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Dynamically driven cellular redox networks power a broad range of physiological cellular processes, and additionally are often dysregulated in various pathologies including cancer and inflammatory diseases. Therefore it is vital to be able to image and to respond to the turnover of the key players in redox homeostasis, to understand their physiological dynamics and to target pathological conditions. However, selective modular probes for assessing specific redox enzyme activities in cells are lacking. Here we report the development of cargo-releasing chemical probes that target the mammalian selenoprotein thioredoxin reductase (TrxR) while being fully resistant to thiol reductants in cells, such as the monothiol glutathione (GSH). We used a rationally oriented cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger that matches the chemistry of the unique TrxR active site, and integrated this reducible trigger into modular probes that release arbitrary cargos upon reduction. The probes' redox biochemistry was evaluated over a panel of thiol-type oxidoreductases, particularly showing remarkable, selenocysteine-dependent sensitivity of the "RX1" probe design to cytosolic TrxR1, with little response to mitochondrial TrxR2. The probe was cross-validated in cells by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and use of TrxR-selective chemical inhibitors, showing excellent TrxR1-dependent cellular performance. The RX1 design is therefore a robust, cellularly-validated, modular probe system for mammalian TrxR1. This sets the stage for in vivo imaging of TrxR1 activity in health and disease; and the thermodynamic and kinetic considerations behind its selectivity mechanism represent a significant advance towards rationally-designed probes for other key players in redox biology.

SummaryIntracellular bacterial pathogens inject effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32Salmonella entericaserovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effector proteins, and quantified PPIs in macrophages and epithelial cells by Affinity-Purification Quantitative Mass-Spectrometry. Thereby, we identified 25 previously described and 421 novel effector-host PPIs. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. Using reciprocal co-immunoprecipitations, we validated 13 out of 22 new PPIs. We then used this host-pathogen physical interactome resource to demonstrate that SseJ and SseL collaborate in redirecting cholesterol to theSalmonellaContaining Vacuole (SCV) via NPC1, PipB directly recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by directly phosphorylating formin-like proteins.

Specialised cellular networks of oxidoreductases coordinate the dithiol/disulfide-exchange reactions that control metabolism, protein regulation, and redox homeostasis. For probes to be selective for redox enzymes and effector proteins (nM to µM concentrations), they must also be able to resist nonspecific triggering by the ca. 50 mM background of non-catalytic cellular monothiols. However, no such selective reduction-sensing systems have yet been established. Here, we used rational structural design to independently vary thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual disulfide reduction trigger units designed for stability to monothiols. We integrated the motifs into modular series of fluorogenic probes that release and activate an arbitrary chemical cargo upon reduction, and compared their performance to that of the literature-known disulfides. The probes were comprehensively screened for biological stability and selectivity against a range of redox effector proteins and enzymes. This design process delivered the first disulfide probes with excellent stability to monothiols, yet high selectivity for the key redox-active protein effector, thioredoxin. We anticipate that further applications of these novel disulfide triggers will deliver unique probes targeting cellular thioredoxins. We also anticipate that further tuning following this design paradigm will deliver redox probes for other important dithiol-manifold redox proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous cellular redox systems.

The cyclic five-membered disulfide 1,2-dithiolane has been used as the key element in numerous chemical biology probes. Contradictory views of this disulfide motif populate the literature: some reports describe it as being nonspecifically reduced, others as a highly specific substrate for thioredoxin reductase (TrxR). We show that 1,2-dithiolanes are nonspecifically reduced by a broad range of thiol reductants and redox-active proteins, and that their cellular performance is barely affected by TrxR inhibition or knockout. We conclude that inhibitor screenings and probe designs treating 1,2-dithiolanes as TrxR-selective substrates should be treated with caution and previous interpretations may need careful re-evaluation. Considering ring-opening polymerisation, and stringently interpreting assays involving the thiophilic gold-based inhibitor auranofin, are critical to assess 1,2-dithiolane’s true behaviour. We present an approach to control against assay misinterpretation with reducible probes, to ensure that future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient redox probe research in the future.