Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically

• Published in: Nature communications Vol. 13; no. 1; pp. 1754 - 13
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 14; 14/34; 140/131; 140/58; 631/45/607/1168; 631/92/96; 639/638/403; 639/638/92/607; Acids; Biology; Disulfides; Disulfides - metabolism; Fluorescence; Humanities and Social Sciences; Localization; Medicin och hälsovetenskap; multidisciplinary; Natural products; Neurosciences; Oxidation-Reduction; Probes; Proteins; Reducing agents; Reductase; Reductases; Ring opening polymerization; Science; Science (multidisciplinary); Selectivity; Substrates; Thioredoxin; Thioredoxin-Disulfide Reductase - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-29136-4

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. Cyclic five-membered disulfides (1,2-dithiolanes) have been reported either as nonspecific redox motifs, or as highly specific cellular probes for thioredoxin reductase (TrxR). Here the authors show that 1,2-dithiolane probes are nonspecifically reduced by a range of thiol reductants and are not sensitive to TrxR modulation, thus they are unsuitable as cellular probes for TrxR.