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Last Updated: March 28, 2024

Claims for Patent: 5,795,713


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Summary for Patent: 5,795,713
Title: Methods for identifying inducers and inhibitors of programmed cell death
Abstract:Methods for identifying inducers and inhibitors of programmed cell death in a cell-free system are described. The methods exploit the finding that programmed cell death is accompanied by shutdown of cellular protein synthesis and by phosphorylation of eIF-2.alpha. and that the dephosphorylation of eIF-2.alpha. prevents the shutdown of protein synthesis.
Inventor(s): Roizman; Bernard (Chicago, IL), He; Bin (Chicago, IL)
Assignee: Arch Development Corporation (Chicago, IL)
Application Number:08/795,470
Patent Claims:1. A method for identifying inhibitors of programmed cell death in a cell-free system, the method comprising the steps of:

(a) preparing a cell-free system comprising phosphorylated eIF-2.alpha. and protein phosphatase 1.alpha. (PP1.alpha.);

(b) introducing into the cell-free system of step (a) a candidate inhibitor of programmed cell death and allowing the resulting mixture to incubate; and,

(c) measuring the level of phosphorylation of eIF-2.alpha. resulting from step (b);

wherein an inhibitor of programmed cell death decreases or prevents increase of the level of phosphorylation of eIF-2.alpha. that accompanies programmed cell death when compared to a control.

2. The method of claim 1 wherein the phosphorylated eIF-2.alpha. is [.sup.32 P]-labeled eIF-2.alpha..

3. The method of claim 1 wherein the cell-free system comprises a post-ribosomal supernatant derived from eukaryotic cells.

4. The method of claim 3 wherein the phosphorylated eIF-2.alpha. is [.sup.32 P]-labeled eIF-2.alpha..

5. The method of claim 3 wherein the post-ribosomal supernatant is derived from mammalian cells infected with a herpes simplex virus lacking expressible .sub..gamma.1 34.5 genes.

6. The method of claim 3 wherein said eukaryotic cells are mammalian cells.

7. The method of claim 1 wherein the phosphorylated eIF-2.alpha. is prepared by reaction in a mixture comprising eIF-2.alpha. , hemin controlled translational repressor, and ATP or an analog thereof.

8. The method of claim 7 wherein the mixture further comprises a buffer.

9. The method of claim 1 wherein the cell-free system of step (a) further comprises a buffer.

10. A method for identifying inducers of programmed cell death in a cell-free system, the method comprising the steps of:

(a) preparing a cell-free system comprising eIF-2.alpha., protein phosphatase 1.alpha. (PP1.alpha.), and ATP or an analog thereof;

(b) introducing into the cell-free system of step (a) a candidate inducer of programmed cell death and allowing the resulting mixture to incubate; and,

(c) measuring the level of phosphorylation of eIF-2.alpha. resulting from step (b);

wherein an inducer of programmed cell death increases or prevents decrease of the level of phosphorylation of eIF-2.alpha. that accompanies programmed cell death when compared to a control.

11. The method of claim 10 wherein the cell-free system further comprises double-strand RNA-dependent protein kinase (PKR).

12. The method of claim 10 wherein the ATP is .sub..gamma..sup.32 P-labeled ATP.

13. The method of claim 10 wherein the cell-free system further comprises a post-ribosomal supernatant derived from eukaryotic cells.

14. The method of claim 13 wherein the cell-free system further comprises double-strand RNA-dependent protein kinase (PKE).

15. The method of claim 13 wherein said eukaryotic cells are mammalian cells.

16. The method of claim 10 wherein the cell-free system of step (a) further comprises a buffer.

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