Accurate DNA replication and repair is essential for proper development, growth and tumor-free survival in all multicellular organisms. A key requirement for the maintenance of genomic integrity is the availability of adequate and balanced pools of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Notably, dNTP pool alterations lead to genomic instability and have been linked to multiple human diseases, including mitochondrial disorders, susceptibility to viral infection and cancer. In this review, we discuss how a key regulator of dNTP biosynthesis in mammals, the enzyme ribonucleotide reductase (RNR), impacts cancer susceptibility and serves as a target for anti-cancer therapies. Because RNR-regulated dNTP production can influence DNA replication fidelity while also supporting genome-protecting DNA repair, RNR has complex and stage-specific roles in carcinogenesis. Nevertheless, cancer cells are dependent on RNR for de novo dNTP biosynthesis. Therefore, elevated RNR expression is a characteristic of many cancers, and an array of mechanistically distinct RNR inhibitors serve as effective agents for cancer treatment. The dNTP metabolism machinery, including RNR, has been exploited for therapeutic benefit for decades and remains an important target for cancer drug development.

The concept of cell signaling in the context of nonenzyme-assisted protein modifications by reactive electrophilic and oxidative species, broadly known as redox signaling, is a uniquely complex topic that has been approached from numerous different and multidisciplinary angles. Our Review reflects on five aspects critical for understanding how nature harnesses these noncanonical post-translational modifications to coordinate distinct cellular activities: (1) specific players and their generation, (2) physicochemical properties, (3) mechanisms of action, (4) methods of interrogation, and (5) functional roles in health and disease. Emphasis is primarily placed on the latest progress in the field, but several aspects of classical work likely forgotten/lost are also recollected. For researchers with interests in getting into the field, our Review is anticipated to function as a primer. For the expert, we aim to stimulate thought and discussion about fundamentals of redox signaling mechanisms and nuances of specificity/selectivity and timing in this sophisticated yet fascinating arena at the crossroads of chemistry and biology.

This protocol describes targetable reactive electrophiles and oxidants (T-REX)—a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE (alkyne)) and the HaloTag-targetable photocaged precursor to HNE (alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t1/2 <1–2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4–24 h, depending on the nature of the pathway and the type of readouts used.

Isozyme-specific posttranslational regulation fine-tunes signaling events. However, sequence/activity redundancy renders links between isozyme-specific modifications and downstream functions uncertain. Methods to study this phenomenon are underdeveloped. A redox-targeting screen unveiled Akt3 as a first-responding isozyme sensing native electrophilic lipids. Electrophile modification of Akt3 modulated downstream pathway responses in cells and Danio rerio (zebrafish) and markedly differed from Akt2-specific oxidative regulation. Digest MS sequencing identified Akt3(C119) as the privileged cysteine that senses 4-hydroxynonenal. The Akt3(C119S) mutant was hypomorphic for all downstream phenotypes shown by wild-type Akt3. The study documents isozyme-specific and chemical redox signal-personalized physiologic responses.

Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized “electrophile toolbox” with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology—T-REX (targetable reactive electrophiles & oxidants)—is established by: (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein—one of several redox-sensitive regulators of the Nrf2–ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2–ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.