Detection of intracellular binding events by measuring protein abundance
AssigneeDiscoveRx Corporation
InventorsWilliam Raab, Thomas S. Wehrman
Life of this patent
Estimating the term…
Abstract
Methods and compositions are provided to measure the binding of a test compound to a target peptide by measuring the effect of the compound on the abundance of the target peptide inside a cell. The target peptide may bind the test compound at an active site or an allosteric site, and it has been found that such binding may stabilize the target peptide against cellular degradation. The target peptide will preferably comprise a destabilizing mutation which shortens the half life of the target peptide within the cell, typically a mammalian cell. Test compounds, including small molecules, have been found to stabilize target peptides. Also provided are systems and kits for use in practicing the methods.
Claims21
A method to assess binding of a compound to a binding site of a target peptide in a cell, comprising:
- (a) providing in the cell a fusion protein comprising (i) a target peptide having a destabilizing mutation and (ii) a labeling peptide;
- (b) incubating a mixture of the cell of (a) and the compound for a period sufficient to allow binding of the compound to the binding site of the target peptide and to allow for stabilization of the fusion protein by the binding, then
- (c) combining the mixture from step (b) with a label that detects an amount of the labeling peptide remaining after incubating, wherein
- a change in the amount of labeling peptide detected indicates binding of the compound to the target peptide.
A method to assess a compound as binding to a binding site on target peptide in a cell, comprising:
- (a) providing cells expressing a fusion protein comprising (i) said target peptide, having therein a destabilizing mutation, and (ii) a labeling peptide;
- (b) incubating a first number of cells from step (a) with said compound and a second number of cells from step (a) without said compound for a period sufficient to allow binding of the compound to the binding site of the target peptide and to allow for stabilization of the fusion protein by the binding of the compound, then
- (c) adding to samples incubated in step (b) a label that detects an amount of the labeling peptide remaining in the cell after said incubating, whereby
- a difference in the amount of labeling peptide between the first number of cells and the second number of cells indicates binding of the compound to the target peptide.
Dependencies are inferred from the claim text.