Method to quantify affinity and selectivity of small molecules for proteins in living cells
AssigneeOhio State Innovation Foundation
InventorBlake Peterson
Life of this patent
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Abstract
This disclosure relates to methods of quantifying affinity and selectivity of compounds for target proteins in living cells.
Claims22
A method for determining binding affinity between a target and a test compound in a cell, the method comprising:
- a. providing a target protein;
- b. providing a first fluorescent molecule;
- c. introducing to the cell a second fluorescent molecule, wherein the second fluorescent molecule is modified so that it interacts with the target protein, and wherein the second fluorescent molecule is spectrally orthogonal to the first fluorescent molecule;
- d. measuring interaction between the second fluorescent molecule and the target protein;
- e. introducing to the cell a test compound;
- f. measuring interaction between the second fluorescent molecule and the target protein in the presence of the test compound;
- and
- g. calculating a difference in interaction of the second fluorescent molecule with the target protein when the test compound is present and when the test compound is not present, thereby determining binding affinity between the target protein and the test compound.
A system for determining binding affinity between a target protein and a test compound,
- the system comprising:
- a. a target protein, wherein the target protein is not fused to a fluorophore;
- b. a first fluorescent molecule;
- and
- c. a second fluorescent molecule, wherein the second fluorescent molecule has been modified so that it can interact with the target protein.
A cell comprising a vector, wherein the vector encodes a first fluorescent molecule and a target protein, wherein a nucleic acid encoding the target protein and a nucleic acid encoding the first fluorescent molecule are separated by an IRES;
- wherein the cell further comprises a second fluorescent molecule, wherein the second fluorescent molecule is modified so that it can interact with the target protein.
A modified probe comprising a compound of Formula A, Formula B, or Formula C:
- wherein Phor is
- Ra is C1-C20 alkyl or C2-C6 alkynyl,
- Rb is hydrogen of C1-C6 alkyl,
- Rc and Rd are each independently C1-C6 alkyl, and
- m is an integer selected from 0 to 20.
Dependencies are inferred from the claim text.