Cyclic Thiosulfonates for Thiol-Mediated Uptake: Cascade Exchangers, Transporters, Inhibitors

Abstract

Thiol-mediated uptake is emerging as a powerful method to penetrate cells. Cyclic oligochalcogenides (COCs) have been identified as privileged scaffolds to enable and inhibit thiol-mediated uptake because they can act as dynamic covalent cascade exchangers, i.e., every exchange produces a new, covalently tethered exchanger. In this study, our focus is on the essentially unexplored COCs of higher oxidation levels. Quantitative characterization of the underlying dynamic covalent exchange cascades reveals that the initial ring opening of cyclic thiosulfonates (CTOs) proceeds at a high speed even at a low pH. The released sulfinates exchange with disulfides in aprotic but much less in protic environments. Hydrophobic domains were thus introduced to direct CTOs into hydrophobic pockets to enhance their reactivity. Equipped with such directing groups, fluorescently labeled CTOs entered the cytosol of living cells more efficiently than the popular asparagusic acid. Added as competitive agents, CTOs inhibit the uptake of various COC transporters and SARS-CoV-2 lentivectors. Orthogonal trends found with different transporters support the existence of multiple cellular partners to account for the diverse expressions of thiol-mediated uptake. Dominant self-inhibition and high activity of dimers imply selective and synergistic exchange in hydrophobic pockets as distinguishing characteristics of thiol-mediated uptake with CTOs. The best CTO dimers with hydrophobic directing groups inhibit the cellular entry of SARS-CoV-2 lentivectors with an IC 50 significantly lower than the previous best CTO, below the 10 μM threshold and better than ebselen. Taken together, these results identify CTOs as an intriguing motif for use in cytosolic delivery, as inhibitors of lentivector entry, and for the evolution of dynamic covalent networks in the broadest sense, with reactivity-based selectivity of cascade exchange emerging as a distinguishing characteristic that deserves further attention.

Anticancer Agents Derived from Cyclic Thiosulfonates: Structure‐Reactivity and Structure‐Activity Relationships

“…The low energy barriers are consistent with the observations of Matile et al . that the ring‐opening of cyclic thiosulfonates proceeds at a high rate (instantaneous on the NMR timescale) in a solution of D 2 O/DMSO‐ d 6 [14a] . Overall, the data obtained by DFT simulations suggest that all cyclic thiosulfonates studied are potentially reactive toward sulfhydryl moieties.…”

Pnictogen-Centered Cascade Exchangers for Thiol-Mediated Uptake: As(III)-, Sb(III)-, and Bi(III)-Expanded Cyclic Disulfides as Inhibitors of Cytosolic Delivery and Viral Entry

“…Without bismuth, the DTT or DTE precursors fail to inhibit thiol-mediated uptake . Their activation as cascade exchangers by oxidation to thiosulfonate 18 gave an IC 50 ≈ 50 μM and inactivity at 10 μM (Figure A). , The inhibition of SARS-CoV-2 lentivector uptake by CAX 18 was thus five times weaker compared to p-CAX 3 with IC 50 ≈ 10 μM. This comparison supported that the activation of cyclic disulfides by ring expansion with pnictogen relays is more powerful than activation by oxidation.…”

“…They are also much better than the popular ebselen, 61 − 63 which inhibits also both thiol-mediated cytosolic delivery with transporter 21 and the entry of SARS-CoV-2 lentivectors, but the latter only at high IC 50 with high toxicity. 60 The availability of privileged scaffolds with and without permanent phenyl substituent on the pnictogen relay is important because they provide access to different cascade exchange chemistries ( Figures 1 C,D, 3 , and 4 ). The availability of privileged scaffolds with As(III) and Bi(III) relays is attractive because their coordination chemistry covers two extremes that provide complementary characteristics in the dynamic covalent exchange cascades related to thiol-mediated uptake ( Figure 4 C,D).…”

Tyrosine Bioconjugation with Hypervalent Iodine

“…41 The ~9-fold increase in uptake efficiency observed between AspA and CTO complexes exceeds the ~3fold difference seen with small molecule fluorophore cargoes, the greater differential perhaps resulting from the additional hydrophobic directing group provided by the CF3-functionalised aromatic unit, which is known to be more important for CTO transporters. 41 In the case of AspA and CTO, a diffuse pattern of fluorescence was observed in spinning disk confocal microscopy (SDCM) images (Figure 1d and e). In line with previous reports, this implied delivery to the cytosol rather than entrapment within endosomes following endocytosis, whereas N3 complex 18 showed a punctate fluorescence indicative of endocytosis (Figure 1c).…”

“…41 Structure-activity relationships for cyclic thiosulfonate transporters and thiol-mediated uptake inhibitors revealed a strong dependence in either case on the presence of hydrophobic directing groups to target aprotic environments such as the cellular membrane and proteins, in which the proticity-dependent cascade exchange of cyclic thiosulfonates is enhanced. 41 The ~9-fold increase in uptake efficiency observed between AspA and CTO complexes exceeds the ~3fold difference seen with small molecule fluorophore cargoes, the greater differential perhaps resulting from the additional hydrophobic directing group provided by the CF3-functionalised aromatic unit, which is known to be more important for CTO transporters. 41 In the case of AspA and CTO, a diffuse pattern of fluorescence was observed in spinning disk confocal microscopy (SDCM) images (Figure 1d and e).…”