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

Abstract

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.

Nanoaggregates of Disulfide-Decorated TrxR Inhibitor Promote Cellular Uptake, Selective Targeting, and Antitumor Efficacy

“…The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.2c02309. DLS, zeta potential, UV−vis spectrum, fluorescence spectrum, 1…”

“…Some of the 1,2dithiolane-based probes and prodrugs have also been reported as selective cellular substrates of TrxR. 1 In previous work, we successfully screened out a novel TrxR inhibitor CPUL1 from the compound library established by our group before, 3−5 but poor water solubility greatly limits its further antitumor application. In recent years, carrier-free nanomedicine completely composed of active pharmaceuticals has attracted a lot of attention.…”

Synthesis and Antimicrobial Evaluation of New Cephalosporin Derivatives Containing Cyclic Disulfide Moieties

“…For example, the “Trojan horse” approach exploits receptor-mediated Fe-uptake mechanisms in bacteria, culminating in the launch of the cephalosporin-based antibiotic cefiderocol on the market. , Alternatively, influx can be enhanced by the self-promoted uptake mechanism with polycationic antibiotics involving the displacement of the divalent cations that stabilize the lipopolysaccharide (LPS) membrane packing, thereby increasing the passage of the promoter into the periplasm . Moreover, some efforts were made to investigate the dithiol-mediated uptake of antibiotics by bacteria in a similar manner to eukaryotic cells; − however, the obtained results have been mixed so far. Another important aspect is the formation of biofilms, which renders the producing bacteria highly recalcitrant to antibiotics .…”

Regioselective, efficient and scalable syntheses of 1,2-thiaselenanes

“…[3][4][5] We have instead exploited 1,2-thiaselenanes as the reducible "triggers" of redox probes that function in live cells with remarkable regioselectivity profiles. 6,7 The 1,2-thiaselenane-4-amine (TSA4) probes are selectively activated by TrxR, without any turn-on by even 10 mM of monothiols over 6 h; whereas their regioisomeric 1,2-thiaselenane-5-amine (TSA5) probes are strongly activated just by glutathione (GSH) and so are not TrxR reporters. This remarkable difference is entirely due to the position of the selenium atom.…”