Hydrogen/Deuterium Exchange and Protein Oxidative Footprinting with Mass Spectrometry Collectively Discriminate the Binding of Small-Molecule Therapeutics to Bcl‑2

Characterizing protein–ligand interactions is
crucial to
understanding cellular metabolism and guiding drug discovery and development.
Herein, we explore complementing hydrogen/deuterium exchange mass
spectrometry (HDX-MS) with a recently developed Fenton chemistry-based
approach to protein oxidative footprinting mass spectrometry (OX-MS)
to discriminate the binding of small-molecule therapeutics. Using
drug-dependent perturbation as the experimental report, this combination
of techniques more clearly differentiates the in-solution binding
profiles of Venetoclax (ABT-199, GDC-0199-AbbVie and Genentech) and
a drug candidate S55746 (Servier) to the apoptotic regulatory protein
Bcl-2 than either technique alone. These results highlight the value
of combining these methods to compare compounds in drug discovery
and development. To better understand the structural context of the
HDX-MS and OX-MS drug-dependent perturbations, we mapped these data
on Bcl-2-Venetoclax and Bcl-2-S55746 cocrystal structures and compared
these results with the structure of apo Bcl-2. HDX-MS shows that Venetoclax
more strongly impacts the protein backbone compared to S55746. OX-MS
reveals oxidation perturbations rationalized by direct side-chain
protection as well as by crystallographically observed drug-induced
protein restructuring. Both methods report the perturbation of some,
but not all, residues mapped within 4 Å of the bound drugs in
the crystal structures. Concordant characterization of backbone and
side-chain accessibility will enhance our understanding of in-solution
protein structure dynamics and protein–ligand interactions
during drug discovery, development, and characterization, particularly
when high-resolution structures are lacking.