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.

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.