Method of screening calcium receptor-active molecules

• US 5,858,684 A
• Filed: 06/07/1995
• Issued: 01/12/1999
• Est. Priority Date: 08/23/1991
• Status: Expired due to Term

• Current Assignee: Brigham and Women's Hospital Incorporated, NPS Pharmaceuticals Incorporated (Shire plc), Susan Brown
• Original Assignee: Brigham and Women's Hospital Incorporated, NPS Pharmaceuticals Incorporated (Shire plc)
• Inventors: Garrett, James E. Jr., Balandrin, Manuel F., Brown, Edward M., Van Wagenen, Bradford C., Del Mar, Eric G., Nemeth, Edward F., Hebert, Steven C.
• Primary Examiner(s): Walsh, Stephen
• Assistant Examiner(s): SORENSEN, KENNETH
• Application Number:
  US08/480,751
• Time in Patent Office: 1,315 Days
• Field of Search: 435/7.2, 435/65.1, 435/325, 435/252.3, 435/320.1, 530/300, 530/350, 530/324, 536/23.1, 536/23.5
• US Class Current: 435/7.2
• CPC Class Codes: A01K 2227/50 Amphibians, e.g. Xenopus
• CPC Class Codes: A61K 31/13 Amines A61K31/04 takes precedence
• CPC Class Codes: A61K 31/135 having aromatic rings , e.g. ketamine, nortriptyline methadone A61K31/137
• CPC Class Codes: A61K 31/137 Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
• CPC Class Codes: A61K 31/165 having aromatic rings, e.g. colchicine, atenolol, progabide
• CPC Class Codes: A61K 31/195 having an amino group
• CPC Class Codes: A61K 31/215 of carboxylic acids
• CPC Class Codes: A61K 31/275 Nitriles; Isonitriles
• CPC Class Codes: A61K 31/335 having oxygen as the only ring hetero atom, e.g. fungichromin
• CPC Class Codes: A61K 31/34 having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
• CPC Class Codes: A61K 31/35 having six-membered rings with one oxygen as the only ring hetero atom
• CPC Class Codes: A61K 31/36 Compounds containing methylenedioxyphenyl groups, e.g. sesamin
• CPC Class Codes: A61K 31/38 having sulfur as a ring hetero atom
• CPC Class Codes: A61K 31/395 having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
• CPC Class Codes: A61K 31/40 having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• CPC Class Codes: A61K 31/415 1,2-Diazoles
• CPC Class Codes: A61K 31/425 Thiazoles
• CPC Class Codes: A61K 31/44 Non condensed pyridines; Hydrogenated derivatives thereof
• CPC Class Codes: A61K 31/55 having seven-membered rings, e.g. azelastine, pentylenetetrazole
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 31/715 : Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• CPC Class Codes: A61K 38/16 : Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61P 19/10 : for osteoporosis
• CPC Class Codes: A61P 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/14 : for calcium homeostasis vitamin D A61P3/02; parathyroid hormones A61P5/18; calcitonin A61P5/22; osteoporosis A61P19/10; bone metastasis A61P35/04
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C07C 211/27 : having amino groups linked to the six-membered aromatic ring by saturated carbon chains
• CPC Class Codes: C07C 211/29 : the carbon skeleton being further substituted by halogen atoms or by nitro or nitroso groups
• CPC Class Codes: C07C 211/30 : the six-membered aromatic ring being part of a condensed ring system formed by two rings
• CPC Class Codes: C07C 211/42 : with six-membered aromatic rings being part of the condensed ring systems
• CPC Class Codes: C07C 215/52 : linked by carbon chains having two carbon atoms between the amino groups and the six-membered aromatic ring or the condensed ring system containing that ring
• CPC Class Codes: C07C 217/58 : with amino groups and the six-membered aromatic ring, or the condensed ring system containing that ring, bound to the same carbon atom of the carbon chain
• CPC Class Codes: C07C 217/60 : linked by carbon chains having two carbon atoms between the amino groups and the six-membered aromatic ring or the condensed ring system containing that ring
• CPC Class Codes: C07C 217/62 : linked by carbon chains having at least three carbon atoms between the amino groups and the six-membered aromatic ring or the condensed ring system containing that ring
• CPC Class Codes: C07C 225/16 : and containing six-membered aromatic rings
• CPC Class Codes: C07C 229/38 : having amino groups bound to acyclic carbon atoms and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
• CPC Class Codes: C07C 233/05 : having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
• CPC Class Codes: C07C 255/58 : containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the carbon skeleton
• CPC Class Codes: C07C 2601/14 : The ring being saturated
• CPC Class Codes: C07C 317/32 : with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
• CPC Class Codes: C07C 323/32 : having at least one of the nitrogen atoms bound to an acyclic carbon atom of the carbon skeleton
• CPC Class Codes: C07D 209/14 : Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 209/16 : Tryptamines
• CPC Class Codes: C07D 209/18 : Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 213/38 : having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
• CPC Class Codes: C07D 233/64 : with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
• CPC Class Codes: C07D 319/18 : Ethylenedioxybenzenes, not substituted on the hetero ring
• CPC Class Codes: C07K 14/705 : Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/106 : Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: G01N 2500/10 : involving cells
• CPC Class Codes: G01N 2800/04 : Endocrine or metabolic disorders
• CPC Class Codes: G01N 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 : for testing non-proliferative effects
• CPC Class Codes: G01N 33/5044 : involving specific cell types
• CPC Class Codes: G01N 33/5091 : for testing the pathological state of an organism
• CPC Class Codes: G01N 33/566 : using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/567 : utilising isolate of tissue or organ as binding agent
• CPC Class Codes: G01N 33/6872 : Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 33/6893 : related to diseases not provided for elsewhere
• CPC Class Codes: G01N 33/84 : involving inorganic compounds or pH

Abstract

The present invention relates to the different roles inorganic ion receptors have in cellular and body processes. The present invention features: (1) molecules which can modulate one or more inorganic ion receptor activities, preferably the molecule can mimic or block an effect of an extracellular ion on a cell having an inorganic ion receptor, more preferably the extracellular ion is Ca²⁺ and the effect is on a cell having a calcium receptor; (2) inorganic ion receptor proteins and fragments thereof, preferably calcium receptor proteins and fragments thereof; (3) nucleic acids encoding inorganic ion receptor proteins and fragments thereof, preferably calcium receptor proteins and fragments thereof; (4) antibodies and fragments thereof, targeted to inorganic ion receptor proteins, preferably calcium receptor protein; and (5) uses of such molecules, proteins, nucleic acids and antibodies.

Claims

1. A method of screening for a compound able to affect one or more activities of a calcium receptor comprising the steps of,

a) contacting a recombinant cell with a test compound, wherein said recombinant cell comprises a recombinant nucleic acid expressing said calcium receptor, provided that said cell does not have functional calcium receptor expression from endogenous nucleic acid, and

b) determining the ability of said test compound to affect one or more activities of said calcium receptor, and comparing said ability with the ability of said test compound to affect said one or more calcium receptor activities in a cell not comprising said recombinant nucleic acid;

wherein said recombinant nucleic acid comprises a calcium receptor nucleic acid selected from the group consisting of;

a) nucleic acid of SEQ ID NO 1,

b) nucleic acid of SEQ ID NO 2,

c) nucleic acid of SEQ ID NO 3,

d) nucleic acid of SEQ ID NO 4,

e) nucleic acid encoding the amino acid of SEQ ID NO 5,

f) nucleic acid encoding the amino acid of SEQ ID NO 6,

g) nucleic acid encoding the amino acid of SEQ ID NO 7,

h) nucleic acid encoding the amino acid of SEQ ID NO 8, and

i) a derivative thereof encoding said calcium receptor, wherein said derivative encodes a receptor having one or more activities of said calcium receptor and comprises at least 20 contiguous nucleotides which can hybridize under stringent hybridization conditions to a complement of at least 20 contiguous nucleotides of the calcium receptor nucleic acid of any of (a), (b), (c), (d), (e), (f), (g), and (h).

3. The method of claim 1, wherein said calcium receptor nucleic acid is a SEQ ID No 1 derivative comprising at least 20 contiguous nucleotides which can hybridize under stringent hybridization conditions to a complement of at least 20 contiguous nucleotides of SEQ ID NO 1.

4. The method of claim 1, wherein said calcium receptor nucleic acid is SEQ ID NO 2.

5. The method of claim 1, wherein said calcium receptor nucleic acid is a SEQ ID NO 2 derivative comprising at least 20 contiguous nucleotides which can hybridize under stringent hybridizations conditions to a complement of at least 20 contiguous nucleotides of SEQ ID NO 2.

6. The method of claim 1, wherein said calcium receptor nucleic acid is SEQ ID NO 3.

7. The method of claim 1, wherein said calcium receptor nucleic acid is a SEQ ID NO 3 derivative comprising at least 20 contiguous nucleotides which can hybridize under stringent hybridizations conditions to a complement of at least 20 contiguous nucleotides of SEQ ID NO 3.

8. The method of claim 1, wherein said calcium receptor nucleic acid is SEQ ID NO 4.

9. The method of claim 1, wherein said calcium receptor nucleic acid is a SEQ ID NO 4 derivative comprising at least 20 contiguous nucleotides which can hybridize under stringent hybridizations conditions to a complement of at least 20 contiguous nucleotides of SEQ ID NO 4.

10. The method of claim 1, wherein said calcium receptor nucleic acid encodes the amino acid sequence of SEQ ID NO 5.

11. The method of claim 1, wherein said calcium receptor nucleic acid encodes the amino acid sequence of a SEQ ID NO 5 derivative comprising least 20 contiguous nucleotides which can hybridize under stringent hybridizations conditions to a complement of at least 20 contiguous nucleotides encoding the amino acid sequence of SEQ ID NO 5.

12. The method of claim 1, wherein said calcium receptor nucleic acid encodes the amino acid sequence of SEQ ID No 6.

13. The method of claim 1, wherein said calcium receptor nucleic acid encodes the amino acid sequence of a SEQ ID NO 6 derivative comprising at least 20 contiguous nucleotides which can hybridize under stringent hybridizations conditions to a complement of at least 20 contiguous nucleotides encoding the amino acid sequence of SEQ ID NO 6.

14. The method of claim 1, wherein said calcium receptor nucleic acid encodes the amino acid sequence of SEQ ID NO 7.

15. The method of claim 1, wherein said calcium receptor nucleic acid encodes the amino acid sequence of a SEQ ID NO 7 derivative comprising at least 20 contiguous nucleotides which can hybridize under stringent hybridizations conditions to a complement of at least 20 contiguous nucleotides encoding the amino acid sequence of SEQ ID NO 7.

16. The method of claim 1, wherein said calcium receptor nucleic acid encodes the amino acid sequence of SEQ ID NO 8.

17. The method of claim 1, wherein said calcium receptor nucleic acid encodes the amino acid sequence of a SEQ ID NO 8 derivative comprising at least 20 contiguous nucleotides which can hybridize under stringent hybridization conditions to a complement of at least 20 contiguous nucleotides encoding the amino acid sequence of SEQ ID NO 8.

18. The method of any one of claims 3, 5, 7, 9, 11, 13, 15, or 17, wherein said derivative comprises at least 27 nucleotides which can hybridize under stringent hybridization conditions to the complement of at least 27 contiguous nucleotides of SEQ ID NO 1, 2, 3, or 4, or to the complement of at least 27 contiguous nucleotides encoding the amino acid sequence of SEQ ID NOs 5, 6, 7 or 8.

19. The method of claim 18, wherein said derivative comprises at least 45 contiguous nucleotides which can hybridize under stringent hybridization conditions to the complement of at least 45 contiguous nucleotides of SEQ ID NO 1, 2, 3, or 4, or to the complement of at least 45 contiguous nucleotides encoding the amino acid sequence of SEQ ID NOs 5, 6, 7 or 8.

20. The method of claim 1, wherein said test compound comprises an aromatic group.

21. The method of claim 20, wherein said aromatic group comprises a moiety selected from the group consisting of:

phenyl, 2-, 3-, or 4-pyridyl, α

- or β

-tetrahydronaphthy, 1- or 2- naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl, and phenoxy.

22. The method of claim 21, wherein said aromatic group comprises a moiety selected from the group consisting of:

phenyl, 2-, 3-, or 4-pyridyl, 1- or 2- naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl, and phenoxy.

23. The method of claim 22, wherein said aromatic group comprises said phenyl moiety.

24. The method of claim 22, wherein said aromatic group comprises said 1- or 2- naphthyl moiety.

25. The method of any one of claims 1-17, wherein said recombinant nucleic acid is an expression vector.

26. The method of any one of claims 1-17, wherein said recombinant nucleic acid comprises an exogenous promoter transcriptionally coupled to said calcium receptor nucleic acid.

2. The method of claim l, wherein said calcium receptor nucleic acid is SEQ ID NO 1.

27. A method of identifying a calcimimetic compound, comprising the steps of:

a) contacting a cell comprising a calcium receptor with a test compound; and

b) determining the ability of said test compound to affect the ability of said calcium receptor to respond to extracellular Ca²⁺ in the presence and, in a separate experiment, in the absence of an activator of Protein Kinase C,

wherein said test compound comprises an aromatic group.

28. The method of claim 27, wherein said step b) involves the use of a Protein Kinase C activator selected from the group consisting of phorbol myristate acetate, mezerein and (-)-indolactam V.

29. The method of claim 27, wherein said method measures mobilization of intracellular Ca²⁺.

30. The method of any one of claims 27-29, wherein said aromatic group is selected from the group consisting of:

phenyl, 2-, 3-, or 4-pyridyl, α

- or β

-tetrahydronaphathyl, 1- or 2- naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl, and phenoxy, and said test compound is not verapamil or D-600.

31. The method of claim 30, wherein said aromatic group comprises a moiety selected from the group consisting of;

phenyl, 2-, 3-, or 4-pyridyl, 1- or 2- naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl, and phenoxy.

32. The method of claim 31, wherein said aromatic group comprises said phenyl moiety.

33. The method of claim 31, wherein said aromatic group comprises said 1- or 2- naphthyl moiety.

34. The method of claim 27, further comprising the step of measuring the ability of said test compound to mimic one or more affects of extracellular Ca²⁺ at a second cell type comprising a calcium receptor.

35. A method of identifying a calcimimetic compound comprising the steps of:

a) contacting a cell comprising a calcium receptor with a test compound; and

b) determining the ability of said test compound to mimic the activity of extracellular Ca²⁺ at said calcium receptor under conditions where calcium influx is blocked,

wherein said test compound comprises an aromatic group.

36. The method of claim 35, wherein said method measures mobilization of intracellular Ca²⁺.

37. The method of any one of claims 35 or 36, wherein said aromatic group is selected from the group consisting of:

phenyl, 2-, 3-, or 4-pyridyl, α

- or β

-tetrahydronaphthyl, 1- or 2-naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl, and phenoxy, and said test compound is not verapamil or D-600.

38. The method of claim 37, wherein said aromatic group comprises a moiety selected from the group consisting of:

phenyl, 2-, 3-, or 4-pyridyl, 1- or 2- naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl, and phenoxy.

39. The method of claim 38, wherein said aromatic group comprises said phenyl moiety.

40. The method of claim 38, wherein said aromatic group comprises said 1- or 2- naphthyl moiety.

48. The method of claim 39, further comprising the step of measuring the ability of said test compound to inhibit one or more effects of extracellular Ca²⁺ at a second cell type comprising a calcium receptor.

41. A method of identifying a calcilytic compound comprising the steps of:

a) contacting a cell comprising a calcium receptor with a test compound; and

b) determining the ability of said test compound to inhibit calcium receptor activity at said calcium receptor,

wherein said test compound comprises an aromatic group.

42. The method of claim 41, wherein said method is carried out under conditions wherein influx of extracellular Ca²⁺ is inhibited.

43. The method of claim 42, wherein said method measures mobilization of intracellular Ca²⁺.

44. The method of any one of claims 41-43, wherein said aromatic group is selected from the group consisting of:

phenyl, 2-, 3-, or 4-pyridyl, α

- or β

-tetrahydronaphthyl, 1- or 2- naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl, and phenoxy, and said test compound is not verapamil or D-600.

45. The method of claim 44, wherein said aromatic group comprises a moiety selected from the group consisting of:

phenyl, 2-, 3-, or 4-pyridyl, 1- or 2- naphthyl, 1- or 2-quinolinyl, 2- or 3-indolyl, benzyl, and phenoxy.

46. The method of claim 45, wherein said aromatic group comprises said phenyl moiety.

47. The method of claim 45, wherein said aromatic group comprises said 1- or 2- naphthyl moiety.