IRE-1α substrates

• US 8,124,343 B2
• Filed: 07/19/2011
• Issued: 02/28/2012
• Est. Priority Date: 11/04/2005
• Status: Active Grant

• Current Assignee: Fosun Orinove Pharmatech, Inc.
• Original Assignee: Mannkind Corporation
• Inventors: Patterson, John Bruce, Volkmann, Kori Shallyn, Brumm, Duane, Stiles, Caryn
• Primary Examiner(s): Babic, Christopher M.
• Application Number:
  US13/185,693
• Publication Number:
  US 20110287952A1
• Time in Patent Office: 224 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

IRE-1α substrates useful for identifying agonists and antagonists of IRE-1α RNase activity.

Claims

1. A substrate for IRE-1α

, comprising an oligonucleotide molecule which consists of (i) an RNA loop comprising a cleavage site for IRE-1α

; and

(ii) a nucleotide stem consisting of 4, 5, 6, 7, 8, 9, or 10 nucleotide base pairs.

2. The substrate of claim 1 wherein the RNA loop consists of a nucleotide sequence selected from the group consisting of:

5′

-CCGAAGC-3′

,5′

-GCGAAGC-3′

,5′

-ACGAAGC-3′

, 5′

-UCGAAGC-3′

,5′

-CCGAAGC-3′

,5′

-CGGAAGC-3′

,5′

-CAGAAGC-3′

,5′

-CUGAAGC-3′

, 5′

-CCGAAGC-3′

,5′

-CCAAAGC-3′

,5′

-CCUAAGC-3′

,5′

-CCGAAGC-3′

,5′

-CCGGAGC-3′

, 5′

-CCGUAGC-3′

,5′

-CCGCAGC-3′

,5′

-CCGAAGC-3′

,5′

-CCGAGGC-3′

,5′

-CCGAUGC-3′

, 5′

-CCGACGC-3′

,5′

-CCGAAGC-3′

,5′

-CCGAAAC-3′

,5′

-CCGAAGC-3′

,5′

-CCGAAGA-3′

, and 5′

-CCGAAGU-3′

.

3. The substrate of claim 1 wherein the RNA loop consists of the nucleotide sequence 5′

-CCGCAGC-3′

.

4. The substrate of claim 1 wherein the oligonucleotide molecule consists of SEQ ID NO:

3.

5. The substrate of claim 1 wherein the nucleotide stem comprises DNA.

6. The substrate of claim 1 wherein the nucleotide stem comprises RNA.

7. The substrate of claim 1 wherein the nucleotide stem comprises a nucleotide analog.

8. The substrate of claim 1, further comprising a detectable label.

9. A method for detecting RNase activity of an IRE-1α

polypeptide, comprising;

(1) contacting the IRE-1α

polypeptide with a substrate for IRE-1α

, comprising an oligonucleotide molecule which consists of (i) an RNA loop comprising a cleavage site for IRE-1α

; and

(ii) a nucleotide stem consisting of 4, 5, 6, 7, 8, 9, or 10 nucleotide base pairs; and

(2) detecting cleavage of the substrate.

10. The method of claim 9 further comprising contacting the IRE-1α

polypeptide with a test compound.

11. The method of claim 9 wherein the IRE-1α

polypeptide and the substrate are in a cell-free system.

12. The method of claim 9 wherein the IRE-1α

polypeptide is a full-length IRE-1α

protein.

13. The method of claim 9 wherein the IRE-1α

polypeptide comprises a kinase domain.

14. The method of claim 9 wherein the RNA loop consists of a nucleotide sequence selected from the group consisting of:

5′

-CCGAAGC-3′

,5′

-GCGAAGC-3′

,5′

-ACGAAGC-3′

, 5′

-UCGAAGC-3′

,5′

-CCGAAGC-3′

,5′

-CGGAAGC-3′

,5′

-CAGAAGC-3′

,5′

-CUGAAGC-3′

, 5′

-CCGAAGC-3′

,5′

-CCAAAGC-3′

,5′

-CCUAAGC-3′

,5′

-CCGAAGC-3′

,5′

-CCGGAGC-3′

, 5′

-CCGUAGC-3′

,5′

-CCGCAGC-3′

,5′

-CCGAAGC-3′

,5′

-CCGAGGC-3′

,5′

-CCGAUGC-3′

, 5′

-CCGACGC-3′

,5′

-CCGAAGC-3′

,5′

-CCGAAAC-3′

,5′

-CCGAAGC-3′

,5′

-CCGAAGA-3′

, and 5′

-CCGAAGU-3′

.

15. The method of claim 9 wherein the RNA loop consists of the nucleotide sequence 5′

-CCGCAGC-3′

.

16. The method of claim 9 wherein the oligonucleotide molecule consists of SEQ ID NO:

3.

17. The method of claim 9 wherein the nucleotide stem comprises DNA.

18. The method of claim 9 wherein the nucleotide stem comprises RNA.

19. The method of claim 9 wherein the nucleotide stem comprises a nucleotide analog.

20. The method of claim 9 wherein the substrate further comprising a detectable label.