HOMOGENOUS THERMAL SHIFT LIGAND BINDING ASSAY

摘要

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.

主权项

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.