Development of inverse electron demand Diels-Alder ligation and TR-FRET assays for the determination of ligand-protein target occupancy in live cells.

ABSTRACT: Determination of target engagement following drug administration under physiological conditions is essential for understanding clinical outcomes of therapeutic candidates. While the list of potential techniques that enable studies of target engagement is continuously expanding, identification of the best method to evaluate interactions between a ligand and its cellular binding partner(s) remains far from straightforward. We developed and compared the applicability of two label-based techniques; inverse electron demand Diels-Alder (IED-DA) ligation-based pull-down and TR-FRET assays for in-cell determination of target occupancy of c-Src kinase and p38-α kinase by the reversible inhibitor Dasatinib. Significantly, none of the assays required engineering proteins-of-interest. Moreover, cellul

SUBMITTER: Marjanovic J 

PROVIDER: S-EPMC6072205 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature