Dual-Probe Activity-Based Protein Profiling Reveals Site-Specific Differences in Protein Binding of EGFR-Directed...

Area of Science:

• Biochemistry
• Chemical Biology
• Pharmacology

Background:

• Understanding drug-target interactions at the amino acid level is vital for drug development.
• Activity-based protein profiling (ABPP) aids in studying drug selectivity and efficacy.
• Site-specific ABPP, like PhosID-ABPP, offers detailed insights into drug engagement.

Purpose of the Study:

• To refine PhosID-ABPP for dual dose-dependent inhibitor competition within a single proteome.
• To comparatively analyze two epidermal growth factor receptor (EGFR) targeted probes, PF-06672131 (PF131) and PF-6422899 (PF899).
• To identify probe-specific binding sites and understand dose-dependent drug-protein interactions at a proteome-wide scale.

Main Methods:

• Utilized a refined PhosID-ABPP strategy on a timsTOF HT mass spectrometer.
• Performed comparative analysis of two EGFR-targeted activity-based probes (ABPs): PF131 and PF899.
• Investigated dose-dependent probe binding to proteinaceous cysteines and off-target interactions.

Main Results:

• Revealed dose-dependent probe-binding preferences for cysteines, even at low nanomolar concentrations.
• PF131 exhibited broader off-target reactivity compared to PF899.
• PF899 showed higher labeling for ERBB2 and bound to catalytic cysteines in other enzymes; PF131 impacted ATP transport by ADP/ATP translocase.

Conclusions:

• The refined PhosID-ABPP enables detailed, dose- and site-specific analysis of drug on- and off-target engagement.
• PF131 demonstrates broader off-target effects and interference with ATP transport, necessitating further investigation.
• PF899 shows specific targeting of ERBB2 and other enzymes, providing insights into its potential therapeutic profile.