Selective, Modular Probes for Thioredoxins Enabled by Rational Tuning of a Unique Disulfide Structure Motif

Abstract

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.

A Fluorescent Probe to Detect Quick Disulfide Reductase Activity in Bacteria

“…In the last two decades, there has been a growing trend towards using fluorescent probes to determine the reactive sulfur species in vivo [ 21 , 22 ]. In recent years, studies have provided important information on the use of fluorescent probes to detect LMW thiols e.g., cysteine [ 23 ], as well as antioxidant enzymes, including mammalian TrxR [ 24 ] and Trx [ 25 , 26 ], whereas, there is a very limited understanding of cellular redox homeostasis in bacteria [ 27 ]. Besides the differences in the cell wall/cell membrane between bacterial and mammalian cells, there are great differences in the composition and function of thiol-dependent systems between two types of organisms.…”

Cyclic 5-Membered Disulfides Are Not Selective Substrates of Thioredoxin Reductase, but Are Opened Nonspecifically

“…By identifying and avoiding problematic and nonselective substrate types such as 1,2-dithiolane, chemical development may instead be oriented towards selective and robust redox chemotypes for bioreductive probe and prodrug research. Indeed, novel reducible motifs with chemotype-based selectivity, as in the recent Trx-selective SS60-PQ 9 and the TrxR-reporting probe RX1, 66 are just now emerging. The modular reductiontriggered phenolic carbamate system we developed for this work already ensures valuable performance in biology, through its zero signal background, excellent hydrolytic robustness, and retained cell-marking signal, which combine to offer high-spatial-resolution imaging and cell-resolved statistics (Fig 4).…”

“…We then tested the TrxR-specificity of cellular activation of both 1,2-dithiolane probes, using the TrxR-independent linear disulfide SS00-PQ 9 and the strongly TrxR-dependent selenenylsulfidecontaining RX1 66 as references to indicate the expected outcomes of selectivity-testing cellular experiments.…”

“…5 mM GSH) compared to low concentrations in plasma or in the extracellular space, linear disulfides undergo irreversible and nonspecific thiol-disulfide interchange and reduction in cells (Fig 1a). 8,9 Linear disulfides are thus used for nonspecific intracellular release and/or activation of appended cargos, exploiting the cellular thiol pool.…”

A fluorescent probe for specifically measuring the overall thioredoxin and glutaredoxin reducing activity in bacterial cells

“…11 So far, cellular small thiol molecules and single antioxidant enzymes have been well characterized with various probes, especially in mammalian cells. 12–18 However, there are few studies to clarify the exact intracellular contribution of various Trxs and Grxs in maintaining the cellular redox balance due to the lack of an effective approach to detect the overall disulfide-reducing activities of both systems. 11,19…”