Screening methods and assays for use with transmembrane proteins, particularly GPCRs.

Abstract

This invention provides methods and tools that can be used for assays, screenings, and drug discovery and development attempts. [Solution] An arrangement and method for screening membrane proteins are provided. The arrangement comprises a first fusion protein containing a membrane protein present in a boundary layer such as the wall of a cell, liposome, or vesicle, fused to one member of a binding pair, and a second fusion protein containing an intracellular ligand of the membrane protein, fused to the other member of the binding pair, the binding pair can generate a detectable signal.

Claims

1. At a minimum, the following components: A boundary layer separating the first environment from the second environment; Translaminar protein; A first ligand present in the first environment for the translayer protein; A second ligand for the translaminar protein present in the second environment; and A bond pair comprising at least a first bond member and a second bond member, capable of generating a detectable signal, An arrangement that includes this.

2. The arrangement according to claim 1, wherein the first binding member of the binding pair is part of a first fusion protein that includes the first binding member which is fused or linked to the translayer protein directly or via a suitable linker or spacer.

3. The arrangement according to claim 1 or 2, wherein the second ligand is a protein, ligand, binding domain, binding unit, or other chemical entity that specifically binds to one or more functional, active, and / or drug-potential conformations of the translaminar protein, induces the formation of one or more functional, active, and / or drug-potential conformations of the translaminar protein and / or stabilizes such conformations (and / or shifts the conformational equilibrium of the translaminar protein toward one or more such conformations), and / or induces the formation of a complex of the translaminar protein with the first ligand and the second ligand and / or stabilizes such complex.

4. The arrangement according to any one of claims 1 to 3, wherein the second ligand is part of a (second) fusion protein comprising the second binding member of the binding pair which is fused or linked to the second ligand directly or via a suitable linker or spacer.

5. The arrangement according to any one of claims 1 to 3, wherein the second ligand is an immunoglobulin monovariable domain.

6. The arrangement according to any one of claims 1 to 3, wherein the second ligand is a naturally occurring ligand of the translaminar protein, or an analog, derivative, or ortholog of such a naturally occurring ligand.

7. The arrangement according to any one of claims 1 to 3 and 6, wherein the second member of the binding pair is part of a (second) fusion protein comprising the second binding member of the binding pair, which is fused or linked to a binding domain or binding unit that can bind directly or via a suitable linker or spacer to the second ligand.

8. The arrangement according to claim 7, wherein the binding domain or binding unit is a single variable immunoglobulin domain.

9. The arrangement according to any one of claims 1 to 3, wherein the second ligand is part of a protein complex comprising the second ligand and one or more further proteins, and the protein complex is bound to or conjugates the translaminar protein.

10. The arrangement according to any one of claims 1 to 3 and 9, wherein the second member of the binding pair is part of a (second) fusion protein comprising the second binding member of the binding pair, which is fused or linked to a binding domain or binding unit that can bind directly or via a suitable linker or spacer to the second ligand.

11. The arrangement according to claim 10, wherein the binding domain or binding unit is an immunoglobulin monovariate domain.

12. The arrangement according to claim 1 or 2, wherein the second binding member of the binding pair is part of a second fusion protein comprising the second binding member of the binding pair, which is fused or linked to a protein that can directly or indirectly bind to the translayer protein, either directly or via a suitable linker or spacer.

13. The arrangement according to any one of claims 1, 2, and 12, wherein the second binding member of the binding pair is part of a second fusion protein comprising the second binding member of the binding pair, which is fused or linked to a protein (the protein being the second ligand) that can directly bind to the translayer protein, either directly or via a suitable linker or spacer.

14. The arrangement according to any one of claims 1, 2, 12, and 13, wherein the second binding member of the binding pair is part of a second fusion protein comprising the second binding member of the binding pair, which is fused or linked directly or via a suitable linker or spacer to a protein (the protein being the second ligand) capable of directly binding to the translaminar protein, and the protein capable of directly binding to the translaminar protein is a protein, ligand, binding domain, binding unit, or other chemical entity that specifically binds to one or more functional, active, and / or drug-potential conformations of the translaminar protein, induces the formation of one or more functional, active, and / or drug-potential conformations of the translaminar protein and / or stabilizes such conformations (and / or shifts the conformational equilibrium of the translaminar protein toward one or more such conformations), and / or induces the formation of a complex of the translaminar protein with the first ligand and the second ligand and / or stabilizes such complex.

15. The arrangement according to claim 13 or 14, wherein the protein that can directly bind to the translayer protein is an immunoglobulin monovariate domain.

16. The arrangement according to any one of claims 1, 2, and 12, wherein the second binding member of the binding pair is part of a second fusion protein comprising the second binding member of the binding pair, which is fused to or linked to a protein that can bind directly or indirectly to the translayer protein via a suitable linker or spacer.

17. The arrangement according to claim 16, wherein the second binding member of the binding pair is part of a second fusion protein comprising the second binding member of the binding pair, which is fused or linked to a binding domain or binding unit protein that can bind directly or via a suitable linker or spacer to the second ligand.

18. The arrangement according to claim 17, wherein the second binding member of the binding pair is part of a second fusion protein comprising the second binding member of the binding pair, which is fused or linked to a binding domain or binding unit protein that can bind directly or via a suitable linker or spacer to the second ligand, and the second ligand is a protein, ligand, binding domain, binding unit or other chemical entity that specifically binds to one or more functional, active and / or drug-potential conformations of the translaminar protein, induces the formation of one or more functional, active and / or drug-potential conformations of the translaminar protein and / or stabilizes such conformations (and / or shifts the conformational equilibrium of the translaminar protein toward one or more such conformations), and / or induces the formation of a complex of the translaminar protein with the first ligand and the second ligand and / or stabilizes the complex.

19. The arrangement according to claim 17 or 18, wherein the protein-binding domain or binding unit protein capable of binding to the second ligand is an immunoglobulin single variable domain.

20. The arrangement according to claim 16, wherein the second binding member of the binding pair is part of a second fusion protein comprising the second binding member of the binding pair, which is fused or linked to a binding domain or binding unit protein that can bind directly or via a suitable linker or spacer to a protein complex comprising the second ligand, and the protein complex is bound to or can bind to the translaminar protein.

21. The arrangement according to claim 20, wherein the protein-binding domain or binding unit protein capable of binding to the protein complex is an immunoglobulin monovariate domain.

22. At a minimum, the following components: A boundary layer separating the first environment from the second environment; Translaminar protein; Ligands for translaminar proteins present in the second environment; and A bond pair comprising at least a first bond member and a second bond member, capable of generating a detectable signal, An arrangement that includes this.

23. The arrangement according to any one of claims 1 to 22, wherein the boundary layer is a cell wall or a cell membrane.

24. The arrangement according to any one of claims 1 to 23, wherein the boundary layer is the wall or membrane of a liposome or vesicle.

25. The arrangement according to any one of claims 1 to 24, wherein the translaminar protein is a GPCR.

26. a) The step of preparing the arrangement described in claim 22, b) The step of adding the first ligand to the first environment of the arrangement. Methods that include...

27. c) The step of measuring the signal generated by the paired pair and / or the step of measuring the change in the signal generated by the paired pair. The method according to claim 26, further comprising:

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