<p>Specialised cellular networks of oxidoreductases coordinate the
dithiol/disulfide-exchange reactions that control metabolism, protein
regulation, and redox homeostasis. For probes to be selective for redox enzymes
and effector proteins (nM to µM concentrations), they must also be able to
resist nonspecific triggering by the ca. 50 mM background of non-catalytic
cellular monothiols. However, no such selective reduction-sensing systems have yet
been established. Here, we used rational structural design to independently vary
thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual
disulfide reduction trigger units designed for stability to monothiols. We integrated
the motifs into modular series of fluorogenic probes that release and activate
an arbitrary chemical cargo upon reduction, and compared their performance to that
of the literature-known disulfides. The probes were comprehensively screened
for biological stability and selectivity against a range of redox effector
proteins and enzymes. This design process delivered the first disulfide probes with
excellent stability to monothiols, yet high selectivity for the key
redox-active protein effector, thioredoxin. We anticipate that further
applications of these novel disulfide triggers will deliver unique probes targeting
cellular thioredoxins. We also anticipate that further tuning following this
design paradigm will deliver redox probes for other important dithiol-manifold redox
proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous
cellular redox systems.</p>
Specialised cellular networks of oxidoreductases coordinate the dithiol/disulfide-exchange reactions that control metabolism, protein regulation, and redox homeostasis. For probes to be selective for redox enzymes and effector proteins (nM to µM concentrations), they must also be able to resist nonspecific triggering by the ca. 50 mM background of non-catalytic cellular monothiols. However, no such selective reduction-sensing systems have yet been established. Here, we used rational structural design to independently vary thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual disulfide reduction trigger units designed for stability to monothiols. We integrated the motifs into modular series of fluorogenic probes that release and activate an arbitrary chemical cargo upon reduction, and compared their performance to that of the literature-known disulfides. The probes were comprehensively screened for biological stability and selectivity against a range of redox effector proteins and enzymes. This design process delivered the first disulfide probes with excellent stability to monothiols, yet high selectivity for the key redox-active protein effector, thioredoxin. We anticipate that further applications of these novel disulfide triggers will deliver unique probes targeting cellular thioredoxins. We also anticipate that further tuning following this design paradigm will deliver redox probes for other important dithiol-manifold redox proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous cellular redox systems.
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.
This comprehensive study features the synthesis, characterization and evaluation of new energetic coordination compounds (ECC) based on two of the most powerful neutral tetrazoles, which have great potential as lead-free primary explosives.
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