Necrosis assay

• US 7,622,106 B1
• Filed: 03/06/2009
• Issued: 11/24/2009
• Est. Priority Date: 03/06/2009
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: He, Sudan, Wang, Xiaodong, Wang, Lai
• Primary Examiner(s): Saoud; Christine J
• Assistant Examiner(s): Seharaseyon; Jegatheesan
• Application Number:
  US12/399,872
• Time in Patent Office: 263 Days
• Field of Search: None
• US Class Current: 424/85.1
• CPC Class Codes: G01N 2333/70575 NGF/TNF-superfamily, e.g. CD70, CD95L, CD153 or CD154
• CPC Class Codes: G01N 2333/96469 Interleukin 1-beta convertase-like enzymes
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

A necrosis assays is performed with a cell expressing RIPK1 and RIP3 by (a) culturing the cell with a smac mimetic, caspase-8 inhibitor and TNF-α; and (b) detecting a resultant necrosis of the cell.

Claims

1. A method of inducing necrosis in a mammalian cell expressing RIPK1 and RIP3, comprising the steps of:

(a) culturing the cell with a smac mimetic, caspase-8 inhibitor and TNF-α

; and

(b) detecting a resultant necrosis of the cell.

2. The method of claim 1, wherein the culturing step comprises contacting the cell with an agent, thereby biasing the resultant necrosis.

3. The method of claim 1, wherein the culturing step comprises contacting the cell with an agent, thereby inhibiting the resultant necrosis, and the detecting step identifies the agent as an inhibitor of necrosis.

4. The method of claim 1, wherein the culturing step comprises contacting the cell with an agent, thereby inhibiting the resultant necrosis, the detecting step identifies the agent as an inhibitor of necrosis, and the method further comprising the step of confirming necrosis inhibitory activity of the agent in a mouse acute pancreatitis model.

5. The method of claim 1, wherein the TNF-α

is autocrine.

6. The method of claim 1, wherein the RIP3 is ectopically expressed.

7. The method of claim 1, wherein the smac mimetic is a smac small molecule dimer 3:

8. The method of claim 1, wherein the caspase-8 inhibitor is z-VAD or Q-VD-OPH.