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Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
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Live-cell target engagement of allosteric MEKi on MEK-RAF/KSR-14-3-3 complexes

William M Marsiglia1,2,3, Arthur Chow4,5,6, Zaigham M Khan4,5

  • 1Department of Oncological Sciences, The Tisch Cancer Institute, Mount Sinai Center for Therapeutic Discovery, Icahn School of Medicine at Mount Sinai, New York, NY, USA. wmmarsig@uab.edu.

Nature Chemical Biology
|November 3, 2023
PubMed

Insights

New NanoBRET assays measure targeted drug engagement with RAS-MAPK pathway complexes in living cells. These assays reveal distinct inhibitor binding profiles and can screen compounds for specific pathway targets.

Area of Science:

  • Molecular biology
  • Cellular signaling
  • Pharmacology

Background:

  • The RAS-mitogen-activated protein kinase (MAPK) pathway is crucial in cell signaling and disease.
  • Targeted therapies, such as allosteric MEK inhibitors (MEKi), show promise but their efficacy depends on interactions with specific pathway complexes.
  • Directly measuring drug interactions within these physiological complexes in live cells has been a significant challenge.

Purpose of the Study:

  • To develop and validate NanoBRET-based assays for quantifying MEK inhibitor (MEKi) engagement with MEK1 and associated complexes in living cells.
  • To characterize MEKi binding profiles across different RAS-MAPK pathway components, including ARAF, BRAF, CRAF, KSR1, and KSR2, with and without 14-3-3.
  • To assess the impact of pathogenic mutations, like BRAF-V600E, on MEKi binding.

Main Methods:

  • Development of NanoBRET assays to measure direct target engagement of MEKi.
  • Quantification of MEKi interactions with MEK1-bound complexes involving ARAF, BRAF, CRAF, KSR1, and KSR2 in live cells.
  • Inclusion of 14-3-3 protein to mimic physiological conditions and assessment of BRAF-V600E mutant influence.

Main Results:

  • Distinct MEKi preferences were observed across various MEK1-bound complexes.
  • Inhibitor binding profiles were generated based on these observed preferences.
  • The assays successfully reported on the influence of the BRAF-V600E mutation on MEKi binding.

Conclusions:

  • The developed NanoBRET assays provide a robust platform for measuring MEKi target engagement in live cells.
  • These assays enable the generation of specific inhibitor binding profiles for RAS-MAPK pathway complexes.
  • This platform can be utilized for screening compounds targeting specific complexes within the RAS-MAPK cascade for therapeutic development.

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