Methods and compositions relating to chimeric nicotinic receptor subunits
(19)AUSTRALIAN PATENT OFFICE (54) Title Methods and compositions relating to chmericnicotinic receptor subunits (51)6 International Patent Classification(s) C07K 001/00 C12N 001/20 G01N 033/53 C07K 014/00 C07H 021/04 C12N 015/74 C07K 017/00 C12N 015/00 C12P 021/06 (21) Application No: 2003254016 (22) Application Date: 2003.07.18 (87) WIPO No: WO04/009775 (30) Priority Data (31) Number (32) Date (33) Country 60/397,380 2002 .07 .19 US (43) Publication Date : 2004 .02.09 (43) Publication Journal Date : 2004 .03.18 (71) Applicant(s) CATHOLIC HEALTHCARE WEST; TARGACEPT, INC. (72) Inventor(s) Lukas, Ronald J.; Bencherif, Merouane (-1-1) Application NoAU2003254016 A8(19)AUSTRALIAN PATENT OFFICE (54) Title Methods and compositions relating to chmericnicotinic receptor subunits (51)6 International Patent Classification(s) C07K 001/00 C12N 001/20 G01N 033/53 C07K 014/00 C07H 021/04 C12N 015/74 C07K 017/00 C12N 015/00 C12P 021/06 (21) Application No: 2003254016 (22) Application Date: 2003.07.18 (87) WIPO No: WO04/009775 (30) Priority Data (31) Number (32) Date (33) Country 60/397,380 2002 .07 .19 US (43) Publication Date : 2004 .02.09 (43) Publication Journal Date : 2004 .03.18 (71) Applicant(s) CATHOLIC HEALTHCARE WEST; TARGACEPT, INC. (72) Inventor(s) Lukas, Ronald J.; Bencherif, Merouane -1- A chimeric nAChR receptor subunit polypeptide having a substitution of at least about 15% of the native amino acid sequence of the subunit in the area of the C-terminal cytoplasmic domain is provided, as well as polynucleotides encoding the polypeptide. Vectors, host cells, and related methods for evaluating compounds are also provided. What is claimed is: 1. A polypeptide comprising a chimeric human nAChR receptor subunit having a substitution of at least a portion of a C-terminal cytoplasmic domain, wherein the substitution comprises at least about 15 % of the native amino acid sequence of the subunit.
2. The polypeptide of claim 1, wherein the substitution comprises at least about 20 % of the native amino acid sequence of the subunit.
3. The polypeptide of claim 1, wherein the substitution comprises at least a portion of a transmembrane domain adjacent to the C-terminal end of the cytoplasmic domain.
4. The polypeptide of claim 3, wherein the substitution further comprises all of the transmembrane domain C-terminal to the large, C-terminal cytoplasmic domain and at least a portion of a C-terminal extracellular domain.
5. The polypeptide of claim 3, wherein the substitution further comprises at least a portion of a transmembrane domain adjacent to the N-terminal end of the cytoplasmic domain.
6. The polypeptide of claim 3, wherein the substitution comprises all of the C-terminal cytoplasmic domain and at least a portion of each transmembrane domain positioned adjacent to the cytoplasmic domain.
7. The polypeptide of claim 3, wherein the substitution further comprises all of the transmembrane domains adjacent to the cytoplasmic domain and all native subunit amino acids C-terminal to the cytoplasmic domain.
8. The polypeptide of claim 1, wherein substitution is an amino acid sequence derived from domain of a receptor subunit of a ligand-gated ion channel superfamily receptor that is structurally analogous to the sequence of the native nAChR polypeptide replaced by the substitution. <Desc/Clms Page number 44> 9. The polypeptide of claim 8, wherein the receptor subunit from which the substituted sequence is derived is characterized by having four transmembrane domains.
10. The polypeptide of claim 9, wherein the subunit is a subunit found in a receptor selected from the group consisting of GABA-A, glycine, seratonin, and nAChR receptors.
11. The polypeptide of claim 10, wherein the receptor is a homopentameric receptor.
12. The polypeptide of claim 1, wherein the substitution is derived from a nAChR receptor subunit.
13. The polypeptide of claim 12, wherein the nAChR receptor subunit is an a7 nAChR subunit.
14. The polypeptide of claim 1, wherein the substitution is derived from a subunit of a serotonin type 3 receptor.
15. The polypeptide of claim 1, wherein the substitution is derived from a subunit selected from the group consisting of GABA-A R a3, GABA-A R pi, glyR a3, and glyR ss.
16. A polypeptide comprising a chimeric nAChR receptor a4 subunit having a substitution of at least a portion of the large C-terminal cytoplasmic domain, wherein the substitution comprises at least about 15 % of the native amino acid sequence of the subunit.
17. The polypeptide of claim 16, wherein the substitution comprises at least about 20 % of the native amino acid sequence of the subunit.
18. The polypeptide of claim 16, wherein the substitution of the native amino acid sequence of the subunit begins in a region represented by, or homologous to, from about amino acid position number P304 to about amino acid position number S362 of SEQ ID NO: 1 and ends in a region represented by, or homologous to, from about amino acid number P562 to about I627 of SEQ ID NO: 1. <Desc/Clms Page number 45> 19. The polypeptide of claim 16, wherein the substitution of the native amino sequence of the subunit begins in a region represented by, or homologous to, from about amino acid position number H331 to about amino acid position number L355 of SEQ ID NO: 1 and ends in a region represented by, or homologous to, from about amino acid number R566 to about R600 of SEQ ID NO : 1.
20. A polypeptide comprising a chimeric p2 nAChR receptor subunit having a substitution of at least a portion of the large C-terminal cytoplasmic domain, wherein the substitution comprises at least about 15 % of the native amino acid sequence of the subunit.
21. The polypeptide of claim 20, wherein the substitution comprises at least about 20 % of the native amino acid sequence of the subunit.
22. The polypeptide of claim 20, wherein the substitution of the native amino acid sequence of the subunit begins in a region represented by, or homologous to, from about amino acid position number P295 to about amino acid position number R353 of SEQ ID NO: 2 and ends in a region represented by, or homologous to, from about amino acid number C422 to about K502 of SEQ ID NO : 2.
23. The polypeptide of claim 20, wherein the substitution of the native amino acid sequence of the subunit begins in a region represented by, or homologous to, from about amino acid position number H322 to about amino acid position number Q350 of SEQ ID NO : 2 and ends in a region represented by, or homologous to, from about amino acid number E426 to about R460 of SEQ ID NO : 2.
24. A polypeptide comprising a chimeric ss4 nAChR receptor subunit having a substitution of at least a portion of the large C-terminal cytoplasmic domain, wherein the substitution comprises at least about 15 % of the native amino acid sequence of the subunit large C- terminal cytoplasmic domain.
25. The polypeptide of claim 24, wherein the substitution comprises at least about 20 % of the native sequence. <Desc/Clms Page number 46> 26. The polypeptide of claim 24, wherein the substitution of the native amino acid sequence of the subunit begins in a region represented by, or homologous to, from about amino acid position number P293 to about amino acid position number G351 of SEQ ID NO: 3 and ends in a region represented by, or homologous to, from about amino acid number Q422 to about D498 of SEQ ID NO : 3.
27. The polypeptide of claim 24, wherein the substitution of the native amino acid sequence of the subunit begins in a region represented by, or homologous to, from about amino acid position number H320 to about amino acid position number K348 of SEQ ID NO : 3 and ends in a region represented by, or homologous to, from about amino acid number E426 to about R460 of SEQ ID NO : 3.
28. A polynucleotide encoding a polypeptide of any one of claims 1-27 and conservative substitutions thereof.
29. A polynucleotide selected from the group consisting of: a polynucleotide encoding a subunit polypeptide as shown SEQ ID NO : 7, SEQ ID NO: 9, or SEQ ID NO : 11 ; and a polynucleotide degenerate to a polynucleotide encoding a subunit polypeptide as shown in SEQ ID NO : 7, SEQ ID NO: 9, or SEQ ID NO: 11.
30. A polynucleotide as shown in SEQ ID NO : 6, SEQ ID NO : 8, or SEQ ID NO : 9.
31. A vector comprising the polynucleotide of any one of claims 28-30.
32. A host cell comprising a vector of claim 31.
33. The host cell of claim 32 wherein the host cell expresses a subunit polypeptide encoded by the vector.
34. A host cell containing at least one polynucleotide of any one of claims 28-30, wherein the cells express at least one subunit polypeptide encoded by the at least one polynucleotide. <Desc/Clms Page number 47> 35. The host cell of claim 34, wherein the cells are selected from the group consisting of bacterial cells, eukaryotic cells, and amphibian oocytes.
36. The cell of claim 34, wherein the cell further comprises at least one polynucleotide encoding a second human subunit nAChR, wherein the subunit can be a chimeric or native amino acid sequence of the subunit and wherein the subunit is a a subunit if the first subunit is a ss subunit and is a P subunit if the first subunit is an a subunit.
37. The cell of claim 34, wherein the cell comprises a receptor having a voltage dependent calcium channel.
38. The cell of claim 34, wherein the cell comprises a functional nAChR that comprises one or more subunit polypeptides encoded by the polynucleotide.
39. A method for identifying compounds that modulate the activity of neuronal nAChR, the method comprising, a) determining the effect of a test compound on the neuronal nAChR activity of a receptor comprising at least one subunit polypeptide of any one of claims 1-27 in test cells, b) comparing the effect determined in a) to the effect of the test compound on control cells that lack any receptors comprising any subunit polypeptides of any one of claims1-27 or to the neuronal nAChR activity of the test cells in the absence of the test compound, wherein a difference in effects of the test compound in the test cells as compared to the control cells indicates that the test compound modulates activity of the nAChR.
40. A method for identifying compounds as candidates for further evaluation as agonists, antagonists, or partial agonists of nAChRs, said method comprising: a) contacting recombinant cells with a test compound, wherein the recombinant cells are produced by introducing a nucleic acid encoding at least one subunit polypeptide of any one of claims 1-27 into the cells; and wherein the recombinant cells express an nAChR comprising at least one subunit encoded by the nucleic acid; and <Desc/Clms Page number 48> b) measuring ion flux, the electrophysiological response of the cells, or binding of the test compound to the nAChR, wherein compounds that are candidates for evaluation as agonists, antagonists, or partial agonists of the nAChR are identified based on an increase or decrease in activity of the nAChR comprising the subunit.