Methods for measuring binding and cellular engagement of ligands with target proteins

1. A method for measuring binding between a compound and a target macromolecule, comprising:

(a) preparing a fluid mixture comprising (i) an intact viable cell expressing a chimeric protein that comprises a target macromolecule linked to a labeling peptide and (ii) a compound being measured for binding to the target macromolecule, wherein said target macromolecule is subject to denaturation;

(b) incubating the fluid mixture of step (a) under conditions permitting binding of said compound to said target macromolecule;

(c) denaturing target macromolecules in the fluid mixture, after incubating in step (b), under conditions that produce a combined mixture of (i) denatured chimeric molecules not bound to the compound and (ii) non-denatured chimeric molecules bound to the compound;

(d) contacting the combined mixture of step (c) with a second label that binds to the labeling peptide to form a complex, wherein the complex occurs less with (i) denatured chimeric molecules not bound to the compound than with (ii) non-denatured chimeric molecules bound to the compound; and

(e) detecting the complex in step (d) to generate a signal that indicates a measurement of binding between a compound and a target macromolecule.

2. The method of claim 1 , wherein the target macromolecule is a protein.

3. The method of claim 2 wherein the protein further comprises an inactive exogenous polypeptide linked to the protein at a terminus distal to the labeling peptide.

4. The method of claim 3 , wherein the labeling peptide is an epitope tag.

5. The method of claim 2 , wherein the labeling peptide is between 10 and 100 amino acids in length.

6. The method of claim 5 , wherein the labeling peptide is an enzyme fragment and the second label is a complementary enzyme fragment which combines with the labeling peptide to create an active enzyme, further comprising a step of lysing the cell.

7. The method of claim 6 , wherein the labeling peptide is an enzyme donor (“ED”) active in enzyme fragment complementation of β-galactosidase and is fused to a terminus of a protein that is the target macromolecule.

8. The method of claim 7 , wherein the ED is one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

9. The method of claim 1 , wherein the compound is a small molecule.

10. The method of claim 9 , wherein the small molecule is one that binds to an active site on the target macromolecule.

11. The method of claim 1 , wherein the step of denaturing target macromolecules in the fluid mixture comprises a single step of heating the fluid mixture to a temperature that is from 25° C. to 100° C., or from 30° C. to 60° C.

12. The method of claim 1 , wherein denaturing target macromolecules in the fluid mixture comprises multiple heating steps for a defined period of time between 0.1 and 5 minutes.

13. The method of claim 12 , wherein said heating step comprises applying heat to the mixture between 40° C. and 60° C., and further comprises multiple steps of heating for a time of 0.1 to 5 minutes.

14. The method of claim 13 , wherein multiple steps of heating comprises individual cooling steps between individual heating steps, of between 10 seconds and 2 minutes in duration.

15. The method of claim 1 , wherein the step of denaturation comprises (a) heating, (b) hydrostatic pressure, (c) an organic solvent, (d) radiation, or any combination thereof, whereby the cell remains intact after denaturation.

16. The method of claim 5 , wherein the organic solvent is alcohol or chloroform.

17. The method of claim 1 wherein steps (a) through (e) are carried out in mixtures containing different dilutions of said compound.

18. The method of claim 17 , wherein repeated steps (a) through (e) may be used to calculate a binding constant (K D ) of binding of compound to the target macromolecule.

19. The method of claim 1 , wherein the target macromolecule is a protein selected from the group consisting of a bromodomain protein, a protein kinase, a hydrolase, and a histone methyltransferase.

20. A method for measuring a binding property between a small molecule compound and a target protein, comprising:

(a) preparing a fluid mixture comprising cells expressing a fusion of a target protein and a labeling peptide that is a β-galactosidase enzyme fragment of between 10 and 100 amino acids in length, said fluid mixture further containing a small molecule compound being measured for binding to the target protein;

(b) incubating the fluid mixture of step (a) under conditions permitting binding of said small molecule compound to target proteins in the cell;

(c) heating the fluid mixture of step (b), whereby denaturation of target proteins not bound to the compound occurs;

(d) lysing cells heated in step (c) and adding to the mixture a second label that is a β-galactosidase fragment that reacts with the labeling peptide on non-denatured target proteins to a greater extent than labeling peptide on denatured target proteins, and further adding a substrate that substrate generates a signal indicative of a degree of denaturation of target proteins, whereby detecting said signal indicates binding between said small molecule and said target protein.

21. The method of claim 20 , wherein a mixture is prepared through steps (a) to (d) in a single container.

22. The method of claim 20 , wherein replicate samples containing different concentrations of compound are prepared.

23. The method of claim 20 , wherein the target macromolecule is selected from the group consisting of a bromodomain protein and an enzyme.