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

DOI: 10.1039/C8SC02754D DOI Help

Authors: Honorine Lebraud (Astex Pharmaceuticals) , Olga Surova (Astex Pharmaceuticals; Karolinska Institute) , Aurélie Courtin (Astex Pharmaceuticals) , Marc O'reilly (Astex Pharmaceuticals) , Chiara R. Valenzano (Astex Pharmaceuticals) , Pär Nordlund (Karolinska Institute) , Tom D. Heightman (Astex Pharmaceuticals)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Chemical Science , VOL 9 , PAGES 8608 - 8618

State: Published (Approved)
Published: November 2018

Open Access Open Access

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.

Subject Areas: Chemistry, Medicine

Instruments: I02-Macromolecular Crystallography


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