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

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
Documents:
c8sc02754d.pdf