DETECTION SYSTEM AND USES THEREFOR

• US 20100021457A1
• Filed: 11/09/2007
• Published: 01/28/2010
• Est. Priority Date: 11/10/2006
• Status: Active Grant

• Current Assignee: Dimerix Bioscience Pty Ltd. (Dimerix Ltd.)
• Original Assignee: Dimerix Bioscience Pty Ltd. (Dimerix Ltd.)
• Inventors: See, Heng Boon, Seeber, Ruth Marie, Eidne, Karin Ann, Pfleger, Kevin Donald George
• Granted Patent: US 8,283,127 B2
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/22 Anxiolytics
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

A system for the detection of molecular associations, the system comprising: i) a first agent, comprising a first interacting group coupled to a first reporter component; ii) a second agent, comprising a second interacting group coupled to a second reporter component; iii) a third agent, comprising a third interacting group; iv) a modulator; and v) a detector; wherein proximity of the first and second reporter components generates a signal capable of detection by the detector; and wherein the modulator modulates the association of the second interacting group with the third interacting group; such that monitoring the signal generated by proximity of the first and second reporter components by the detector constitutes monitoring the association of the first and third agents.

Claims

1. A system for the detection of molecular associations, the system comprising:

i). a first agent, comprising a first interacting group coupled to a first reporter component;

ii). a second agent, comprising a second interacting group coupled to a second reporter component;

iii). a third agent, comprising a third interacting group;

iv). a modulator; and

v). a detector;

wherein proximity of the first and second reporter components generates a signal capable of detection by the detector; and

wherein the modulator modulates the association of the second interacting group with the third interacting group;

such that monitoring the signal generated by proximity of the first and second reporter components by the detector constitutes monitoring the association of the first and third agents.

2. A system according to claim 1 characterised in that the system further comprises a reporter component initiator, wherein proximity of the first and second reporter components generates a signal capable of detection by the detector only in the presence of the reporter component initiator.

6. A system or method according to any one of the preceding claims characterised in that the first, second and third agents are provided by way of co-expression in a cell, to which the modulator is introduced.

22. A system, method or kit as claimed in any one of the preceding claims characterised in that the modulator increases the propensity of the second interacting group to associate with the third interacting group, such that detection of the signal generated by proximity of the first and second reporter components by the detector constitutes detection of the association of the first and third agents.

23. A system, method or kit as claimed in any one of the preceding claims characterised in that the modulator decreases the propensity of the second interacting group to associate with the third interacting group, such that detection of a reduction of the signal generated by proximity of the first and second reporter components by the detector constitutes detection of the dissociation of the first and third agents.

24. A system, method or kit as claimed in any one of the preceding claims characterised in that the first interacting group differs from the third interacting group, such that detection of the signal generated by the proximity of the first and second reporter components constitutes detection of the hetero-dimerisation and/or -oligomerisation of the first and third interacting groups.

25. A system, method or kit as claimed in any one of the preceding claims characterised in that the third agent does not comprise a component capable of generating a signal that substantially interferes with and/or contributes to the signal generated by the proximity of the first and second reporter components, such that detection of the signal generated by the proximity of the first and second reporter components is facilitated.

26. A system, method or kit as claimed in any one of the preceding claims characterised in that the third agent is selected from the group:

receptors, ion channels, enzymes, carriers, transporters, integral membrane proteins, cytoskeletal proteins, adhesion molecules, signalling proteins, scaffolding proteins, accessory proteins, trafficking proteins, transcription factors, nuclear co-factors and nucleic acid molecules.

27. A system, method or kit as claimed in any one of the preceding claims characterised in that the modulator is a ligand or an enzyme.

28. A system, method or kit as claimed in any one of the preceding claims characterised in that the third agent is produced by expression in cells, including whole cells, cellular fractions or in cell-free systems.

29. A system, method or kit as claimed in any one of the preceding claims characterised in that the first and third interacting groups are provided in the form of receptors, and the modulator is provided in the form of a ligand that modulates the receptor of the third interacting group, such that modulation of the signal generated by proximity of the first and second reporter components by the modulator is indicative of constitutive association of the receptors of the first and third interacting groups.

3. A method for the detection of the molecular associations, the method comprising the steps of:

i). Providing a first agent, comprising a first interacting group coupled to a first reporter component;

ii). Providing a second agent, comprising a second interacting group coupled to a second reporter component;

iii). Providing a third agent, comprising a third interacting group;

iv). Providing a modulator;

 

wherein;

proximity of the first and second reporter components generates a signal; and

wherein the modulator modulates the association of the second interacting group with the third interacting group;

then

v). Detecting and/or monitoring any signal generated by proximity of the first and second reporter components.

such that monitoring the signal generated by proximity of the first and second reporter components by the detector constitutes monitoring the association of the first and third agents.

4. A method according to claim 3 characterised in that, before the step of detecting and/or monitoring any signal generated by proximity of the first and second reporter components, the method further comprises the step of providing a reporter component initiator, wherein proximity of the first and second reporter components generates a signal capable of detection by the detector only in the presence of the reporter component initiator.

5. A method according to claim 4 characterised in that the steps of providing a reporter component initiator and providing a modulator occur after the steps of providing the first, second and third agents.

7. A method for determining whether and/or the extent to which a test compound interacts with a second protein when the second protein is associated with a first protein, the method comprising the steps of:

a). contacting said test compound with a system comprising;

i). a first agent, comprising the first protein coupled to a first reporter component;

ii). a second agent, comprising an interacting group coupled to a second reporter component;

iii). a third agent, comprising the second protein;

 

wherein proximity of the first and second reporter components generates a signal; and

wherein the modulator modulates the association of the interacting group with the second protein;

b). determining the signal as a determination of whether and/or the extent to which said test compound interacts with a second protein when the second protein is associated with a first protein.

8. A method according to claim 7 characterised in that the second protein is a receptor, such that the method for determining whether and/or the extent to which a test compound interacts with a second protein when the second protein is associated with a first protein comprises a method for determining whether and/or the extent to which a test compound is an agonist of the second protein when the second protein is associated with the first protein, and the step of determining the signal as a determination of whether and/or the extent to which said test compound interacts with a second protein when the second protein is associated with a first protein more specifically comprises the step of detecting an increase in the signal as a determination of whether the test compound is an agonist of the second protein when the second protein is associated with the first protein.

9. A method according to claim 8 characterised in that the first protein is also a receptor.

10. A method according to claim 7 characterised in that the second protein is a receptor, such that the method for determining whether and/or the extent to which a test compound interacts with a second protein when the second protein is associated with a first protein comprises a method for determining whether and/or the extent to which a test compound is an antagonist or partial agonist of the second protein when the second protein is associated with the first protein, the method comprising the steps of:

a). contacting said test compound with a system comprising;

i). a first agent, comprising the first protein coupled to a first reporter component;

ii). a second agent, comprising an interacting group coupled to a second reporter component;

iii). a third agent, comprising the second protein;

iv). an agonist of the second protein;

 

wherein proximity of the first and second reporter components generates a signal; and

wherein the modulator modulates the association of the interacting group with the second protein;

b). detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist or partial agonist of the second protein when the second protein is associated with the first protein.

11. A method according to claim 10 characterised in that the first protein is also a receptor.

12. A method according to claim 7 characterised in that the second protein is a constitutively active receptor, such that the method for determining whether and/or the extent to which a test compound interacts with a second protein when the second protein is associated with a first protein comprises a method for determining whether and/or the extent to which a test compound is an inverse agonist of the second protein when the second protein is associated with the first protein, and the step of determining the signal as a determination of whether and/or the extent to which said test compound interacts with a second protein when the second protein is associated with a first protein more specifically comprises the step of detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the second protein when the second protein is associated with the first protein.

13. A method according to claim 12 characterised in that the first protein is also a receptor.

14. A method according to claim 7 characterised in that, the first and second proteins are both receptors, such that the method for determining whether and/or the extent to which a test compound interacts with a second protein when the second protein is associated with a first protein comprises a method for screening a test compound for first protein/second protein hetero-dimer/-oligomer selective activity, the method comprising the steps of:

a) determining whether, and/or the extent to which, the test compound interacts with the second protein while the second protein is associated with the first protein; and

b) if the test compound interacts with the second protein while the second protein is associated with the first protein, determining whether, or the extent to which the test compound interacts with the second protein in the absence of the first protein;

such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the second protein while the second protein is associated with the first protein is selective for the first protein/second protein hetero-dimer/-oligomer.

15. A method according to claim 7 characterised in that the first and second proteins are both receptors, and the method comprises a method for screening a test compound for first protein/second protein hetero-dimer/-oligomer selective antagonism or partial agonism, the method comprising the steps of:

a) determining whether, and/or the extent to which, the test compound is an antagonist or partial agonist of the first protein/second protein hetero-dimer/-oligomer, by contacting said test compound with a system comprising;

i). a first agent, comprising the first protein coupled to a first reporter component;

ii). a second agent, comprising an interacting group coupled to a second reporter component;

iii). a third agent, comprising the second protein;

iv). an agonist of the first protein, the second protein and/or the first protein/second protein hetero-dimer/-oligomer;

 

wherein proximity of the first and second reporter components generates a signal; and

wherein the modulator modulates the association of the interacting group with the second protein;

b). detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist or partial agonist of the first protein/second protein hetero-dimer/-oligomer;

c) if the test compound is an antagonist or partial agonist of the first protein/second protein hetero-dimer/-oligomer, determining whether, or the extent to which the test compound interacts with the second protein in the absence of the first protein and the first protein in the absence of the second protein;

such that a test compound that exhibits greater agonistic or partial agonistic properties when interacting with the first protein/second protein hetero-dimer/-oligomer is selective for the first protein/second protein hetero-dimer/-oligomer.

16. A method according to claim 7 characterised in that the first and second proteins are both receptors, and the method comprises a method for screening a test compound for first protein/second protein hetero-dimer/-oligomer selective antagonism or partial agonism, the method comprising the steps of:

a) determining whether, and/or the extent to which, the test compound is an antagonist or partial agonist of the first protein/second protein hetero-dimer/-oligomer, by contacting said test compound with a system comprising;

i). a first agent, comprising the second protein coupled to a first reporter component;

ii). a second agent, comprising an interacting group coupled to a second reporter component;

iii). a third agent, comprising the first protein;

iv). an agonist of the second protein, the first protein and/or the first protein/second protein hetero-dimer/-oligomer;

 

wherein proximity of the first and second reporter components generates a signal; and

wherein the modulator modulates the association of the interacting group with the first protein;

b). detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist or partial agonist of the first protein/second protein hetero-dimer/-oligomer;

c) if the test compound is an antagonist or partial agonist of the first protein/second protein hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an antagonist or partial agonist of the first protein in the absence of the second protein and the second protein in the absence of the first protein;

such that a test compound that exhibits greater antagonistic or partial agonistic properties when interacting with the first protein/second protein hetero-dimer/-oligomer is selective for the first protein/second protein hetero-dimer/-oligomer.

17. A method according to claim 7 characterised in that the first and second proteins are both receptors, and the method comprises a method for screening a test compound for first protein/second protein hetero-dimer/-oligomer selective inverse agonism, the method comprising the steps of:

a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the first protein/second protein hetero-dimer/-oligomer, by contacting said test compound with a system comprising;

i). a first agent, comprising the first protein coupled to a first reporter component;

ii). a second agent, comprising an interacting group coupled to a second reporter component;

iii). a third agent, comprising a constitutively active second protein;

 

wherein proximity of the first and second reporter components generates a signal; and

wherein the modulator modulates the association of the interacting group with the second protein;

b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the first protein/second protein hetero-dimer/-oligomer;

c) if the test compound is an inverse agonist of the first protein/second protein hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an inverse agonist of the first protein in the absence of the second protein and the second protein in the absence of the first protein;

such that a test compound that exhibits greater inverse agonistic properties when interacting with the first protein/second protein hetero-dimer/-oligomer is selective for the first protein/second protein hetero-dimer/-oligomer.

18. A method according to claim 7 characterised in that the first and second proteins are both receptors, there is provided a method for screening a test compound for first protein/second protein hetero-dimer/-oligomer selective inverse agonism, the method comprising the steps of:

a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the first protein/second protein hetero-dimer/-oligomer, by contacting said test compound with a system comprising;

i). a first agent, comprising the second protein coupled to a first reporter component;

ii). a second agent, comprising an interacting group coupled to a second reporter component;

iii). a third agent, comprising a constitutively first protein;

 

wherein proximity of the first and second reporter components generates a signal; and

wherein the modulator modulates the association of the interacting group with the first protein;

b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the first protein/second protein hetero-dimer/-oligomer;

c) if the test compound is an inverse agonist of the first protein/second protein hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an inverse agonist of the second protein in the absence of the first protein and the first protein in the absence of the second protein;

such that a test compound that exhibits greater inverse agonistic properties when interacting with the first protein/second protein hetero-dimer/-oligomer is selective for the first protein/second protein hetero-dimer/-oligomer.

44. An agonist of the second protein, when the second protein is associated with the first protein, identified by the method of claims 8 or 9.

45. An antagonist or partial agonist of the second protein, when the second protein is associated with the first protein, identified by the method of claim 10 or 11.

46. An inverse agonist of the second protein, when the second protein is associated with the first protein, identified by the method of claim 12 or 13.

47. Selective agonists and/or antagonists and/or inverse agonists of the first protein-second protein hetero-dimer/-oligomer identified by the method according to any one of claim 14 to 18.

19. A kit for the detection of the molecular associations, the kit comprising:

i). a first agent, comprising a first interacting group coupled to a first reporter component;

ii). a second agent, comprising a second interacting group coupled to a second reporter component;

iii). a third agent, comprising a third interacting group;

iv). a modulator; and

wherein proximity of the first and second reporter components generates a signal capable of detection; and

wherein the modulator modulates the association of the second interacting group with the third interacting group.

20. A kit according to claim 19, characterised in that the kit further comprises a reporter component initiator, and proximity of the first and second reporter components generates a signal capable of detection only in the presence of the reporter component initiator.

21. A method for screening a test compound for selective activity against a heterodimer of a first protein and a second protein, the method comprising the steps of:

a) contacting said test compound at increasing concentrations with a system comprising;

i). a first agent, comprising the first protein coupled to a first reporter component;

ii). a second agent, comprising an interacting group coupled to a second reporter component;

iii). a third agent, comprising the second protein;

 

wherein proximity of the first and second reporter components generates a signal; and

wherein the modulator modulates the association of the interacting group with the second protein;

b). determining signal as a determination of whether said test compound modulates said association of the interacting group with the second protein at each concentration to produce a dose-response curve;

c). determining the Hill slope of the dose-response curve, wherein, a Hill slope in excess of 1 indicates interaction of the test compound with the hetero-dimer.

30. A first protein-second protein hetero-dimer/-oligomer identified by any one of the systems, methods or kits of the preceding claims.

31. A hetero-dimeric or hetero-oligomeric receptor, comprising at least one first receptor subunit associated with at least one second receptor subunit, identified by any one of the systems, methods or kits of the preceding claims.

32. A method for the treatment of a patient suffering from a first receptor-related ailment by administering a therapeutically effective amount of a second receptor agonist, inverse agonist or antagonist, wherein the first and second receptors form a hetero-dimer/-oligomer identified by any one of the systems, methods or kits of the preceding claims.

33. A method according to claim 32 characterised in that the second receptor agonist, inverse agonist or antagonist is co-administered with an first receptor agonist, inverse agonist or antagonist.

34. A method for the treatment of a patient suffering from a second receptor-related ailment by administering a therapeutically effective amount of an first receptor agonist, inverse agonist or antagonist, wherein the first and second receptors form a hetero-dimer/-oligomer identified by any one of the systems, methods or kits of the preceding claims.

35. A method according to claim 34 characterised in that the first receptor agonist, inverse agonist or antagonist is co-administered with a second receptor agonist, inverse agonist or antagonist.

36. A method for the manufacture of a medicament for the treatment of a patient suffering from an first receptor-related ailment comprising use of a therapeutically effective amount of a second receptor agonist, inverse agonist or antagonist, wherein the first and second receptors form a hetero-dimer/-oligomer identified by any one of the systems, methods or kits of the preceding claims.

37. A method according to claim 36 characterised in that the medicament contains a first receptor agonist, inverse agonist or antagonist.

38. A method for the manufacture of a medicament for the treatment of a patient suffering from a second receptor-related ailment comprising use of a therapeutically effective amount of an first receptor agonist, inverse agonist or antagonist, wherein the first and second receptors form a hetero-dimer/-oligomer identified by any one of the systems, methods or kits of the preceding claims.

39. A method according to claim 38 characterised in that the medicament contains a second receptor agonist, inverse agonist or antagonist.

40. A method for the treatment of a patient suffering from a first receptor-related ailment by administering a therapeutically effective amount of a selective second receptor agonist, antagonist or inverse agonist binding agent, or fragment thereof, wherein the first and second receptors form a hetero-dimer/-oligomer identified by any one of the systems, methods or kits of the preceding claims.

41. A method according to claim 40 characterised in that the selective second receptor agonist, antagonist or inverse agonist binding agent is an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-idiotypic antibody.

42. A method for the treatment of a patient suffering from a second receptor-related ailment by administering a therapeutically effective amount of a selective first receptor agonist, antagonist or inverse agonist binding agent, or fragment thereof, wherein the first and second receptors form a hetero-dimer/-oligomer identified by any one of the systems, methods or kits of the preceding claims.

43. A method according to claim 42 characterised in that the selective first receptor agonist, antagonist or inverse agonist binding agent may be an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-idiotypic antibody.