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

Abstract

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.

Advances in covalent drug discovery

“…ABPP principles and ‘reverse-polarity’ probes have been employed to study electrophilic post-translational modifications, such as N-terminal pyruvoyl and glyoxylyl modifications, that can react with hydrazine-containing compounds 197 . Hydrazine-based probes have also been used to help identify and characterize ligands for proteins with electrophilic cofactors or post-translational modifications 198 , 199 .…”

Spectroscopic and in vitro Investigations of Fe2+/α‐Ketoglutarate‐Dependent Enzymes Involved in Nucleic Acid Repair and Modification

“…For instance, point mutations or deletion variants are very commonly included in activity studies to give indication for the function of particular amino acid residues. Also, hydrazines have recently been introduced as novel, versatile nucleophilic in situ probes for the study of (among others) AlkBH and related enzymes by activity‐based protein profiling (ABPP) [28] . While there are examples of these methods briefly introduced in several of the following sections where appropriate, this frequent element is not described as a technique in the context of this review in detail.…”

“…Also, hydrazines have recently been introduced as novel, versatile nucleophilic in situ probes for the study of (among others) AlkBH and related enzymes by activity-based protein profiling (ABPP). [28] While there are examples of these methods briefly introduced in several of the following sections where appropriate, this frequent element is not described as a technique in the context of this review in detail. Further, the mutual dependence between detailed in vitro investigation of an enzyme and experiments in vivo (e. g., by gene deletion studies to learn about its physiological function) is not addressed as in vivo studies are beyond the scope of this article.…”

Flipping the Switch: Innovations in Inducible Probes for Protein Profiling

“…In 2021, Matthews and co-workers developed a novel class of organohydrazine mechanism-based probes. 119 Previous studies had demonstrated that organohydrazine probes could be used as nucleophiles for reverse polarity protein profiling; 120 however detection of some unexplained adducts suggested a secondary labeling pathway. Examination revealed that enzymes which bound oxidizing cofactors at their active sites could oxidize organohydrazine probes to form a carbon centered radical for covalent protein–probe cross-linking ( Figure 5 f).…”

“…In 2021, Matthews and co-workers developed a novel class of organohydrazine mechanism-based probes . Previous studies had demonstrated that organohydrazine probes could be used as nucleophiles for reverse polarity protein profiling; however detection of some unexplained adducts suggested a secondary labeling pathway.…”