Isoform-selective activity-based profiling of ERK signaling

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Mitogen-activated protein kinase (MAPK) pathway dysregulation, particularly ERK signaling, is central to cancer development.
• Resistance to existing RAF and MEK inhibitors necessitates direct targeting of ERK1 and ERK2 for effective cancer therapy.
• Current methods for assessing ERK inhibitor activity rely on recombinant proteins, limiting direct profiling of native enzyme function.

Purpose of the Study:

• To develop a quantitative chemoproteomic strategy for isoform-specific profiling of native ERK1 and ERK2 activity.
• To enable independent evaluation of ERK1 versus ERK2 activity across diverse biological samples.
• To assess the potency and selectivity of ERK inhibitors and correlate target engagement with cellular anti-proliferative effects.

Main Methods:

• Utilized active-site directed probes for quantitative chemoproteomic analysis.
• Exploited subtle differences in substrate binding sites for isoform-specific activity-based profiling of ERK1 and ERK2.
• Applied the strategy to evaluate academic (VX-11e) and clinical (Ulixertinib) ERK inhibitors in various cell types, tissues, and species.

Main Results:

• Developed a chemoproteomic assay capable of distinguishing and quantifying native ERK1 and ERK2 activity.
• Determined the potency and selectivity profiles of VX-11e and Ulixertinib against native ERK isoforms.
• Established that >90% inactivation of both ERK1 and ERK2 is required for observed cellular anti-proliferative activity of the tested inhibitors.

Conclusions:

• Introduced a novel chemoproteomic approach for direct, isoform-specific assessment of native ERK activity.
• This method provides a valuable tool for advancing drug discovery targeting the oncogenic MAPK pathway.
• Demonstrated the critical need for substantial ERK1/ERK2 inhibition to achieve therapeutic effects in cancer cells.