Methods of modulating and of identifying agents that modulate intracellular calcium

• US 20040009537A1
• Filed: 01/13/2003
• Published: 01/15/2004
• Est. Priority Date: 01/11/2002
• Status: Abandoned Application

• Current Assignee: TorreyPines Therapeutics, Inc. /Old/
• Original Assignee: Comerica Bank (Comerica Incorporated)
• Inventors: Velicelebi, Gonul, Roos, Jack, Stauderman, Kenneth
• Application Number:
  US10/342,844
• Publication Number:
  US 20040009537A1
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

Methods are provided for identifying agents that modulate intracellular calcium. Also provided are methods of modulating calcium within cells and treating disease by modulating intracellular calcium. Also provided are methods of screening for nucleic acids encoding candidate ion transport proteins and methods of identifying nucleic acids encoding ion transport proteins.

Claims

1. A method of identifying an agent that modulates intracellular calcium, comprising:

contacting one or more test cells or a portion thereof comprising one or more proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry with a test agent, wherein the portion of the cell comprises the one or more proteins;

monitoring the effect(s) of the test compound on store-operated calcium entry; and

identifying a test agent as an agent if it has an effect on store-operated calcium entry.

2. The method of claim 1, wherein the portion of the cell comprises a cell membrane, a cell organelle or a cell organelle membrane.

3. The method of claim 1, wherein:

the step of monitoring the effects, comprises;

comparing store-operated calcium entry into one or more test cell(s) in the presence and absence of the test agent or comparing store-operated calcium entry into a test cell and into a control cell in the presence of the test agent, wherein;

the control cell is substantially similar cell to a test cell, but contains a different amount, including none, of the one or more proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the cell comprises; and

the step of identifying comprises identifying a test agent as an agent that modulates intracellular calcium if store-operated calcium entry into the one or more test cells differs in the presence and absence of the test agent or if store-operated calcium entry into a test cell and the control cell differs.

4. The method of claim 1, wherein:

monitoring the effect(s) of the test agent on store-operated calcium entry, comprises;

reducing the calcium level in an intracellular calcium store of one or more test cells; and

monitoring the effect of test agent on intracellular and/or extracellular a) ion movement, b) ion flux or c) ion levels of the one or more test cells; and

identifying comprises identifying a test agent as an agent if it has an effect on a), b) or c), wherein;

the step of reducing the calcium levels in an intracellular calcium store is performed before, simultaneously with or after the step of contacting one or more test cells with the test agent.

5. The method of claim 4, wherein:

monitoring the effect(s) of test agent on store-operated calcium further comprises;

comparing a), b) or c) in the presence and absence of the test agent or comparing a), b) or c) of a test cell and a control cell in the presence of the test agent; and

identifying further comprises;

identifying a test agent as an agent that modulates intracellular calcium if a), b) or c) of a test cell differ in the presence and absence of the test agent or if a), b) or c) of a test cell and the control cell differ, wherein;

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises.

6. The method of claim 4, wherein:

monitoring the effects of test agent on store-operated calcium comprises;

determining intracellular or cytoplasmic calcium levels of one or more test cells; and

identifying comprises;

identifying a test agent as an agent if it has an effect on intracellular or cytoplasmic calcium levels of one or more test cells.

7. The method of claim 6, wherein:

monitoring the effect(s) of test agent on store-operated calcium further comprises;

comparing the intracellular calcium levels of one or more test cells in the presence and absence of the test agent or the intracellular calcium levels of a test cell and of a control cell in the presence of the test agent; and

identifying comprises;

identifying a test agent as an agent that modulates intracellular calcium if the intracellular calcium levels of a test cell differ in the presence and absence of the test agent or if the intracellular calcium levels of a test cell and the control cell differ, wherein

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises.

8. The method of claim 4, wherein:

monitoring the effects of test agent on store-operated calcium comprises;

monitoring intracellular calcium levels over time in one or more test cells for fluctuations in intracellular calcium levels; and

identifying comprises;

identifying a test agent as an agent if it has an effect on intracellular calcium level time courses.

9. The method of claim 8, wherein:

monitoring the effect(s) of test agent on store-operated calcium further comprises;

comparing the intracellular calcium level time courses of a test cell in the presence and absence of the test agent or the intracellular calcium level time courses of a test cell and a control cell in the presence of the test agent; and

identifying comprises;

identifying a test agent as an agent that modulates intracellular calcium if the intracellular calcium level time courses of a test cell differ in the presence and absence of the test agent or if the intracellular calcium level time courses of a test cell and the control cell differ, wherein

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises.

10. The method of claim 4, wherein:

monitoring the effects of test agent on store-operated calcium comprises;

assessing comprises assessing ion flux across the cell plasma membrane of a test cell; and

identifying comprises;

identifying a test agent as an agent if it has an effect on ion flux across the cell plasma membrane of a test cell.

11. The method of claim 10, wherein:

monitoring the effect(s) of test agent on store-operated calcium further comprises;

comparing the ion flux across the cell plasma membrane of a test cell in the presence and absence of the test agent or the ion flux across plasma membrane of a test cell and a control cell in the presence of the test agent, wherein

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises; and

identifying further comprises identifying a test agent as an agent that modulates intracellular calcium if the ion flux across the cell plasma membrane of a test cell differs in the presence and absence of the test agent or if the ion flux across the plasma membrane of a test cell and the control cell differs.

12. The method of claim 1, wherein a protein that is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry is selected from the group consisting of a calcium transport protein 1 (CaT protein), an ECaC protein, an epithelial calcium channel, a CaT2 protein, an olfactory channel, a stretch-activated channel protein, a vanilloid receptor-related osmotically activated channel protein, a vanilloid receptor type 1-like protein, a vanilloid receptor subtype 1 protein, a vanilloid receptor 1 protein, a vanilloid receptor-like protein, a capsaicin receptor protein, an olfactory trp C4 (OTRPC4) protein, a growth factor-regulated calcium channel protein, a transient receptor potential protein 12 (trp 12), a transient receptor potential protein V3 (TRPV3), a stretch-inhibitable nonselective channel (SIC) protein.

13. The method of claim 3, wherein a protein that is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry is selected from the group consisting of a calcium transport protein 1 (CaT protein), an ECaC protein, an epithelial calcium channel, a CaT2 protein, an olfactory channel, a stretch-activated channel protein, a vanilloid receptor-related osmotically activated channel protein, a vanilloid receptor type 1-like protein, a vanilloid receptor subtype 1 protein, a vanilloid receptor 1 protein, a vanilloid receptor-like protein, a capsaicin receptor protein, an olfactory trp C4 (OTRPC4) protein, a growth factor-regulated calcium channel protein, a transient receptor potential protein 12 (trp12), a transient receptor potential protein V3 (TRPV3), a stretch-inhibitable nonselective channel (SIC) protein.

14. The method of claim 4, wherein a protein that is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry is selected from the group consisting of a calcium transport protein 1 (CaT protein), an ECaC protein, an epithelial calcium channel, a CaT2 protein, an olfactory channel, a stretch-activated channel protein, a vanilloid receptor-related osmotically activated channel protein, a vanilloid receptor type 1-like protein, a vanilloid receptor subtype 1 protein, a vanilloid receptor 1 protein, a vanilloid receptor-like protein, a capsaicin receptor protein, an olfactory trp C4 (OTRPC4) protein, a growth factor-regulated calcium channel protein, a transient receptor potential protein 12 (trp12), a transient receptor potential protein V3 (TRPV3) and a stretch-inhibitable nonselective channel (SIC) protein.

15. The method of any of claims 1-10, wherein a protein that is homologous to the protein encoded by the Drosophila gene is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 50%, 51% or 52% of the encoded protein and that provides for store-operated calcium entry with a test agent.

16. The method of any of claims 1-10, wherein a protein that is homologous to the protein encoded by the Drosophila gene is at least about 41% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 50%, 51% or 52% of the encoded protein and that provides for store-operated calcium entry with a test agent.

17. The method of any of claims 1-10, wherein a protein homologous to the protein encoded by the Drosophila gene does not contain the contiguous sequence of amino acids EWKFAR (SEQ ID 114) and/or EXD(E)CR(K)GXYXXYE (SEQ ID 115), wherein X is any amino acid and an amino acid residue in parentheses is an alternative to the residue immediately preceding it.

27. The method of claim 31, wherein:

monitoring the effects of test agent on store-operated calcium comprises;

assessing comprises assessing ion flux across the cell plasma membrane of a test cell; and

identifying comprises;

identifying a test agent as an agent if it has an effect on ion flux across the cell plasma membrane of a test cell.

28. The method of claim 27, wherein:

monitoring the effect(s) of test agent on store-operated calcium further comprises;

comparing the ion flux across the cell plasma membrane of a test cell in the presence and absence of the test agent or the ion flux across plasma membrane of a test cell and a control cell in the presence of the test agent; and

identifying further comprises identifying a test agent as an agent that modulates intracellular calcium if the ion flux across the cell plasma membrane of a test cell differs in the presence and absence of the test agent or if the ion flux across the plasma membrane of a test cell and the control cell differs, wherein

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises.

30. The method of claim 1 or claim 18, wherein a protein that provides for store-operated calcium entry is a mammalian protein.

31. The method of claim 30, wherein the mammalian protein is a human protein.

35. The method of claim 1 or claim 18, wherein the protein that provides for store-operated calcium entry is a component of a store-operated calcium entry channel.

36. The method of claim 1 or claim 18, wherein the proteins that is (are) at least about 35% homologous to the protein encoded by a Drosophila gene over about 40% of the encoded protein are selected from among ion transport proteins.

37. The method of claim 1 or claim 18, wherein the cell is a recombinant cell and at least one of the one or more proteins that is (are) at least about 35% homologous to a protein encoded by a Drosophila gene over about 40% of the encoded protein is heterologous to the cell.

38. The method of claims 1 or claim 18, wherein the test cell is a mammalian cell.

39. The method of claims 37, wherein the test cell is a mammalian cell.

40. The method of claim 1 or claim 18, wherein the agent is a drug for treatment of a neurodegenerative disease.

41. The method of claim 1 or claim 18, wherein a protein that is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry is a calcium transport protein 1 (CaT protein) or an ECaC protein.

18. A method of identifying an agent that modulates intracellular calcium, comprising:

monitoring the effects of an agent on store-operated calcium entry, wherein the agent modulates the activity of, the interaction of, the level of or binds to a protein that provides for store-operated calcium entry and that is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein; and

identifying a test agent as an agent that modulates intracellular calcium if it has an effect on store-operated calcium entry.

19. The method of claim 18, wherein the effect on store-operated calcium entry is tested on a test cell or a portion thereof that comprises the protein that is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry.

20. The method of claim 19, wherein:

the step of monitoring the effects, comprises;

comparing store-operated calcium entry into one or more test cell(s) in the presence and absence of the test agent or comparing store-operated calcium entry into a test cell and into a control cell in the presence of the test agent, wherein;

the control cell is substantially similar cell to a test cell, but contains a different amount, including none, of the one or more proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the cell comprises; and

the step of identifying comprises identifying a test agent as an agent that modulates intracellular calcium if store-operated calcium entry into the one or more test cells differs in the presence and absence of the test agent or if store-operated calcium entry into a test cell and the control cell differs.

21. The method of claim 19, wherein:

monitoring the effect(s) of the test agent on store-operated calcium entry, comprises;

reducing the calcium level in an intracellular calcium store of one or more test cells; and

monitoring the effect of test agent on intracellular and/or extracellular a) ion movement, b) ion flux or c) ion levels of the one or more test cells; and

identifying comprises identifying a test agent as an agent if it has an effect on a), b) or c), wherein;

the step of reducing the calcium levels in an intracellular calcium store is performed before, simultaneously with or after the step of contacting one or more test cells with the test agent.

22. The method of claim 21, wherein:

monitoring the effect(s) of test agent on store-operated calcium further comprises;

comparing a), b) or c) in the presence and absence of the test agent or comparing a), b) or c) of a test cell and a control cell in the presence of the test agent, wherein

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises; and

identifying further comprises;

identifying a test agent as an agent that modulates intracellular calcium if a), b) or c) of a test cell differ in the presence and absence of the test agent or if a), b) or c) of a test cell and the control cell differ, wherein;

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises.

23. The method of claim 19, wherein:

monitoring the effects of a test agent on store-operated calcium comprises;

determining intracellular or cytoplasmic calcium levels of one or more test cells; and

identifying comprises;

identifying a test agent as an agent if it has an effect on intracellular or cytoplasmic calcium levels of one or more test cells.

24. The method of claim 23, wherein:

monitoring the effect(s) of test agent on store-operated calcium further comprises;

comparing the intracellular calcium levels of one or more test cells in the presence and absence of the test agent or the intracellular calcium levels of a test cell and of a control cell in the presence of the test agent, wherein

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises; and

identifying comprises;

identifying a test agent as an agent that modulates intracellular calcium if the intracellular calcium levels of a test cell differ in the presence and absence of the test agent or if the intracellular calcium levels of a test cell and the control cell differ.

25. The method of claim 19, wherein:

monitoring the effects of test agent on store-operated calcium comprises;

monitoring intracellular calcium levels over time in one or more test cells for fluctuations in intracellular calcium levels; and

identifying comprises;

identifying a test agent as an agent if it has an effect on intracellular calcium level time courses.

26. The method of claim 25, wherein:

monitoring the effect(s) of test agent on store-operated calcium further comprises;

comparing the intracellular calcium level time courses of a test cell in the presence and absence of the test agent or the intracellular calcium level time courses of a test cell and a control cell in the presence of the test agent, wherein

a control cell is substantially similar to a test cell, but contains a different amount, including none, of the proteins that is (are) at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein that the test cell comprises; and

identifying comprises;

identifying a test agent as an agent that modulates intracellular calcium if the intracellular calcium level time courses of a test cell differ in the presence and absence of the test agent or if the intracellular calcium level time courses of a test cell and the control cell differ.

29. The method of claim 18, wherein a protein that is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry is selected from the group consisting of a calcium transport protein 1 (CaT protein), an ECaC protein, an epithelial calcium channel, a CaT2 protein, an olfactory channel, a stretch-activated channel protein, a vanilloid receptor-related osmotically activated channel protein, a vanilloid receptor type 1-like protein, a vanilloid receptor subtype 1 protein, a vanilloid receptor 1 protein, a vanilloid receptor-like protein, a capsaicin receptor protein, an olfactory trp C4 (OTRPC4) protein, a growth factor-regulated calcium channel protein, a transient receptor potential protein 12 (trp 12), a transient receptor potential protein V3 (TRPV3), a stretch-inhibitable nonselective channel (SIC) protein.

32. The method of claim 18 wherein a protein that is homologous to the protein encoded by the Drosophila gene is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 50%, 51% or 52% of the encoded protein and that provides for store-operated calcium entry with a test agent.

33. The method of claim 18, wherein a protein that is homologous to the protein encoded by the Drosophila gene is at least about 41% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 50%, 51% or 52% of the encoded protein and that provides for store-operated calcium entry with a test agent.

34. The method of claim 18, wherein a protein homologous to the protein encoded by the Drosophila gene does not contain the contiguous sequence of amino acids EWKFAR (SEQ ID 114) and/or EXD(E)CR(K)GXYXXYE (SEQ ID 115), wherein X is any amino acid and an amino acid residue in parentheses is an alternative to the residue immediately preceding it.

42. A method of modulating store-operated calcium entry, comprising modulating the level of, expression of, activity of or molecular interactions of a protein in a cell that has altered store-operated calcium entry, wherein the protein is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry, and whereby store-operated calcium transport into the cell is modulated.

43. The method of claim 42, wherein a protein that is at least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry is selected from the group consisting of a calcium transport protein 1 (CaT protein), an ECaC protein, an epithelial calcium channel, a CaT2 protein, an olfactory channel, a stretch-activated channel protein, a vanilloid receptor-related osmotically activated channel protein, a vanilloid receptor type 1-like protein, a vanilloid receptor subtype 1 protein, a vanilloid receptor 1 protein, a vanilloid receptor-like protein, a capsaicin receptor protein, an olfactory trp C4 (OTRPC4) protein, a growth factor-regulated calcium channel protein, a transient receptor potential protein 12 (trp12), a transient receptor potential protein V3 (TRPV3), a stretch-inhibitable nonselective channel (SIC) protein.

44. The method of claim 43, wherein the protein is a CaT1 or EcAc protein.

45. A method of identifying a molecule that provides for store-operated calcium entry, comprising identifying a molecule that interacts with a protein that is least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein and that provides for store-operated calcium entry, thereby identifying molecules involved in modulating store-operated calcium entry.

46. The method of claim 45, further comprising eliminating, altering or reducing the expression of a gene encoding the protein in a cell and evaluating intracellular calcium.

47. The method of claim 45, wherein the protein that is least about 35% homologous to the protein encoded by Drosophila gene CG4536 or CG5842 over at least about 40% of the encoded protein is a mammalian protein.

48. The method of claim 46, wherein the cell in which intracellular calcium is evaluated is a mammalian cell.