Methods of treatment and screening assays for HIF-1α regulation
The present invention relates to a method of treating a patient for a condition where blood flow to a tissue or organ is interrupted. This method involves administering to a patient with a hypoxic condition where blood flow to a tissue or organ is interrupted, or at risk thereof, a compound that reduces the rate of HIF-1α inactivation in cells affected by the condition, thereby treating the patient for the condition. Also disclosed is a method of treating a tumor in a patient. In addition, the present invention relates to methods of identifying compounds as candidate drugs for treatment of hypoxic conditions and for treating tumors in a subject.
1. A method of identifying compounds as candidate drugs for treatment of hypoxic conditions, said method comprising:
providing a cell expressing Hypoxia-Inducible-Factor-1alpha (HIF-1α) wherein HIF-1α undergoes site-specific cleavage that generates paired HIF-1α fragments;
contacting the cell with a compound to be evaluated;
determining the ability of the compound to inhibit the site-specific cleavage of HIF-1α, wherein said determining involves detecting the presence of one or both of the paired HIF-1α fragments generated, wherein a decrease in the number of one or both of the paired HIF-1α fragments in the presence of the compound compared to when the compound is absent indicates the compound has the ability to inhibit the site-specific cleavage of HIF-1α; and
selecting, based on said determining, compounds capable of reducing site-specific cleavage of HIF-1α as candidate drugs for treatment of hypoxic conditions.
2. The method according to claim 1 , wherein the cell further expresses Mitogen activated protein Kinase Phosphatase-1 (MKP-1).
3. The method according to claim 2 , wherein compounds are further selected based on their ability to enhance or suppress MKP-1 activity/expression in the cell.
4. The method according to claim 1 , wherein the cell further expresses HIF-1α targets.
5. The method according to claim 4 , wherein compounds are further selected based on their ability to down-regulate activity of the pro-apototic HIF-1α targets BNIP3 and Noxa.
6. The method according to claim 1 , wherein the cell further expresses a protease known to cleave HIF-1α.
7. The method according to claim 1 , wherein said site-specific cleavage occurs at two sites in HIF-1α.
8. The method according to claim 7 , wherein said site-specific cleavage occurs between (i) amino acid residues 300-350 of HIF-1α and (ii) amino acid residues 477-539 of HIF-1α.
9. The method according to claim 7 , wherein the paired HIF-1α fragments comprise, in one pair, a fragment of approximately 75 kDa, and in a second pair, a fragment of approximately 45 kDa.
10. The method according to claim 9 , wherein said determining involves detecting the presence of the approximately 75 kDa fragment or a corresponding fragment of approximately 35 kDa.
11. The method according to claim 9 , wherein said determining involves detecting the presence of the approximately 45 kDa fragment or a corresponding fragment of approximately 65 kDa.
12. The method according to claim 1 , wherein said detecting is carried out using an antibody to one of the fragments.
13. The method according to claim 1 , wherein said detecting involves Western blot analysis.