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

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.

Fig. 1. (A) Examples of ERK1/2 inhibitors classified in two categories: Type 1 and pERK modulating inhibitors. (B) Co-crystal structure of SCH772984 with ERK2 (pdb:4QTA). The inhibitor binds to an altered conformation of ERK2 allowing interaction with the Tyr64 side chain. The two sites identified to extend the molecule and insert the TCO tag are highlighted (red arrows). The structural design of the two probes, TCO-SCH and SCH-TCO are shown. (C) Co-crystal structure of GDC-0994 with ERK2 (pdb:; 5K4I) showing the vector for the extension of the molecule (red arrow) and structural design of the two probes, TCO-GDC-1 and TCO-GDC-2.

Fig. 2. (A) Co-crystal structure of TCO-SCH with ERK2 (pdb:6GJD). The binding mode of the inhibitor core was conserved compared with the unmodified inhibitor SCH772984. (B) The amide linker of TCO-SCH likely encountering a steric clash with the protein is highlighted. (C) Co-crystal structure of TCO-GDC-2 with ERK2 (pdb:; 6GJB). The more slender alkyne linker provides a suitable vector to position the TCO tag in the solvent with a minimal steric clash with the protein.

Fig. 3. (A) Immunoblot analysis of ERK1/2 from the pulled down fractions obtained after competition experiments between SCH-TCO and a range of concentrations of selected ERK1/2 inhibitors in HCT116 cells. (B) Graphs showing the target occupancies of LY3214996, GDC-0994 and SCH772984 determined in competition mode with SCH-TCO in HCT116 cells.

Fig. 4. In-cell ERK1 and ERK2 occupancies of SCH772984, GDC-0994 and LY3214996 determined by ITDR-CETSA. HCT116 cells in suspension were treated with the appropriate ERK inhibitor for 1 h at 37 °C followed by heating at either +52° or +58 °C for 3 min.