Redox regulation is a phenomenon in which signals are relayed through oxidative and electrophilic modifications of specific redox-responsive proteins. These chemical events in turn orchestrate unique cytoprotective responses downstream. The deregulation of redox signaling pathways underlies many human diseases, including cancers, Alzheimer's, and cardiovascular disorders. However, the links between disease and individual redox signaling events remain shrouded in mystery. Currently, decoding the consequences of redox-linked modifications of specific proteins in cells induced by a given reactive small molecule is impossible. Conventional strategies limited to whole-cell bathing with reactive entities target simultaneously many, if not most, re- dox-sensitive proteins in cells. Although these "multihit" approaches model oxidative stress, they do not allow study of physiological redox signaling. My laboratory has developed a chemistry-driven innovation-targetable reactive electrophiles and oxidants (T-REX)-ultimately aimed at directly linking individual downstream biological responses to the chemical redox alteration of specific target proteins. T-REX mimics endogenous signaling, enabling selective and spatiotemporally controlled perturbation of any redox-responsive protein in cells with any reactive entity. Toward this ultimate goal, we recently showed proof of concept in which ligand-directed intramolecular delivery enabled selective targeting of a bioactive lipid electrophile, 4-hydroxynonenal (HNE), to two distinct redox-responsive proteins of biomedical interest in living cells. We herein exploit T-REX to probe mechanistic relationships between specific protein-electrophile perturbation and downstream signal propagation within a major disease-implicated redox signaling cascade. The proposed experiments will (1) profile the target-specific mechanisms by which HNE and analogous electrophiles operate in cells, (2) define the extent to which physiologic HNEylation prompts response in an otherwise native cell, and (3) pinpoint specific residues of the upstream target genuinely responsible for electrophile sensing. A multidisciplinary combination of synthetic chemistry, chemical biology, mechanistic biochemistry, mammalian cell biology and structural biology approaches will be used. Success will establish T-REX as the first chemical biology platform capable of mimicking non-enzyme-mediated posttranslational modifications in redox signaling. The utility of T-REX is far reaching: the strategy described herein can perturb any disease-implicated redox-responsive protein with any bioactive small-molecule inducer such that sophisticated redox-information-processing mechanisms within individual redox-modulated pathways can be clearly understood. T-REX is an ambitious yet transformative approach to understanding redox regulation. As with phosphoregulatory pathways wherein mechanistic understanding of temporal dynamics within individual phosphoryl transfer events has yielded multiple medical breakthroughs, T-REX strategy has the same long-term potential in impacting modern biomedical research.

Public Health Relevance

Targetable Reactive Electrophiles and Oxidants technology allows time-resolved and controlled perturbation of specific proteins in cells with reactive small molecules, enabling dissection of individual redox-modulated path- ways. If successful, this chemistry-driven innovation will be a novel gateway to elucidating the basic biology of redox signaling underlying etiologies of cancer, neurodegeneration and cardiovascular diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
NIH Director’s New Innovator Awards (DP2)
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Special Emphasis Panel (ZRG1-MOSS-C (56))
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Reddy, Michael K
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Cornell University
Schools of Arts and Sciences
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Long, Marcus J C; Poganik, Jesse R; Aye, Yimon (2016) On-Demand Targeting: Investigating Biology with Proximity-Directed Chemistry. J Am Chem Soc 138:3610-22
Wisitpitthaya, Somsinee; Zhao, Yi; Long, Marcus J C et al. (2016) Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition. ACS Chem Biol 11:2021-32
Parvez, Saba; Fu, Yuan; Li, Jiayang et al. (2015) Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response. J Am Chem Soc 137:10-3
Lin, Hong-Yu; Haegele, Joseph A; Disare, Michael T et al. (2015) A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling. J Am Chem Soc 137:6232-44