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Issue 4, 2017
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Development of inverse electron demand Diels–Alder ligation and TR-FRET assays for the determination of ligand–protein target occupancy in live cells

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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, cellular TR-FRET assay emerged as a very promising platform for the determination of target occupancy of specific protein in a high-throughput manner. Our studies suggest that both IED-DA assay and TR-FRET assay should be considered as methods of choice for the determination of target engagement of small molecule protein binders in live cells.

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

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Publication details

The article was received on 30 Jan 2017, accepted on 20 Feb 2017 and first published on 21 Feb 2017


Article type: Research Article
DOI: 10.1039/C7MD00049A
Citation: Med. Chem. Commun., 2017,8, 789-795
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    Development of inverse electron demand Diels–Alder ligation and TR-FRET assays for the determination of ligand–protein target occupancy in live cells

    J. Marjanovic, A. Baranczak, V. Marin, H. Stockmann, P. L. Richardson and A. Vasudevan, Med. Chem. Commun., 2017, 8, 789
    DOI: 10.1039/C7MD00049A

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