DERMACENTOR VARIABILIS GABA-GATED CHLORIDE CHANNELS

• US 20030208041A1
• Filed: 09/25/2002
• Published: 11/06/2003
• Est. Priority Date: 03/31/2000
• Status: Active Grant

• Current Assignee: Merial Limited (C.H. Boehringer Sohn AG & Co. KG)
• Original Assignee: Merck & Co., Inc.
• Inventors: Cully, Doris F., Ludmerer, Steve W., Zheng, Yingcong
• Granted Patent: US 7,220,832 B2
• Time in Patent Office: Days
• US Class Current: 530/350
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 33/14 Ectoparasiticides, e.g. scabicides
• CPC Class Codes: C07K 14/43527 from ticks
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68

Abstract

The present invention features Dermacentor variabilis GABA-gated chloride channel polypeptides and nucleic acids, and uses of such polypeptides and nucleic acids. D. variabilis is a widely distributed tick associated with different diseases. A preferred use of the present invention is to obtain compounds for preventing or treating a tick infestation.

Claims

1. A purified polypeptide comprising a unique amino acid region of SEQ. ID. NO. 1, SEQ. ID. NO. 2, or SEQ. ID. NO. 3 that is at least 9 contiguous amino acids in length, wherein said unique region is not present in SEQ. ID. NO. 7.

2. The polypeptide of claim 1, wherein said unique region comprises an amino acid sequence selected from the group consisting of:

3. The polypeptide of claim 1, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2, and SEQ. ID. NO. 3.

4. The polypeptide of claim 3, wherein said polypeptide consists of an amino acid sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2, and SEQ. ID. NO. 3.

5. The polypeptide of claim 1, wherein said polypeptide consists of an amino acid sequence having a sequence identity of at least 70% with SEQ. ID. NO. 1.

6. A purified nucleic acid comprising a nucleotide sequence encoding for the polypeptide of claim 1.

7. A purified nucleic acid comprising a nucleotide sequence encoding for the polypeptide of claim 5.

8. A purified nucleic acid comprising a unique nucleotide sequence region of SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6 or the complement thereof, that is at least 18 contiguous nucleotides in length, wherein said unique region is not present in SEQ. ID. NO. 8 or the complement thereof.

9. The purified nucleic acid of claim 8, wherein said unique region comprises a sequence selected from the group consisting of:

10. The purified nucleic acid of claim 8, wherein said nucleic acid comprises a sequence selected from the group consisting of the nucleotide sequence of SEQ. ID. NO. 4, SEQ. ID. NO. 5 and SEQ. ID. NO. 6.

11. The purified nucleic acid of claim 10, wherein said nucleic acid consists of a sequence selected from the group consisting of the nucleotide sequence of SEQ. ID. NO. 4, SEQ. ID. NO. 5 and SEQ. ID. NO. 6.

12. An expression vector comprising a recombinant nucleotide sequence encoding for a unique amino acid region of SEQ. ID. NO. 1, SEQ. ID. NO. 2, or SEQ. ID. NO. 3 that is at least 9 contiguous amino acids in length, wherein said unique region is not present in SEQ. ID. NO. 7.

13. The expression vector of claim 12, wherein said recombinant nucleotide sequence encodes for a polypeptide comprising a sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3.

14. A recombinant cell comprising the expression vector of claim 12, wherein said recombinant nucleotide sequence is functionally coupled to a promoter recognized by said cell.

15. A recombinant cell comprising the expression vector of claim 13, wherein said recombinant nucleotide sequence is functionally coupled to a promoter recognized by said cell.

16. A recombinant cell made by a process comprising the step of introducing into said cell the expression vector of claim 12, wherein said recombinant nucleotide sequence is functionally coupled to a promoter recognized by said cell.

17. A purified antibody preparation comprising an antibody that binds a polypeptide selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3.

18. The antibody of claim 17, wherein said polypeptide consists of the amino acid sequence of SEQ. ID. NO. 19 (LGKRITMRKTRCQQLAKLAEQ HRQR).

19. A method of preparing a GABA-gated chloride polypeptide comprising the step of incubating a cell comprising a recombinant nucleotide sequence encoding for a unique amino acid region of SEQ. ID. NO. 1, SEQ. ID. NO. 2, or SEQ. ID. NO. 3 that is at least 9 contiguous amino acids in length, wherein said unique region is not present in SEQ. ID. NO. 7, under conditions wherein said polypeptide is expressed.

20. A method for assaying the binding of a compound to a D. variabilis GABA-gated chloride channel comprising the steps of:

a) expressing from a recombinant nucleic acid a polypeptide comprising a unique amino acid region of SEQ. ID. NO. 1, SEQ. ID. NO. 2, or SEQ. ID. NO. 3 that is at least 9 contiguous amino acids in length, wherein said unique region is not present in SEQ. ID. NO. 7;

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

c) measuring the ability of said test preparation to bind to said polypeptide.

21. The method of claim 20, wherein said polypeptide consists of 9 contiguous amino acids up to the full-length sequence provided in SEQ. ID. NOs. 1, 2, or 3.

22. The method of claim 21, wherein said steps (b) and (c) are performed in vitro.

23. The method of claim 21, wherein said steps (a), (b) and (c) are performed using a whole cell.

24. The method of claim 21, wherein said polypeptide is expressed from an expression vector.

25. The method of claim 24, wherein said polypeptide consists of a sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3.

26. A method of measuring GABA-gated chloride channel activity comprising the steps of:

a) contacting a recombinant cell with a compound, wherein said recombinant cell comprises a recombinant nucleotide sequence expressing a functional GABA-gated chloride channel that comprises a unique amino acid region of SEQ. ID. NO. 1, SEQ. ID. NO. 2, or SEQ. ID. NO. 3 that is at least 9 contiguous amino acids in length, provided that said unique region is not present in SEQ. ID. NO. 7; and

b) measuring GABA-gated chloride channel activity.

27. The method of claim 26, wherein said functional GABA-gated chloride channel comprises an amino sequence having a sequence identity of at least 70% with SEQ. ID. NO. 1.

28. The method of claim 26, wherein said functional GABA-gated chloride channel comprises a sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3.

29. The method of claim 26, wherein said functional GABA-gated chloride channel consists of a sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3.

30. A method of decreasing or preventing a tick infestation comprising the steps of:

a) identifying a compound that modulates a D. variabilis GABA-gated chloride channel activity; and

b) using said compound to decrease or prevent a tick infestation.

31. The method of claim 30, wherein said step (a) is performed by (i) contacting a recombinant cell with said compound, wherein said recombinant cell comprises a recombinant nucleotide sequence expressing a functional GABA-gated chloride channel that comprises a unique amino acid region of SEQ. ID. NO. 1, SEQ. ID. NO. 2, or SEQ. ID. NO. 3 that is at least 9 contiguous amino acids in length, provided that said unique region is not present in SEQ. ID. NO. 7 and (ii) measuring GABA-gated chloride channel activity.

32. The method of claim 31, wherein said functional GABA-gated chloride channel comprises a sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2, and SEQ. ID. NO. 3.

33. The method of claim 32, wherein said functional GABA-gated chloride channel consists of a sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2, and SEQ. ID. NO. 3.