6GJD

Erk2 signalling protein

Summary for 6GJD

• Entry DOI: 10.2210/pdb6gjd/pdb
• Descriptor: Mitogen-activated protein kinase 1, SULFATE ION, DIMETHYL SULFOXIDE, ... (6 entities in total)
• Functional Keywords: erk2, inhibitor, reversible probe, signaling protein
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 1
• Total formula weight: 44314.72

Authors

O'Reilly, M. (deposition date: 2018-05-16, release date: 2019-01-02, Last modification date: 2024-10-23)

Primary citation

Lebraud, H.,Surova, O.,Courtin, A.,O'Reilly, M.,Valenzano, C.R.,Nordlund, P.,Heightman, T.D.
Quantitation of ERK1/2 inhibitor cellular target occupancies with a reversible slow off-rate probe.
Chem Sci, 9:8608-8618, 2018

PubMed 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, 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 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 EC values measured by both methods, the results were compared with IC 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.

PubMed: 30568786
DOI: 10.1039/c8sc02754d

Experimental method

X-RAY DIFFRACTION (1.58 Å)