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

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 populate the literature: some reports describe it as being nonspecifically reduced, others as a highly specific substrate for thioredoxin reductase (TrxR). We here show that 1,2-dithiolane probes 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 "TRFS" probes that have used 1,2-dithiolanes as TrxRselective substrates should be treated with caution, and may need re-evaluation. Understanding 1,2-dithiolanes’ behaviour needs consideration of probe localisation and environmentdependent fluorescence, reduction-independent ring-opening polymerisation, thiol-dependent cellular uptake, and caution when applying thiophilic inhibitors. We present an approach controlling against assay misinterpretation with reducible probes, to ensure that future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research.