Akt-based inducible survival switch
AssigneeBaylor College of Medicine
InventorDavid Spencer
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Abstract
The present invention relates to the field of apoptosis and programmed cell-death. More particularly, it relates to expression vectors, pharmaceutical compositions and methods for inhibiting cell-death using the expression vectors and/or pharmaceutical compositions. Yet further, the present invention also relates to methods of using the expression vector to screen for additional regulators of an anti-apoptotic gene.
Claims40
An expression vector comprising an inducible chimeric protein, wherein said protein comprises a mutant Akt polypeptide fused to a ligand-binding domain.
A fusion protein comprising a mutant Akt sequence and at least one ligand-binding domain.
A method of modulating apoptosis comprising the steps of:
- administering to a cell susceptible to apoptosis an expression vector encoding an inducible chimeric protein comprising a mutant Akt polypeptide fused to a ligand-binding domain;
- administering to the cell a second expression vector encoding a second ligand-binding domain fused to a membrane-targeting region;
- and
- modulating apoptosis by administering to the cell a chemical ligand, wherein the ligand results in activation of the mutant Akt.
A method of modulating apoptosis comprising the steps of:
- administering to a cell susceptible to apoptosis an expression vector encoding an inducible chimeric protein comprising a mutant Akt polypeptide fused to a ligand-binding domain and a second chimeric protein comprising a ligand-binding domain fused to a membrane-targeting region;
- and
- modulating apoptosis by administering to the cell a chemical ligand, wherein the ligand results in activation of the mutant Akt.
A method of modulating hypoxia-induced apoptosis comprising the steps of:
- administering to a cell suspected of hypoxia-induced apoptosis an expression vector encoding an inducible chimeric protein comprising a mutant Akt polypeptide fused to a ligand-binding domain;
- administering to the cell a second expression vector encoding a second ligand-binding domain fused to a membrane-targeting region;
- and
- modulating hypoxia-induced apoptosis by administering to the cell a chemical ligand, wherein the chemical ligand results in activation of the mutant Akt.
A method of modulating tissue damage following ischemia-reperfusion comprising the steps of:
- administering to a tissue suspected of tissue damage an expression vector encoding an inducible chimeric protein comprising a mutant Akt polypeptide fused to a ligand-binding domain;
- administering to the tissue a second expression vector encoding a second ligand-binding domain fused to a membrane-targeting region;
- and
- modulating tissue damage by administering to the tissue a chemical ligand, wherein the ligand results in activation of the mutant Akt.
A method of treating myocardial infarction comprising the step of:
- administering to a subject in need of such treatment an inducible Akt molecule in an amount effective to reduce cardiac tissue necrosis in the subject.
A method of modulating tissue damage during transplantation comprising the steps of:
- administering to a tissue suspected of tissue damage an expression vector encoding an inducible chimeric protein comprising a mutant Akt polypeptide fused to a ligand-binding domain;
- administering to the tissue a second expression vector encoding a second ligand-binding domain fused to a membrane-targeting region;
- and
- modulating tissue damage by administering to the tissue a chemical ligand, wherein the chemical ligand results in activation of the mutant Akt.
A method of screening compounds to identify a modulator of Akt comprising the steps of:
- providing a cell expressing iAkt;
- contacting said cell with a candidate compound;
- admixing rapamycin analogs to induce activation of Akt;
- measuring the level of activation of Akt;
- and
- comparing said Akt activation in the presence of said candidate compound with the activation of Akt in the absence of said candidate compound;
- wherein a difference in the activation of Akt in the presence of said candidate compound, as compared with the activation of Akt in the absence of said candidate compound, identifies said candidate compound as a modulator of Akt activation.
A method of screening compounds to identify a modulator of Akt comprising the steps of:
- providing a cell expressing iAkt;
- contacting said cell with a candidate compound;
- admixing rapamycin analogs to induce activation of Akt;
- measuring the level of phosphorylation of Akt;
- and
- comparing said Akt phosphorylation in the presence of said candidate compound with the Akt phosphorylation in the absence of said candidate compound;
- wherein a difference in the phosphorylation of Akt in the presence of said candidate compound, as compared with the phosphorylation of Akt in the absence of said candidate compound, identifies said candidate compound as a modulator of Akt phosphorylation.
A method of screening compounds to identify a modulator of Akt comprising the steps of:
- providing a cell expressing iAkt;
- contacting said cell with a candidate compound;
- admixing rapamycin analogs to induce activation of Akt;
- measuring Akt activity;
- and
- comparing said Akt activity in the presence of said candidate compound with the Akt activity in the absence of said candidate compound;
- wherein a difference in the activity of Akt in the presence of said candidate compound, as compared with the activity of Akt in the absence of said candidate compound, identifies said candidate compound as a modulator of Akt activity.
A method of treating a disease by screening compounds to identify a modulator of Akt comprising the steps of:
- providing a cell expressing iAkt;
- contacting said cell with a candidate compound;
- admixing rapamycin analogs to induce activation of Akt;
- measuring Akt activity;
- comparing said Akt activity in the presence of said candidate compound with the Akt activity in the absence of said candidate compound;
- wherein a difference in the activity of Akt in the presence of said candidate compound, as compared with the activity of Akt in the absence of said candidate compound, identifies said candidate compound as a modulator of Akt activity;
- and
- administering to a subject suffering from the disease the modulator of Akt activity.
Dependencies are inferred from the claim text.