Modulation of phosphatidylinositol-5-phosphate-4-kinase activity
The invention features methods for identifying compounds that modulate the activity of phosphatidylinositol 5-phosphate 4-kinase (PI5P4K). Inhibitors of PI5P4K can be used in, for example, the treatment or prevention of cell proliferation disorders (e.g., the prevention of tumor cell growth in p53 mutated cancers).
1. A method for identifying a compound that inhibits phosphatidylinositol-5-phosphate-4-kinase (PI5P4K), said method comprising:
(a) providing a cell expressing a phosphatidylinositol-5-phosphate-4-kinase (PI5P4K) and comprising a p53-inactive mutation or a p53-deletion and a substrate;
(b) contacting said cell with a candidate compound;
(c) detecting the activity of said PI5P4K; and
(d) determining if said candidate compound inhibits said PI5P4K;
wherein said candidate compound is a preferred candidate for treating a p53-mutated cancer if said candidate compound inhibits said PI5P4K, and wherein said cell is not a leukemia cell.
2. The method of claim 1 , wherein said substrate is guanosine-5′-triphosphate (GTP).
3. The method of claim 1 , wherein said PI5P4K is the PI5P4Kβ isoform.
4. The method of claim 1 , wherein said substrate is adenosine-5′-triphosphate (ATP).
5. The method of claim 1 , wherein said PI5P4K is the PI5P4Kα isoform.
6. The method of claim 1 , wherein said method further comprises comparing the activity detected in step (c) with the detected activity of PI5P4K in a medium not contacted with said candidate compound of step (b), wherein decreased activity of PI5P4K in the presence of said candidate compound of step (b) identifies said candidate compound as said preferred candidate.
7. The method of claim 1 , wherein said method further comprises comparing the activity detected in step (c) with the activity observed in a medium where said PI5P4K is absent.
8. The method of claim 1 , wherein said PI5P4K is human recombinant PI5P4K.
9. The method of claim 1 , wherein said cell is a mouse embryonic fibroblast (MEF).
10. The method of claim 9 , wherein said MEF is an immortalized MEF.
11. The method of claim 10 , wherein said activity detected in step (c) is compared with the activity observed in a medium where said PI5P4K is absent.
12. The method of claim 11 , wherein said activity detected in step (c) is the production of cellular phosphatidylinositol-5-phosphate (PI5P).
13. The method of claim 11 , wherein said medium where said PI5P4K is absent comprises an immortalized MEF prepared from PI5P4Kα −/− β −/− knockout mice.
14. The method of claim 12 , wherein the levels of said cellular phosphatidylinositol-5-phosphate (PI5P) increase compared to the levels of PI5P in MEFs prepared from PI5P4Kα −/− β −/− knockout mice.
15. The method of claim 1 , wherein said activity detected in step (c) is AKT activity.
16. The method of claim 15 , wherein said AKT activity is detected using an AKT specific antibody.
17. The method of claim 16 , wherein said AKT specific antibody is the phospho-T308-AKT or phospho-S473-AKT antibody.
18. The method of claim 1 , wherein, said activity detected is step (c) is the consumption of GTP.
19. The method of claim 1 , wherein said PI5P4K activity is detected using absorbance, fluorescence, Raman scattering, phosphorescence, luminescence, luciferase activity, or radioactivity; and said PI5P4K activity is optionally quantified using absorbance, fluorescence, Raman scattering, phosphorescence, luminescence, luciferase activity, or radioactivity.
20. The method of claim 1 , wherein said cell has a p53 inactive mutation.
21. The method of claim 20 , wherein said cell is a p53-mutated cancer cell.
22. The method of claim 1 , wherein said cell is a p53-null cell.
23. The method of claim 1 , wherein said cell is a breast cancer cell.
24. The method of claim 1 , wherein said activity detected in step (c) is detected by a cell proliferation assay.