Alternatively spliced isoforms of checkpoint kinase 1 (CHK1)

• US 20050266469A1
• Filed: 05/25/2005
• Published: 12/01/2005
• Est. Priority Date: 05/25/2004
• Status: Abandoned Application

• Current Assignee: Rosetta Inpharmatics LLC (Merck & Co., Inc.)
• Original Assignee: Rosetta Inpharmatics LLC (Merck & Co., Inc.)
• Inventors: Garrett-Engele, Philip W., Raymond, Christopher K., Castle, John C.
• Application Number:
  US11/137,315
• Publication Number:
  US 20050266469A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Abstract

The present invention features nucleic acids and polypeptides encoding two novel splice variant isoforms of checkpoint kinase 1 (CHK1). The polynucleotide sequences of CHK1sv1 and CHK1sv2 are provided by SEQ ID NO 3 and SEQ ID NO 5, respectively. The amino acid sequences for CHK1sv1 and CHK1sv2 are provided by SEQ ID NO 4 and SEQ ID NO 6, respectively. The present invention also provides methods for using CHK1sv1 and CHK1sv2 polynucleotides and proteins to screen for compounds that bind to CHK1sv1 and CHK1sv2, respectively.

Claims

1. A purified human nucleic acid comprising SEQ ID NO 3, or the complement thereof.

2. The purified nucleic acid of claim 1, wherein said nucleic acid comprises a region encoding SEQ ID NO 4.

3. The purified nucleic acid of claim 1, wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 4.

4. A purified polypeptide comprising SEQ ID NO 4.

5. The polypeptide of claim 4, wherein said polypeptide consists of SEQ ID NO 4.

6. An expression vector comprising a nucleotide sequence encoding SEQ ID NO 4, wherein said nucleotide sequence is transcriptionally coupled to an exogenous promoter.

7. The expression vector of claim 6, wherein said nucleotide sequence encodes a polypeptide consisting of SEQ ID NO 4.

8. The expression vector of claim 6, wherein said nucleotide sequence comprises SEQ ID NO 3.

9. The expression vector of claim 6, wherein said nucleotide sequence consists of SEQ ID NO 3.

10. A method of screening for compounds able to bind selectively to CHK1sv1 comprising the steps of:

(a) providing a CHK1sv1 polypeptide comprising SEQ ID NO 4;

(b) providing one or more CHK1 isoform polypeptides that are not CHK1sv1;

(c) contacting said CHK1sv1 polypeptide and said CHK1 isoform polypeptide that is not CHK1sv1 with a test preparation comprising one or more compounds; and

(d) determining the binding of said test preparation to said CHK1sv1 polypeptide and to said CHK1 isoform polypeptide that is not CHK1sv1, wherein a test preparation that binds to said CHK1sv1 polypeptide, but does not bind to said CHK1 polypeptide that is not CHK1sv1, contains a compound that selectively binds said CHK1sv1 polypeptide.

11. The method of claim 10, wherein said CHK1sv1 polypeptide is obtained by expression of said polypeptide from an expression vector comprising a polynucleotide encoding SEQ ID NO 4.

12. The method of claim 11, wherein said polypeptide consists of SEQ ID NO 4.

13. A method for screening for a compound able to bind to or interact with a CHK1sv1 protein or a fragment thereof comprising the steps of:

(a) expressing a CHK1sv1 polypeptide comprising SEQ ID NO 4 or fragment thereof from a recombinant nucleic acid;

(b) providing to said polypeptide a labeled CHK1 ligand that binds to said polypeptide and a test preparation comprising one or more compounds; and

(c) measuring the effect of said test preparation on binding of said labeled CHK1 ligand to said polypeptide, wherein a test preparation that alters the binding of said labeled CHK1 ligand to said polypeptide contains a compound that binds to or interacts with said polypeptide.

14. The method of claim 13, wherein said steps (b) and (c) are performed in vitro.

15. The method of claim 13, wherein said steps (a), (b) and (c) are performed using a whole cell.

16. The method of claim 13, wherein said polypeptide is expressed from an expression vector.

17. The method of claim 13, wherein said CHK1sv1 ligand is a CHK1 inhibitor.

18. The method of claim 16, wherein said expression vector comprises SEQ ID NO 3 or a fragment of SEQ ID NO 3.

19. The method of claim 16, wherein said polypeptide comprises SEQ ID NO 4 or a fragment of SEQ ID NO 4.

20. The method of claim 16, wherein said test preparation contains one compound.

21. A method of screening for CHK1sv1 activity comprising the steps of:

(a) contacting a cell expressing a recombinant nucleic acid encoding CHK1sv1 comprising SEQ ID NO 4 with a test preparation comprising one or more test compounds; and

(b) measuring the effect of said test preparation on protein kinase activity.

22. A method of screening for compounds able to bind selectively to one or more CHK1 isoform polypeptides but not to CHK1sv1 comprising the steps of:

(a) providing a CHK1 isoform polypeptide that is not CHKsv1;

(b) providing a CHK1sv1 polypeptide comprising SEQ ID NO 4;

(c) contacting said CHK1 isoform polypeptide that is not CHKsv1 and said CHKsv1 polypeptide with a test preparation comprising one or more compounds; and

(d) determining the binding of said test preparation to said CHK1 isoform polypeptide that is not CHK1sv1 and to said CHK1sv1 polypeptide, wherein a test preparation that binds to said CHK1 isoform polypeptide that is not CHK1sv1, but does not bind to CHK1sv1 polypeptide, contains a compound that selectively binds said CHK1 isoform polypeptide that is not CHK1sv1.

23. A method for screening compounds able to inhibit or alter the interaction between CHK1sv1 polypeptide and CHK1 ligands comprising the steps of:

(a) providing a CHK1sv1 polypeptide comprising SEQ ID NO 4 or fragment thereof;

(b) providing to said polypeptide a CHK1 ligand that binds to said polypeptide and a test preparation comprising one or more compounds; and

(c) measuring the effect of said test preparation on the interaction of said CHK1 ligand with said CHK1sv1 polypeptide, wherein a test preparation that alters the interaction between said CHK1 ligand with said CHK1sv1 polypeptide contains a compound that inhibits or alters the interaction between said CHK1sv1 polypeptide and said CHK1 ligand.

24. The method of claim 23, wherein said CHK1 ligand is selected from the group consisting of:

CDC25, p53, and TLK1/2.

25. A method for screening compounds able to modulate the kinase activity of CHK1sv1 comprising the steps of:

(a) providing a CHK1sv1 polypeptide comprising SEQ ID NO 4 or fragment thereof;

(b) providing to said CHK1sv1 polypeptide a kinase substrate and a test preparation comprising one or more compounds; and

(c) determining the effect of said test preparation on protein kinase activity of said CHK1sv1 polypeptide to thereby identify a compound that modulates the activity of the said CHK1sv1 polypeptide.

26. The method of claim 25, wherein said kinase substrate is selected from the group consisting of:

CDC25, p53, and TLK1/2.