Selective, Modular Probes for Thioredoxins Enabled by Rational Tuning of a Unique Disulfide Structure Motif

Abstract

<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>

Selective cellular probes for mammalian thioredoxin reductase TrxR1: rational design of RX1, a modular 1,2-thiaselenane redox probe

“…(c) Topology considerations: linear topology dichalcogenides (shown: disulfides) are irreversibly committed to cargo release after monothiol exchange, whereas for cyclic topology dichalcogenides, the exchange / reduction pathways is reversible: for full mechanism, see Fig S3 . As disulfides are their native substrates, chemocompatible disulfide-based probes have long been explored for their ability to report on these redox-active enzyme systems. Disulfide trigger-cargo constructs 23 are a conceptually simple, modular turn-on design. Ideally, the cargo is chosen and attached to mask a key structural element, such that (i) the intact probe is fully deactivated, but (ii) trigger reduction causes a cascade that irreversibly restores activity by unmasking, often by simply liberating the cargo (Fig S1c).…”

“…29 Thus, although some reports have used linear disulfide probes (Fig S1a), we believed that selective reporters would require a different design. 23 Cyclic-topology disulfide probes can, however, resist triggering by monothiols in two ways. They can reform the disulfide after the initial thiol-disulfide exchange by expelling the attacking monothiol, or after reduction to the dithiol they can be re-oxidised by other e e e disulfides in their environment: both of which prevent them from committing to cargo release (Fig S3b ; full discussion in ref.…”

“…This means that in the cellular context, they cannot be selective for dithiol proteins. This rules out cyclic 7-membered, 23 ETP-type-6membered, 33 and cyclic 5-membered disulfides 29,34 as substrates that can be cellularly selective for distinct dithiol-based enzymes. Although probes based on cyclic 5-membered disulfides have been published and commercialised as TrxR-selective (Fig S1a), [35][36][37] the unselectivity and kinetic problems of such disulfides have been shown historically as well as recently (Fig S1b,c).…”

“…In support of this view, Matile has shown the lability of cyclic 5-membered diselenide probes 39 to nonspecific thiol exchange, mirroring that of the corresponding disulfides (Fig S1b) . We therefore expected that only cyclic 6-membered dichalcogenides would be stable enough to avoid irreversible triggering in cells. Selecting them is only a partial solution, as this only addresses the overall challenge of ensuring that monothiol attack has the potential to be reversible (discussion in 23 ). As highly reducing dithiol proteins Trx and Grx are present at ca.…”

“…Therefore, to meaningfully characterise the response to reductant challenges, we titrated reductants up to supraphysiological concentrations, and acquired full timecourse rather than endpoint data for more rigorous interpretation. 23,29 We began with GSH challenge tests. These were highly encouraging: over 6 h, A-type probes showed zero fluorescence response up to 10 mM GSH, indicating outstanding monothiol resistance (Fig 4a The comparison probe types behaved as expected.…”