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

Honorine Lebraud1, Olga Surova1,2, Aurélie Courtin1

  • 1Astex Pharmaceuticals , 436 Cambridge Science Park , Cambridge , CB4 0QA , UK . Email: honorine.lebraud@astx.com ;

Chemical Science
|December 21, 2018
PubMed

Insights

Researchers developed a novel clickable probe to measure target engagement for ERK1/2 inhibitors in cells. This method allows for efficient protein pull-down, aiding drug discovery and development.

Area of Science:

  • Drug Discovery and Development
  • Chemical Biology
  • Molecular Pharmacology

Background:

  • Target engagement measurement is crucial for drug efficacy and toxicity assessment.
  • Low success rates in clinical drug development are partly due to inadequate target occupancy data.
  • Quantifying target engagement in situ remains a significant technical challenge.

Purpose of the Study:

  • To develop a novel non-covalent clickable probe for measuring target engagement of ERK1/2 inhibitors.
  • To enable efficient in situ quantification of target occupancy in relevant cells.
  • To validate the probe's performance against established methods like cellular thermal shift assay.

Main Methods:

  • Development of a clickable probe based on the slow off-rate ERK1/2 inhibitor SCH772984.
  • Utilizing click chemistry with tetrazine-tagged agarose beads for ERK1/2 protein pull-down.
  • Employing a competition assay to determine relative target occupancy by inhibitors.
  • Comparison with cellular thermal shift assay (CTSA) and functional RSK phosphorylation inhibition.

Main Results:

  • The developed probe successfully enabled efficient pull-down of ERK1/2 protein from cells.
  • The probe facilitated the measurement of relative target occupancy for various ERK1/2 inhibitors.
  • Results from the probe assay correlated well with CTSA and functional inhibition data (RSK phosphorylation).

Conclusions:

  • A slow off-rate reversible probe can be effectively used for cellular protein pull-down.
  • This approach extends target engagement measurement capabilities beyond covalent or photoaffinity probes.
  • The developed method offers a valuable tool for quantitative target engagement studies in drug discovery.

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