Activity-Based Hydrazine Probes for Protein Profiling of Electrophilic Functionality in Therapeutic Targets

Most known probes
for activity-based protein profiling (ABPP) use
electrophilic groups that tag a single type of nucleophilic amino
acid to identify cases in which its hyper-reactivity underpins function.
Much important biochemistry derives from electrophilic enzyme cofactors,
transient intermediates, and labile regulatory modifications, but
ABPP probes for such species are underdeveloped. Here, we describe
a versatile class of probes for this less charted hemisphere of the
proteome. The use of an electron-rich hydrazine as the common chemical
modifier enables covalent targeting of multiple, pharmacologically
important classes of enzymes bearing diverse organic and inorganic
cofactors. Probe attachment occurs by both polar and radicaloid mechanisms,
can be blocked by molecules that occupy the active sites, and depends
on the proper poise of the active site for turnover. These traits
will enable the probes to be used to identify specific inhibitors
of individual members of these multiple enzyme classes, making them
uniquely versatile among known ABPP probes.