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1. WO2020221768 - CHIMERIC PROTEINS AND METHODS TO SCREEN FOR COMPOUNDS AND LIGANDS BINDING TO GPCRS

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[ EN ]

C L A I M S

1. Chimeric GPCR having the structure:

[N-terminal sequence]-[TMl]-[ICl]-[TM2]-[ECl]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6] - [EC3 ] -[TM7] -[C-terminal sequence]

in which the extracellular loops are derived from a first GPCR and the intracellular loops that are derived from a second GPCR (different from the first).

2. Chimeric GCPR according to claim 1, in which the extracellular binding domain of said chimeric GPCR is derived from said first GPCR.

3. Chimeric GCPR according to claim 1 or 2, in which the TMs are derived from said first GPCR.

4. Chimeric GCPR according to any of claims 1 to 3, in which the first GPCR and the second GPCR both belong to class A.

5. Composition comprising a chimeric GPCR according to any of claims 1 to 4 and a binding domain or binding unit that can bind to at least one of the intracellular loops that are derived from said second GPCR.

6. Composition according to claim 5, in which said binding domain or binding unit is ligand is capable of stabilizing and/or inducing a functional and/or active

conformational state of the chimeric GPCR upon binding to the chimeric GPCR.

7. Composition according to claim 5 or 6, in which said binding domain or binding unit is an immunoglobulin single variable domain.

8. Composition according to any of claims 4 to 6, which is a cellular

composition.

9. Method of forming a complex of a chimeric GPCR, a binding domain or binding unit, and a compound or ligand that is capable of binding to an extracellular binding site (as defined herein) on said chimeric GPCR, said method comprising the steps of:

c) providing a composition according to any of claims 6 to 8; and

d) contacting said composition with one or more test compounds or ligands under conditions that (i) allow said binding domain or binding unit to bind to said binding site on the chimeric GPCR that comprises at least one of said intracellular loops; and (ii) allow said test compounds to bind to the extracellular binding site of said chimeric GPCR.

10. Method of identifying and/or generating compounds or ligands that are capable of binding to an extracellular binding site (as defined herein) of a GPCR, said method comprising the steps of:

a) providing a composition according to any of claims 6 to 8, in which the chimeric GPCR comprises at least the extracellular loops of said GPCR;

b) contacting said composition with one or more test compounds or ligands under conditions that (i) allow said binding domain or binding unit to bind to said binding site on the chimeric GPCR that comprises at least one of said intracellular loops; and (ii) allow said test compounds to bind to the extracellular binding site of said chimeric GPCR;

c) evaluating whether each of the test compounds or ligands (and/or which of said test

compounds or ligands) binds to the chimeric GPCR; in said composition; and optionally d) selecting the test compounds or ligands that bind to the chimeric GPCR in said

composition.

11. Method of identifying and/or generating compounds or ligands that are capable of binding to an active conformation of a GPCR, said method comprising the steps of:

a) providing a composition according to any of claims 6 to 8, in which the chimeric GPCR comprises at least the extracellular loops of said GPCR and in which the binding domain or binding unit is ligand is capable of stabilizing and/or inducing an active conformational state of the chimeric GPCR upon binding to the chimeric GPCR.;

b) contacting said composition with one or more test compounds or ligands under conditions that (i) allow said binding domain or binding unit to bind to said binding site on the chimeric GPCR that comprises at least one of said intracellular loops; and (ii) allow said test compounds to bind to the extracellular binding site of said chimeric GPCR;

c) evaluating whether each of the test compounds or ligands (and/or which of said test

compounds or ligands) binds to the chimeric GPCR; in said composition; and optionally

d) selecting the test compounds or ligands that bind to the chimeric GPCR in said composition.

12. Arrangement that comprises at least the following elements:

- a boundary layer that separates a first environment from a second environment;

- a chimeric GPCR according to any of claims 1 to 4;

- a first ligand for the chimeric GPCR that is present in the first environment;

- a second ligand for the chimeric GPCR that is present in the second environment, which second ligand is a binding domain or binding unit that can bind to at least one of the intracellular loops on said chimeric GPCR; and

- a binding pair that consists of at least a first binding member and a second binding

member, which binding pair is capable of generating a detectable signal.

13. Arrangement according to claim 12, in which the chimeric GPCR is fused or linked, either directly or via a suitable spacer or linker, to the first binding member of said binding pair and in which the second ligand is fused or linked, either directly or via a suitable spacer or linker, to the first binding member of said binding pair.

14. Fusion protein comprising a chimeric GPCR according to any of claims 1 to 4 that is fused or linked, either directly or via a suitable spacer or linker, to a binding domain or binding unit that can bind to at least one of the intracellular loops of said chimeric GPCR.

15. Fusion protein according to claim 14, in which said binding domain or binding unit is ligand is capable of stabilizing and/or inducing a functional and/or active

conformational state of the chimeric GPCR upon binding to the chimeric GPCR.