells or one or more members thereof ex vivo comprising providing whole cells from a mammal;

contacting the whole cells with a cell-permeable pan-substrate or isotype-specific substrate for the protein deacetylase family or the one or more members thereof, wherein deacetylation of the substrate by the protein deacetylase family or the one or more members thereof generates a detectable reporter molecule; and

quantitating the detectable reporter molecule.

2. The method of claim 1, wherein the quantity of the detectable reporter molecule is measured against a control standard for the protein deacetylase family or the one or more member thereof.

3. The method of claim 1, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

4. The method of claim 1, wherein the protein deacetylase family is a Sir-2 family.

5. A method for assessing isotype-specific activity of one or more member of a protein deacetylase family from whole cells ex vivo, wherein the one or more isotype of the protein deacetylase family provides a majority of the total deacetylase activity, comprising providing whole cells from a mammal;

contacting the whole cells with a cell-permeable pan-substrate for the protein deacetylase family or a cell permeable isotype-specific inhibitor of the one or more member of the protein deacetylase family, wherein deacetylation of the substrate by the protein deacetylase generates a detectable reporter molecule;

contacting a first aliquot of the cells with an isotype-specific inhibitor of the one or more protein deacetylase that provides a majority of the total deacetylase activity;

providing a second aliquot of the whole cells which is not contacted with the isotype-specific inhibitor of the one or more protein deacetylase that provides a majority of the total deacetylase activity;

quantitating the detectable reporter molecule in the first and second aliquots; and

comparing the detectable reporter molecule in the first and second aliquots.

6. The method of claim 5, wherein the quantity of the detectable reporter molecule is measured against a control standard for the protein deacetylase family or the one or more member thereof.

7. The method of claim 5, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

8. The method of claim 5, wherein the protein deacetylase family is a Sir-2 family.

9. The method of claim 5, wherein the whole cells have been transfected with a gene or genes expressing the one or more isotype prior to quantitating the detectable reporter molecule.

11. The method of claim 9, wherein the quantity of the detectable reporter molecule is measured against a control standard for the protein deacetylase family or the one or more members therof.

12. The method of claim 9, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

13. The method of claim 9, wherein the protein deacetylase family is a Sir-2 family.

15. The method of claim 13, wherein the quantity of the detectable reporter molecule is measured against a control standard for the protein deacetylase family or the one or more members thereof.

16. The method of claim 13, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

17. The method of claim 13, wherein the protein deacetylase family is a Sir-2 family.

19. The method of claim 17 wherein the quantity of the detectable reporter molecule is measured against a control standard for the protein deacetylase family or the one or more members thereof.

20. The method of claim 17, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

21. The method of claim 17, wherein the protein deacetylase family is a Sir-2 family.

23. The method of claim 21, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

24. The method of claim 21, wherein the protein deacetylase family is a Sir-2 family.

26. The method of claim 24, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

27. The method of claim 24, wherein the protein deacetylase family is a Sir-2 family.

29. The method of claim 27, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

30. The method of claim 27, wherein the protein deacetylase family is a Sir-2 family.

32. The method of claim 30, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

33. The method of claim 30, wherein the protein deacetylase family is a Sir-2 family.

35. The method of claim 33, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

36. The method of claim 33, wherein the protein deacetylase family is a Sir-2 family.

38. The method according to claim 36, wherein the protein deacetylase family is the histone deacetylase (HDAC) family.

39. The method according to claim 36, wherein the protein deacetylase family is the Sir2 family.

41. The method according to claim 39, wherein the protein deacetylase family is the histone deacetylase (HDAC) family.

42. The method according to claim 39, wherein the protein deacetylase family is the Sir2 family.

44. The method of claim 42, wherein the protein deacetylase family is a histone deacetylase (HDAC) family.

45. The method according to claim 42, wherein the protein deacetylase family is the Sir2 family.

10. A method for assessing the isotype-specific activity of one or more member of a protein deacetylase family ex vivo, comprising providing whole cells from a mammal;

contacting the whole cells with a cell-permeable isotype-specific substrate for the one or more member of a protein deacetylase family, wherein deacetylation of the substrate by the one or more protein deacetylase generates a detectable reporter molecule; and

quantitating the detectable reporter molecule.

14. A method for assessing the activity of a candidate pan-inhibitor of a protein deacetylase family or one or more members thereof in whole cells ex vivo, comprising providing whole cells from a mammal;

contacting the whole cells with a cell-permeable pan-substrate for the protein deacetylase family or an isotype-specific substrate, wherein deacetylation of the substrate by the protein deacetylase family or the one or more members thereof generates a detectable reporter molecule;

contacting a first aliquot of the cells with a candidate pan-inhibitor of the protein deacetylase family;

providing a second aliquot of the cells which is not contacted with the candidate pan-inhibitor of the protein deacetylase family;

quantitating the detectable reporter molecule in the first and second aliquots; and

comparing the quantity of detectable reporter molecule in the first aliquot and the second aliquot.

18. A method for assessing isotype-specific activity of a candidate inhibitor of one or more member of a protein deacetylase family from whole cells ex vivo, wherein the one or more isotype of the protein deacetylase family provides a majority of the total deacetylase activity;

the method comprising providing whole cells from a mammal;

contacting the whole cells with a cell-permeable pan-substrate for the protein deacetylase family or a cell permeable isotype-specific inhibitor of the one or more member of the protein deacetylase family, wherein deacetylation of the substrate by the protein deacetylase generates a detectable reporter molecule;

contacting a first aliquot of the whole cells with the candidate isotype-specific inhibitor of the one or more protein deacetylase that provides a majority of the total deacetylase activity;

providing a second aliquot of the cells which is not contacted with with the candidate isotype-specific inhibitor of the one or more protein deacetylase that provides a majority of the total deacetylase activity;

quantitating the detectable reporter molecule in the first and second aliquots; and

comparing the quantity of the detectable reporter molecule for each aliquot.

22. A method for assessing the efficacy of a pan-inhibitor of a protein deacetylase family or one or more members thereof in vivo, comprising providing whole cells from a mammal;

contacting the whole cells with a pan-substrate for the protein deacetylase family or an isotype-specific substrate, wherein deacetylation of the substrate by the protein deacetylase family or the one or more members thereof generates a detectable reporter molecule;

quantitating the reporter molecule;

administering to the mammal the pan-inhibitor;

providing whole cells from the mammal;

contacting the whole cells with the pan-substrate or isotype-specific substrate;

quantitating the reporter molecule; and

comparing the quantity of the reporter molecule in the whole cells from the mammal before administration of the pan-inhibitor with the quantity of the reporter molecule in the whole cells after administration of the pan-inhibitor.

25. A method for assessing the efficacy of an isotype-specific inhibitor of a protein deacetylase family in vivo, providing whole cells from a mammal;

contacting the whole cells with an isotype-specific substrate for one or more member of a protein deacetylase family, wherein deacetylation of the substrate by the one or more protein deacetylase generates a detectable reporter molecule;

quantitating the reporter molecule;

administering to the mammal the isotype-specific inhibitor;

providing whole cells from the mammal;

contacting the whole cells with the isotype-specific substrate;

quantitating the reporter molecule; and

comparing the quantity of the reporter molecule from the whole cells after administration of the isotype-specific inhibitor with the quantity of the reporter molecule in the whole cells from the mammal before administration of the isotype-specific inhibitor.

28. A method for assessing the efficacy of a pan-activator of a protein deacetylase family or one or more members thereof in vivo, comprising providing whole cells from a mammal;

contacting the whole cells with a pan-substrate for the protein deacetylase family or an isotype-specific substrate, wherein deacetylation of the substrate by the protein deacetylase family or the one or more members thereof generates a detectable reporter molecule;

quantitating the reporter molecule;

administering to the mammal the pan-activator;

providing whole cells from the mammal;

contacting the whole cells with the pan-substrate or isotype-specific substrate;

quantitating the reporter molecule; and

comparing the quantity of the reporter molecule in the whole cells from the mammal before administration of the pan-activator with the quantity of the reporter molecule in the whole cells after administration of the pan-inhibitor.

31. A method for assessing the efficacy of an isotype-specific activator of a protein deacetylase family in vivo, providing whole cells from a mammal;

contacting the whole cells with an isotype-specific substrate for one or more member of a protein deacetylase family, wherein deacetylation of the substrate by the one or more protein deacetylase generates a detectable reporter molecule;

quantitating the reporter molecule;

administering to the mammal the isotype-specific activator;

providing whole cells from the mammal;

contacting the whole cells with the isotype-specific substrate;

quantitating the reporter molecule; and

comparing the quantity of the reporter molecule from the whole cells after administration of the isotype-specific activator with the quantity of the reporter molecule in the whole cells from the mammal before administration of the isotype-specific activator.

34. A method for assessing the efficacy of a pan-inhibitor of total protein deacetylase of a mammal or of one or more members thereof in vivo comprising administering to the mammal a cell-permeable pan-substrate for a protein deacetylase family, wherein deacetylation of the pan-substrate or isotype-specific substrate generates a detectable reporter molecule;

obtaining bodily fluids from the mammal;

determining the quantity of the detectable reporter molecule in the bodily fluids;

administering to the mammal a pan-inhibitor of the protein deacetylase family;

administering to the mammal the pan-substrate or the isotype-specific substrate;

obtaining bodily fluids from the mammal;

determining the quantity of the detectable reporter molecule in the bodily fluids; and

comparing the quantity of detectable reporter molecule in bodily fluids obtained prior to administration of the pan-inhibitor with the quantity of the detectable reporter molecule in bodily fluids after administration of the pan-inhibitor.

37. A method for assessing the efficacy of an isotype-specific inhibitor of one or more member of a protein deacetylase family in a mammal in vivo comprising administering to the mammal a cell-permeable isotype-specific substrate for one or more member of a protein deacetylase family, wherein deacetylation of the isotype-specific substrate generates a detectable reporter molecule;

obtaining bodily fluids from the mammal;

determining the quantity of the detectable reporter molecule in the bodily fluids;

administering to the mammal an isotype-specific inhibitor of the one or more member of a protein deacetylase family;

administering to the mammal the isotype-specific substrate;

obtaining bodily fluids from the mammal;

determining the quantity of the detectable reporter molecule in the bodily fluids; and

comparing the quantity of detectable reporter molecule in bodily fluids obtained prior to administration of the isotype-specific inhibitor with the quantity of the detectable reporter molecule in bodily fluids after administration of the isotype-specific inhibitor.

40. A method for assessing the efficacy of a pan-activator of total activity of a protein deacetylase family in a mammal or one or more members thereof in vivo comprising administering to the mammal a cell-permeable pan-substrate for a protein deacetylase family or an isotype-specific substrate, wherein deacetylation of the pan-substrate or isotype-specific substrate generates a detectable reporter molecule;

obtaining bodily fluids from the mammal;

determining the quantity of the detectable reporter molecule in the bodily fluids;

administering to the mammal a pan-activator of the protein deacetylase family;

administering to the mammal the pan-substrate or the isotype-specific substrate;

obtaining bodily fluids from the mammal;

determining the quantity of the detectable reporter molecule in the bodily fluids; and

comparing the quantity of detectable reporter molecule in bodily fluids obtained prior to administration of the pan-activator with the quantity of the detectable reporter molecule in bodily fluids after administration of the pan-activator.

43. A method for assessing the efficacy of an isotype-specific activator of one or more member of a protein deacetylase family in a mammal in vivo comprising administering to the mammal a cell-permeable isotype-specific substrate for one or more member of a protein deacetylase family, wherein deacetylation of the isotype-specific substrate generates a detectable reporter molecule;

obtaining bodily fluids from the mammal;

determining the quantity of the detectable reporter molecule in the bodily fluids;

administering to the mammal an isotype-specific activator of the one or more member of the protein deacetylase family;

administering to the mammal the isotype-specific substrate;

obtaining bodily fluids from the mammal;

determining the quantity of the detectable reporter molecule in the bodily fluids; and

comparing the quantity of detectable reporter molecule in bodily fluids obtained prior to administration of the isotype-specific activator with the quantity of the detectable reporter molecule in bodily fluids after administration of the isotype-specific activator.

46. A method for assessing the activity of a candidate pan-activator of a protein deacetylase family or one or more members thereof in whole cells ex vivo, comprising providing whole cells from a mammal;

contacting the whole cells with a cell-permeable pan-substrate for the protein deacetylase family or an isotype-specific substrate, wherein deacetylation of the substrate by the protein deacetylase family or the one or more members thereof generates a detectable reporter molecule;

contacting a first aliquot of the cells with a candidate pan-activator of the protein deacetylase family;

providing a second aliquot of the cells which is not contacted with the candidate pan-actvator of the protein deacetylase family;

quantitating the detectable reporter molecule in the first and second aliquots; and

comparing the quantity of detectable reporter molecule in the first aliquot and the second aliquot.

47. A method for assessing the activity of a candidate isotype-specififc activator of a protein deacetylase family or one or more members thereof in whole cells ex vivo, comprising providing whole cells from a mammal;

contacting the whole cells with a cell-permeable pan-substrate for the protein deacetylase family or an isotype-specific substrate, wherein deacetylation of the substrate by the protein deacetylase family or the one or more members thereof generates a detectable reporter molecule;

contacting a first aliquot of the cells with a candidate isotype-specific activator of one or more member of the protein deacetylase family;

providing a second aliquot of the cells which is not contacted with the candidate isotype-specific activator of the one or more member of the protein deacetylase family;

quantitating the detectable reporter molecule in the first and second aliquots; and

comparing the quantity of detectable reporter molecule in the first aliquot and the second aliquot.