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Allosteric Kinase Inhibitors Reshape MEK1 Kinase Activity Conformations in Cells and In Silico

Jakob Fleischmann1, Andreas Feichtner1, Louis DeFalco2

  • 1Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.

Biomolecules
|April 3, 2021
PubMed

Insights

This study introduces a novel biosensor to track MEK1 kinase activity, crucial in cancer signaling. The biosensor effectively monitors MEK inhibitor efficacy in real-time within cells, aiding drug development.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Oncology

Background:

  • Mitogen-activated protein kinase (MAPK) pathway mutations drive cancer by activating kinases like MEK1/2.
  • MEK1/2 are key regulators, transmitting signals to ERK1/2, making them targets for cancer therapy.
  • Understanding MEK1/2 conformational changes is vital for developing effective MEK inhibitors (MEKi).

Purpose of the Study:

  • To develop and validate a kinase conformation (KinCon) biosensor for real-time MEK1 activity monitoring.
  • To investigate MEK1 conformational dynamics in response to activating mutations and MEKi.
  • To assess the potential of the MEK1 KinCon biosensor for evaluating MEKi efficacy in intact cells.

Main Methods:

  • Extension of the KinCon biosensor platform to specifically detect MEK1 activity dynamics.
  • Introduction of phosphorylation-mimetic mutations (S218D/S222D) to mimic MEK1 activation.
  • Treatment of cells with allosteric MEK inhibitors (trametinib, cobimetinib, refametinib, selumetinib) and analysis of biosensor response.
  • Confirmation of biosensor dynamics through molecular dynamics simulations of MEK1 structure.

Main Results:

  • The MEK1 KinCon biosensor successfully recorded activity dynamics, showing an 'open' conformation upon activating mutations (S218D/S222D).
  • Allosteric MEK inhibitors effectively reverted the activated MEK1 KinCon reporter to a 'closed', inactive conformation.
  • Molecular dynamics simulations revealed increased flexibility in the S218D/S222D mutant, particularly in the A-helix and alpha-C helix regions.
  • The biosensor accurately reflected drug engagement in a patient-derived melanoma cell line (A2058) with a BRAF V600E mutation.

Conclusions:

  • The developed MEK1 KinCon biosensor platform is a valuable tool for studying MEK1 kinase dynamics.
  • This biosensor enables real-time assessment of MEK1 conformational changes induced by mutations and MEK inhibitors.
  • The MEK1 KinCon biosensor shows significant potential for validating MEK inhibitor efficacy in a cellular context.
Keywords:
ERKKinCon reporterMAPK pathwayRAFRASRAS–RAF–ERK pathwayallosteric inhibitorbiosensorcancer mutationskinase inhibitorpersonalized therapyprotein-fragment complementation assaystructure dynamicsstructure simulations

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