Discovery of Potent and Selective Inhibitors against Protein-Derived Electrophilic Cofactors

Electrophilic cofactors are widely
distributed in nature and play
important roles in many physiological and disease processes, yet they
have remained blind spots in traditional activity-based protein profiling
(ABPP) approaches that target nucleophiles. More recently, reverse-polarity
(RP)-ABPP using hydrazine probes identified an electrophilic N-terminal glyoxylyl (Glox) group for the first time in
secernin-3 (SCRN3). The biological function(s) of both the protein
and Glox as a cofactor has not yet been pharmacologically validated
because of the lack of selective inhibitors that could disrupt and
therefore identify its activity. Here, we present the first platform
for analyzing the reactivity and selectivity of an expanded nucleophilic
probe library toward main-chain carbonyl cofactors such as Glox and
pyruvoyl (Pyvl) groups. We first applied the library proteome-wide
to profile and confirm engagement with various electrophilic protein
targets, including secernin-2 (SCRN2), shown here also to possess
a Glox group. A broadly reactive indole ethylhydrazine probe was used
for a competitive in vitro RP-ABPP assay to screen
for selective inhibitors against such cofactors from a set of commercially
available nucleophilic fragments. Using Glox-containing SCRN proteins
as a case study, naphthyl hydrazine was identified as a potent and
selective SCRN3 inhibitor, showing complete inhibition in cell lysates
with no significant cross-reactivity detected for other enzymes. Moving
forward, this platform provides the fundamental basis for the development
of selective Glox inhibitors and represents a starting point to advance
small molecules that modulate electrophile-dependent function.