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Felber, Jan G.; Poczka, Lena; Scholzen, Karoline C.; Zeisel, Lukas; Maier, Martin S.; Busker, Sander; Theisen, Ulrike; Brandstädter, Christina; Becker, Katja; Arnér, Elias S. J.; Thorn-Seshold, Julia; Thorn-Seshold, Oliver ORCID: 0000-0003-3981-651X (2022): Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically. Nature Communications, 13: 1754. ISSN 2041-1723

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The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as “TRFS” probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes’ complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research.

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