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

Comparative, dose-dependent analysis of interactions
between small
molecule drugs and their targets, as well as off-target interactions,
in complex proteomes is crucial for selecting optimal drug candidates.
The affinity of small molecules for targeted proteins is largely dictated
by interactions between amino acid side chains and these drugs. Thus,
studying drug–protein interactions at an amino acid resolution
provides a comprehensive understanding of the drug selectivity and
efficacy. In this study, we further refined the site-specific activity-based
protein profiling strategy (ABPP), PhosID-ABPP, on a timsTOF HT mass
spectrometer. This refinement enables dual dose-dependent competition
of inhibitors within a single cellular proteome. Here, a comparative
analysis of two activity-based probes (ABPs), developed to selectively
target the epidermal growth factor receptor (EGFR), namely, PF-06672131
(PF131) and PF-6422899 (PF899), facilitated the simultaneous identification
of ABP-specific binding sites at a proteome-wide scale within a cellular
proteome. Dose-dependent probe-binding preferences for proteinaceous
cysteines, even at low nanomolar ABP concentrations, could be revealed.
Notably, in addition to the intrinsic affinity of the electrophilic
probes for specific sites in targeted proteins, the observed labeling
intensity is influenced by several other factors. These include the
efficiency of cellular uptake, the stability of the probes, and their
intracellular distribution. While both ABPs showed comparable labeling
efficiency for EGFR, PF131 had a broader off-target reactivity profile.
In contrast, PF899 exhibited a higher labeling efficiency for the
ERBB2 receptor and bound to catalytic cysteines in several other enzymes,
which is likely to disrupt their catalytic activity. Notably, PF131
effectively labeled ADP/ATP translocase proteins at a concentration
of just 1 nm, and we found this affected ATP transport. Analysis of
the effect of PF131 and its parent inhibitor Afatinib on murine translocase
SLC25A4 (ANT1)-mediated ATP transport strongly indicated that PF131
(10 μM) partially blocked ATP transport. Afatinib was less efficient
at inhibiting ATP transport by SLC25A4 than PF131, and the reduction
of ATP transport by Afatinib was not significant. Follow-up analysis
is required to evaluate the affinity of these inhibitors for ADP/ATP
translocase SLC25A4 in more detail. Additionally, the analysis of
different binding sites within the EGF receptor and the voltage-dependent
anion channel 2 revealed secondary binding sites of both probes and
provided insights into the binding poses of inhibitors on these proteins.
Insights from the PhosID-ABPP analysis of these two ABPs serve as
a valuable resource for understanding drug on- and off-target engagement
in a dose- and site-specific manner.