Pharmaceuticals and methods for treating hypoxia and screening methods therefor
Light-generating fusion proteins having a ligand binding site and a light-generating polypeptide moiety and their use as diagnostics, in drug screening and discovery, and as therapeutics, are disclosed. The light-generating fusion protein has a feature where the bioluminescence of the polypeptide moiety changes upon binding of a ligand at the ligand binding site. Tie ligand may be, for example, an enzyme present in an environment only under certain conditions, e.g., ubiquitin ligase in a hypoxic state, such that the light-generating fusion protein is “turned on” only under such conditions.
1. A method of identifying prolyl hydroxylase modulators, comprising the steps of:
a) contacting a prolyl hydroxylase, a putative prolyl hydroxylase modulator and a light-generating fusion protein comprising an HIFα polypeptide moiety having binding character for prolyl hydroxylase and a light-generating polypeptide moiety, wherein the light generation of said light-generating polypeptide moiety changes upon binding of a prolyl hydroxylase to said HIFα polypeptide moiety, under conditions favorable for binding of a prolyl hydroxylase to a HIFα to form a test sample; and
b) determining the ability of said putative prolyl hydroxylase modulator to modulate prolyl hydroxylase by measuring the light generated in said test sample.
2. An assay for a modulator of prolyl hydroxylase, comprising
a) bringing into contact a prolyl hydroxylase, a HIF1α polypeptide moiety and a putative modulator compound under conditions where prolyl hydroxylase, in the absence of modulator, is capable of acting on said HIF1α polypeptide moiety; and
b) measuring the degree of prolyl hydroxylase inhibition caused by said modulator compound.
3. The method of claim 2 , wherein said test sample further comprises pVHL.
4. The method of claim 2 , wherein said HIF1α polypeptide moiety comprises the amino acid sequence Y-X 1 -Leu-X 2 -Pro h -X 3 -X 4 -X 5 -X 6 -Y′, wherein
a) Pro h is hydroxylated proline;
b) X 1 , X 2 , X 4 , X 5 , and X 6 are independently Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp;
c) X 3 is Ser, Thr, or Tyr; and
d) Y and Y′ are independently present or absent and, if present, independently comprise a peptide having from 1 to 600 amino acids.
5. The method of claim 2 , wherein said HIF1α polypeptide moiety comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
6. The method of claim 2 , wherein said HIF1α polypeptide moiety comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 402 is hydroxylated proline.
7. The method of claim 2 wherein said HIF1α polypeptide moiety comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α, numbered in accordance with wild-type HIF1α, wherein either or both of residues 402 and 564 are hydroxylated proline.
8. The method of claim 2 , wherein said HIF1α polypeptide moiety comprises a 4 to 12 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
9. The method of claim 2 , wherein said HIF1α polypeptide moiety comprises a 12 to 14 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
10. The method of claim 2 , wherein said HIF1α polypeptide moiety comprises a 20 to 30 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
11. The method of claim 2 , wherein said HIF1α polypeptide moiety comprises a 80 to 120 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
12. The method of claim 1 , whereupon ligand binding to said ligand binding site alters the light generation of said light-generating fussion protein without altering the phosphorylational state of said light-generating fusion protein.
13. The method of claim 2 , wherein said HIF1α polypeptide moiety comprises amino acids 555-575 of wild type HIFα, numbered in accordance with wild type HIFα.
14. HIFα polypeptide comprising amino acids 555-575 of wild type HIFα, numbered in accordance with wild type HIFα.
15. A method of treating or preventing a hypoxic or ischemic related disorder in a subject, comprising administering to a subject in need thereof a compound identified by the method of claim 1 which decreases prolyl hydroxylase expression or activity, such that said hypoxic or ischemic related disorder is treated.
16. The method of claim 15 , wherein said compound is a prolyl hydroxylase antibody, or a nucleic acid that decreases the expression of a nucleic acid that encodes a prolyl hydroxylase polypeptide.
17. The method of claim 16 , wherein the nucleic acid is a prolyl hydroxylase anti-sense nucleic acid.
18. The method of claim 15 , wherein the hypoxic or ischemic related disorder is an acute event selected from the consisting of myocardial infarction, stroke, cancer, and diabetes.
19. The method of claim 15 , wherein the hypoxic or ischemic related disorder is a chronic event not caused by tissue scarring.
20. The method of claim 15 , wherein the hypoxic or ischemic related disorder is a chronic event selected from the group consisting of deep vein thrombosis, pulmonary embolus, and renal failure.
21. The method of claim 15 , wherein the half life of HIF in said subject is increased compared to a subject not exposed to said compound.
22. A method of increasing angiogenesis or vascularization in a subject, comprising administering to a subject in need thereof a compound identified by the method of claim 1 which decreases prolyl hydroxylase expression or activity.
23. A method of treating cancer in a subject, comprising administering to a subject in need thereof a compound identified by the method of claim 1 which increases prolyl hydroxylase expression or activity.
24. A method of treating a cell-proliferating disorder by administering to a subject an effective amount of a fusion protein comprising a HIFα polypeptide moiety having a binding character for prolyl hydroxylase, and a suicide polypeptide moiety, such that said cell-proliferating disorder is treated.
25. A method of treating a hypoxic or ischemic disorder by administering to a subject an effective amount of a fusion protein comprising a HIFα polypeptide moiety having a binding character for prolyl hydroxylase, and a suicide polypeptide moiety, such that said hypoxic or ischemic disorder is treated.
26. A method of killing hypoxic tumor cells, comprising administration of an effective amount of a fusion protein to a subject, said fusion protein comprising a HIFα polypeptide moiety having a binding character for prolyl hydroxylase, and a suicide polypeptide moiety, such that said hypoxic tumor cells are killed.
27. A method of monitoring the treatment of a cell-proliferating disorder by
a) administering to a subject an effective amount of a fusion protein comprising a HIFα polypeptide moiety having a binding character for prolyl hydroxylase, a suicide polypeptide moiety, and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of prolyl hydroxylase to said HIFα polypeptide moiety, such that said cell-proliferating disorder is treated, and
b) monitoring the ability of said fusion protein to inhibit cell proliferation by measuring the light generated by said light-generating fusion protein.
28. A method of treating a cell-proliferating disorder by administering to a subject an effective amount of a fusion protein comprising a cyclin/cdk binding site and a suicide protein polypeptide moiety, such that said cell-proliferating disorder is treated.
29. A method of monitoring the treatment of a cell-proliferating disorder by
a) administering to a subject an effective amount of a light-generating fusion protein comprising a cyclin/cdk binding site, a suicide protein polypeptide moiety, and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a cyclin to said cyclin/cdk binding site, such that said cell-proliferating disorder is treated; and
b) monitoring the ability of said fusion protein to inhibit cell proliferation by measuring the light generated by said light-generating fusion protein.
30. A method of screening candidate compounds that inhibit cell proliferation, comprising the steps of:
a) contacting cells with a candidate compound and a light-generating fusion protein comprising a cyclin/cdk binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a cyclin at said cyclin binding site, said cyclin binding indicative of cancerous tissue; and
b) determining the ability of said candidate compound to inhibit cell proliferation by measuring the luminescence of said cells.
31. A cell proliferation-inhibiting compound identified by the method of claim 30 .
32. A method for screening for a modulator of activity or latency of, or predisposition to a disorder, said method comprising:
a) administering a test compound to a test animal at increased risk for a disorder, wherein said test animal recombinantly expresses a light-generating fusion protein comprising a ligand binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ligand at said ligand binding site, said ligand binding site recognizing a ligand on an entity associated with a disorder, or a product of said disorder;
b) allowing for localization of said light-generating fusion protein and an entity, wherein contact between said ligand binding site and a ligand associated with said disorder causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety;
c) detecting the luminescence of said light-generating polypeptide moiety in said test animal after administering the compound of step (a); and
d) comparing the luminescence of said light-generating polypeptide moiety in said test animal with the luminescence of said light-generating polypeptide moiety in a control animal not administered said compound, wherein a change in the activity of said light-generating polypeptide moiety in said test animal relative to said control animal indicates the test compound is a modulator of latency of or predisposition to, a disorder.
33. The method of claim 32 , wherein the disorder is a hypoxia-related disorder.
34. The method of claim 32 , wherein the disorder is selected from the group consisting of cancer, diabetes, heart disease and stroke.
35. A non-invasive method for determining the effects of an anti-hypoxic compound in vivo, comprising:
a) administering to a subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, comprising an ubiquitin ligase binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a ubiquitin ligase at said ubiquitin ligase binding site, said ubiquitin ligase binding site recognizing a ubiquitin ligase present in hypoxic conditions in hypoxic tissue;
b) allowing for localization of said light-generating fusion protein or cell in hypoxic tissue in said subject, wherein contact between said ubiquitin ligase binding site and a ubiquitin ligase causes a modification of a colinear effector site which alters the light generation of said light-generating polypeptide moiety; and;
c) determining the ability of said candidate compound to inhibit hypoxia by measuring the luminescence of said localized light-generating fusion protein.
36. The method of claim 35 , wherein said ligand binding site comprises the amino acid sequence Y-X 1 -Leu-X 2 -Pro h -X 3 -X 4 -X 5 -X 6 -Y′, wherein
a) Pro h is hydroxylated proline;
b) X 1 , X 2 , X 4, X 5 , and X 6 are independently Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, or Trp;
c) X 3 is Ser, Thr, or Tyr; and
d) Y and Y′ are independently present or absent and, if present, independently comprise a peptide having from 1 to 600 amino acids.
37. The method of claim 35 , wherein said ligand binding site comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
38. The method of claim 35 , wherein said ligand binding site comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 402 is hydroxylated proline.
39. The method of claim 35 , wherein said ligand binding site comprises the amino acid sequence corresponding to the N-terminal residues 1-600 of HIF1α, numbered in accordance with wild-type HIF1α, wherein either or both of residues 402 and 564 are hydroxylated proline.
40. The method of claim 35 , wherein said ligand binding site comprises a 4 to 12 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
41. The method of claim 35 , wherein said ligand binding site comprises a 12 to 14 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
42. The method of claim 35 , wherein said ligand binding site comprises a 20 to 30 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
43. The method of claim 35 , wherein said ligand binding site comprises a 80 to 120 amino acid sequence corresponding to the residues adjacent to and/or surrounding residue 564, inclusive, of HIF1α, numbered in accordance with wild-type HIF1α, wherein residue 564 is hydroxylated proline.
44. A noninvasive method for detecting the effects of an anti-cell proliferation compound under study in a mammalian subject, comprising:
a) administering to a subject a light-generating fusion protein, or a cell expressing said light-generating fusion protein, comprising a cyclin/cdk binding site and a light-generating polypeptide moiety, wherein the light generation of said light-generating fusion protein changes upon binding of a cyclin at said cyclin binding site, said cyclin binding indicative of cell-proliferating tissue;
b) allowing for localization of said light-generating fusion protein or cell in cell-proliferating tissue in said subject, wherein contact between said cyclin binding site and a cyclin causes a modification of a colinear effector site which alters the sight generation of said light-generating polypeptide moiety; and
c) determining the ability of said candidate compound to inhibit cell proliferation by measuring the luminescence from said localized light-generating fusion protein.
45. A method of treating a hypoxic or ischemic related disorder in a subject, comprising administering to a subject in need thereof a compound which modulates prolyl hydroxylation of HIF, such that said hypoxic or ischemic related disorder is treated.
46. The method of claim 45 , wherein said compound decreases prolyl hydroxylation of HIF.
47. The method of claim 46 , wherein said compound increases prolyl hydroxylation of HIF.
48. A method of regulating HIF turnover in a subject, comprising administering to a subject in need thereof a compound which modulates prolyl hydroxylation of HIF, such that HIF turnover is regulated.
49. The method of claim 48 , wherein said compound increases prolyl hydroxylation of HIF.
50. The method of claim 49 , wherein said compound decreases prolyl hydroxylation of HIF.
51. A method of treating a HIF-related disorder in a subject, comprising administering to a subject in need thereof a compound which modulates prolyl hydroxylation of HIF, such that said HIF-related disorder is prevented, reversed or stabilized.
52. The method of claim 51 , wherein said compound increases prolyl hydroxylation of HIF.
53. The method of claim 52 , wherein said compound decreases prolyl hydroxylation of HIF.
54. The method of claim 52 , wherein said HIF-related disorder is selected from the group consisting of myocardial infarction, stroke, cancer, and diabetes.