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

Abstract

<div> <div> <div> <p>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. </p> </div> </div> </div>

Selective cellular probes for mammalian thioredoxin reductase TrxR1: rational design of RX1, a modular 1,2-thiaselenane redox probe

“…This makes linear topology probes nonspecifically labile to cellular monothiols, [26][27][28] and therefore prevents them from being enzyme-selective reporters in the cellular context. 29 Thus, although some reports have used linear disulfide probes (Fig S1a), we believed that selective reporters would require a different design. 23 Cyclic-topology disulfide probes can, however, resist triggering by monothiols in two ways.…”

“…They can reform the disulfide after the initial thiol-disulfide exchange by expelling the attacking monothiol, or after reduction to the dithiol they can be re-oxidised by other e e e disulfides in their environment: both of which prevent them from committing to cargo release (Fig S3b ; full discussion in ref. 29 ). The geometry of the cyclic disulfide is critical for its tendency to perform these reactions, and therefore determines the cellular performance of cyclic-disulfide-based probes.…”

“…This means that in the cellular context, they cannot be selective for dithiol proteins. This rules out cyclic 7-membered, 23 ETP-type-6membered, 33 and cyclic 5-membered disulfides 29,34 as substrates that can be cellularly selective for distinct dithiol-based enzymes. Although probes based on cyclic 5-membered disulfides have been published and commercialised as TrxR-selective (Fig S1a), [35][36][37] the unselectivity and kinetic problems of such disulfides have been shown historically as well as recently (Fig S1b,c).…”

“…Although probes based on cyclic 5-membered disulfides have been published and commercialised as TrxR-selective (Fig S1a), [35][36][37] the unselectivity and kinetic problems of such disulfides have been shown historically as well as recently (Fig S1b,c). 22,29,34,[38][39][40] Alicyclic 6membered disulfides (1,2-dithianes), however, are stable against monothiols. We recently characterised monocyclic and annelated bicyclic 1,2-dithianes for their performance as reductive triggers, showing that they resist GSH 28 as well as TrxR 41 , and can be harnessed in trigger-cargo constructs that are selective for Trx (Fig S1c) (see Supporting Note 1).…”

“…We used a cyclic disulfide (S-type) and a cyclic diselenide (Se3) as reducible controls with different thermodynamic and kinetic sensitivity profiles that we anticipated would make them not TrxR-selective. Linear disulfide SS00 29 (Fig S5 ) was used as our mechanistic control, since according to our hypotheses it ought to access the irreversible pathway and so should be thiol-labile. (see Supporting Note 2).…”