Homogenous thermal shift ligand binding assay
View Patent ↗Disclosed are methods for detecting and quantitatively measuring a binding property of a compound to a target macromolecule, wherein the target macromolecule is subject to denaturation and is linked to a labeling peptide, such as a short enzyme fragment. The method uses a fluid mixture comprising (i) a chimeric molecule comprising a target macromolecule linked to the labeling peptide and (ii) a compound being measured for binding to the target macromolecule, wherein said target macromolecule is subject to denaturation. After allowing for binding of the compound (e.g. a small molecule inhibitor of the target macromolecule), one detects a signal from the labeling peptide, such as by enzyme fragment complementation. This signal indicates a differential between denatured and non-denatured target macromolecules and thereby indicates a differential between target macromolecules not bound to the compound and target macromolecules bound to the compound, respectively.
1. A method for measuring a binding property between a compound and a target macromolecule, comprising:
(a) preparing a fluid mixture comprising (i) a chimeric molecule comprising a target macromolecule subject to denaturation linked to a labeling peptide and (ii) a compound being measured for binding to the target macromolecule;
(b) incubating the fluid mixture of step (a) under conditions permitting binding of said compound to target macromolecules in the mixture;
(c) treating the fluid mixture of step (b) under conditions that cause a differential denaturation between chimeric molecules bound to the compound and chimeric molecules not bound to the compound; and
(d) generating a signal from chimeric molecules subjected to said differential denaturation in step (c), by adding to the mixture a second label that reacts with the labeling peptide in the chimeric molecule, wherein,
(e) the signal in step (d) is detected and indicates a binding property between the compound and the target macromolecule.
2. The method of claim 1 , wherein said differential denaturation comprises heating the fluid mixture in a step that is that one of (a) multiple heating steps and at least one cooling step between heating steps and (b) a single heating step.
3. The method of claim 2 , wherein the target macromolecule is a protein.
4. The method of claim 3 , wherein the labeling peptide is an epitope tag.
5. The method of claim 3 , 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.
7. The method of claim 6 , wherein the labeling peptide is an enzyme donor (“ED”) active in enzyme fragment complementation of β-galactosidase and being 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, SEQ ID NO: 3, or SEQ ID NO: 4.
9. The method of claim 5 , 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 5 , wherein said differential denaturation is a step of heating the mixture to a temperature that is one of between 30° C. and 50° C.
12. The method of claim 11 , wherein said heating 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 45° 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 steps comprises an individual cooling step between individual heating steps, of being between 10 seconds and 2 minutes in duration.
15. The method of claim 14 , comprising between three and ten cooling steps.
16. The method of claim 15 , wherein said a temperature of the mixture during a cooling step remains greater than a temperature in a previous cooling step.
17. The method of claim 14 , wherein said cooling steps comprise actively cooling the mixture.
18. The method claim 1 , wherein the step of denaturation comprises the addition of a strong acid or base, a concentrated inorganic salt, an organic solvent, or exposure to radiation.
19. The method of claim 18 , wherein the organic solvent is alcohol or chloroform.
20. A method for measuring a binding property between a compound and a target macromolecule that is a protein, comprising:
(a) preparing a fluid mixture comprising (i) a chimeric molecule comprising a protein target macromolecule that is subject to denaturation linked to a labeling peptide and (ii) a compound being measured for binding to the target macromolecule;
(b) incubating the fluid mixture of step (a) under heating conditions permitting binding of said compound to target macromolecules in the mixture;
(c) treating the fluid mixture of step (b) under conditions that cause a differential denaturation between chimeric molecules bound to the compound and chimeric molecules not bound to the compound;
(d) generating a signal from chimeric molecules subjected to said differential denaturation using enzyme fragment complementation between the labeling peptide and a complementary enzyme fragment;
(e) repeating steps (a) through (d) with mixtures containing different dilutions of the compound, wherein
the signals in step (e) is detected and indicate a binding property between the compound and the target macromolecule, comprising a dose-response curve of the compound.
21. The method of claim 20 wherein repeated steps (a) through (e) are used to calculate a binding constant (K D ) of binding of compound to the target macromolecule.
22. The method of claim 20 , wherein the target macromolecule is a protein which is one of a bromodomain protein, a protein kinase or a histone methyltransferase.