Defining the precise biological impacts of localized reactive electrophilic metabolites and related small-molecule drugs has proven to be highly challenging. Traditional approaches to study such non-enzymatic modifications had relied on in vitro work and/or swamping cells with excess electrophiles. However, these conditions often do not recapitulate signaling behaviors, and teasing apart on- vs. off-target responses, against issues such as toxicity, metabolic conversion, etc., remains complex. Latest technologies thus pursued target profiling; yet, a major question mark remained as to how important modifications of these protein-hits are, in terms of measured phenotypic or pharmacological output. My laboratory proposed that for a protein to sense and respond to an electrophile often manifesting short half-life in cells, the protein should have elevated reactivity for that electrophile. We thus devised a strategy, later termed T-REX, to present in close proximity such proteins with a native, unfettered electrophile, and assay: (1) to what extent the protein reacts with this freely-diffusible electrophile; and (2) should some degree of labeling occur, to what extent such substoichiometric modification is sufficient to elicit a functional signaling ouput. Over the past 8 years, T-REX, and a related development, G-REX electrophile-responsivity profiling in living systems, have enabled us to identify bona fide first responders that interact with native signaling electrophiles under close to endogenous redox signaling conditions (i.e., “kcat/Km”-like). Our data show that these first responders lie at nexuses between electrophile- and canonical-signaling pathways. Thus, these proteins translate information encoded by electrophiles to, for instance, phosphate or ubiquitin to reroute signaling pathway flux often conserved across evolution, even at the organismal level. We have also shown recently that the electrophile-sensing characteristics that are unique to native electrophiles can be transferred to custom-designed inhibitors.

Illuminating decision making shaped by on-target reactive electrophile modification

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.