Biological inspiration. Ability to directly interrogate on-target redox responses in vivo promises to advance a mechanistic understanding of specific redox pathways and precision therapeutic intervention. RES/ROS, reactive electrophilic/oxygen species. “SH” designates a thiol group of the cysteine residue on the target sensor protein. “R” represents a generic chemical modification. Inset: representative natural and synthetic reactive small electrophilic signaling mediators and their associated bioactivities. Blue shades within the chemical structures highlight electrophilic motifs.

Time is of the essence. Deconvoluting precise impacts of target-specific redox responses remains a challenge because on-target binding and responses are often overwhelmed by off-target mass action of these covalent modifications by highly diffusible and reactive (often bifunctional; see , inset) small electrophilic signaling mediators that can react with many targets nonspecifically. The red dot designates a RES signal such as HNE. Inset: dual-reactivity manifested in LDEs such as HNE, potentially resulting in protein cross-links through Schiff base formation (dotted arrow) in addition to conjugate addition (block arrow). Note: Conjugate addition can also occur, but to a lesser extent, with a histdine or lysine (Lys, shown) residue, in addition to with the more nucleophilic residue cysteine (Cys) as shown.

Evolution of RES signaling concepts. Multihit paradigm necessitates modifications of many localized sensor targets in order to elicit a response downstream, such as modulation of transcriptional response (this illustration). By contrast, the latest findings in the field initially suggest and subsequently provide direct evidence that redox responses operate similarly to canonical signal transduction wherein low-stoichiometry modest modifications of a single target are capable of driving a functional response. See text for details. The double-head and blunt-end arrows, respectively, indicate direct/indirect activation and inhibition. Arrows highlighted in red designate the nuclear signaling trajectory of interest.

Single-target RES modifications are individual events that drive functional redox response. Inset: ability to directly and precisely flip a single redox switch in living systems by T-REX offers a lens to understand functional on-target redox responses on demand (see text for details). Red dot, alkyne-functionalized HNE photouncaged from its photocaged precursor (pink dot) covalently bound to HaloTag. T-REX shows that (a) Keap-1-alone electrophilic modification is sufficient to stabilize Nrf2 and activate transcriptional antioxidant response in a way similar to canonical gain-of-function signaling; and (b) Pten-specific electrophilic modification modulates cellular phosphoinositide levels through a conventional mechanism of dominant loss-of-function inhibitory cell signaling. (RTK, receptor tyrosine kinase; PI3K, phosphoinositide 3-kinase; PI[(3,)4,5]P2(3), phosphatidylinositol (3,)4,5-bis(tris)phosphate. (c) A potential model subcategorizing the types of RES modifications that drive on-target redox signal propagation as a gradient of occupancy (e.g., “LDEylation stoichiometry”) and time/dose of RES. See text for details.