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Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe

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Abstract

Target engagement is a key concept in drug discovery and its direct measurement can provide a quantitative understanding of drug efficacy and/or toxicity. Failure to demonstrate target occupancy in relevant cells and tissues has been recognised as a contributing factor to the low success rate of clinical drug development. Several techniques are emerging to quantify target engagement in cells; however, in situ measurements remain challenging, mainly due to technical limitations. Here, we report the development of a non-covalent clickable probe, based on SCH772984, a slow off-rate ERK1/2 inhibitor, which enabled efficient pull down of ERK1/2 protein via click reaction with tetrazine tagged agarose beads. This was used in a competition setting to measure relative target occupancy by selected ERK1/2 inhibitors. As a reference we used the cellular thermal shift assay, a label-free biophysical assay relying solely on ligand-induced thermodynamic stabilization of proteins. To validate the EC50 values measured by both methods, the results were compared with IC50 data for the phosphorylation of RSK, a downstream substrate of ERK1/2 used as a functional biomarker of ERK1/2 inhibition. We showed that a slow off-rate reversible probe can be used to efficiently pull down cellular proteins, significantly extending the potential of the approach beyond the need for covalent or photoaffinity warheads.

Graphical abstract: Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe

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Publication details

The article was received on 22 Jun 2018, accepted on 15 Sep 2018 and first published on 17 Sep 2018


Article type: Edge Article
DOI: 10.1039/C8SC02754D
Citation: Chem. Sci., 2018, Advance Article
  • Open access: Creative Commons BY-NC license
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    Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe

    H. Lebraud, O. Surova, A. Courtin, M. O'Reilly, C. R. Valenzano, P. Nordlund and T. D. Heightman, Chem. Sci., 2018, Advance Article , DOI: 10.1039/C8SC02754D

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