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Review
. 2021 May 3;1(6):710-728.
doi: 10.1021/jacsau.1c00128. eCollection 2021 Jun 28.

Thiol-Mediated Uptake

Quentin Laurent  1 , Rémi Martinent  1 , Bumhee Lim  1 , Anh-Tuan Pham  1 , Takehiro Kato  1 , Javier López-Andarias  1 , Naomi Sakai  1 , Stefan Matile  1
Affiliations
Review

Thiol-Mediated Uptake

Quentin Laurent et al. JACS Au. .

Abstract

This Perspective focuses on thiol-mediated uptake, that is, the entry of substrates into cells enabled by oligochalcogenides or mimics, often disulfides, and inhibited by thiol-reactive agents. A short chronology from the initial observations in 1990 until today is followed by a summary of cell-penetrating poly(disulfide)s (CPDs) and cyclic oligochalcogenides (COCs) as privileged scaffolds in thiol-mediated uptake and inhibitors of thiol-mediated uptake as potential antivirals. In the spirit of a Perspective, the main part brings together topics that possibly could help to explain how thiol-mediated uptake really works. Extreme sulfur chemistry mostly related to COCs and their mimics, cyclic disulfides, thiosulfinates/-onates, diselenolanes, benzopolysulfanes, but also arsenics and Michael acceptors, is viewed in the context of acidity, ring tension, exchange cascades, adaptive networks, exchange affinity columns, molecular walkers, ring-opening polymerizations, and templated polymerizations. Micellar pores (or lipid ion channels) are considered, from cell-penetrating peptides and natural antibiotics to voltage sensors, and a concise gallery of membrane proteins, as possible targets of thiol-mediated uptake, is provided, including CLIC1, a thiol-reactive chloride channel; TMEM16F, a Ca-activated scramblase; EGFR, the epithelial growth factor receptor; and protein-disulfide isomerase, known from HIV entry or the transferrin receptor, a top hit in proteomics and recently identified in the cellular entry of SARS-CoV-2.

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Conflict of interest statement

The authors declare the following competing financial interest(s): A U.S. Patent Application has been filed covering the antiviral activity of cyclic thiosulfonates (No. 63/073,863).

Figures

Figure 1
Figure 1
Thiol-mediated uptake (a) operates with the dynamic covalent chemistry (DCC) of chalcogenide exchange before or during cellular entry by direct translocation, endocytosis, or fusion, (b) usually involves thiol/disulfide exchange, and (c) can be inhibited with the same DCC.
Figure 2
Figure 2
Timeline with milestones for the emergence of thiol-mediated uptake.
Figure 3
Figure 3
Shift of attention from CPPs 7 to CPDs 4, which are accessible in situ from monomers 8 and depolymerizable in the cytosol. Initiators 9–13 (see Figure 4) and terminators 14, e.g., iodoacetamide (LG, leaving group).
Figure 4
Figure 4
Selected CPD initiators for CPD interfacing (9–13), cyclization (15, to give 16), traceless release (17), and cryopolymerization on proteins (e.g., GFP, 18).
Figure 5
Figure 5
Artificial metalloenzymes (19), quantum dots (QD, 20, 21), and MPSNPs (22, 23) as selected more complex systems delivered with CPDs. (a) Microscopy images show the poor colocalization of QDs and dextran, indicating the nonendosomal localization (left), and the intracellular aggregate 21 formation by the interaction between anti-GFP nanobody (GBP) and GFP, loaded on different sets of QDs (right). Adapted from ref (81). Copyright 2017 American Chemical Society.
Figure 6
Figure 6
Some cyclic oligochalcogenides (COCs), highlighting (a) CXXC dihedral angles of (b) privileged scaffolds with pKa’s of the ring-opened form, (c) their cellular uptake efficiencies,, and (d) their occurrence in natural products and drugs.
Figure 7
Figure 7
Cys-X-Cys γ-turns 31 as privileged COC scaffold (R = peptide, R′ usually H), compatibility of COCs and AOCs with automated oligomer synthesis, and use of COCs for sensing and liposome delivery.
Figure 8
Figure 8
Competitive inhibitors inactivate thiols and disulfides on cell surfaces, usually by dynamic covalent chalcogenide exchange, with selected inhibitors of thiol-mediated uptake of fluorescent BPS (B, 28, R = anionic fluorophore) and ETP (E, 25, R = anionic fluorophore) and with indication of E/B selectivity (MICs against 25/28).
Figure 9
Figure 9
Redox-switched dynamic covalent sulfur exchange cascades of (a) disulfide COCs, covering (b) dithiols, (c) thiosulfinates, and (d) thiosulfonates. (a) Disulfides 5 can exchange with thiols, then disulfides (A), then thiols, etc (B); (b) dithiols 43, perhaps also phosphorothioates 44, with disulfides, etc (C, B); (c) thiosulfinates 38 with neighboring thiols (D–F); and (d) thiosulfonates 6 with thiols (G), then disulfides (H), then thiols, etc (I).
Figure 10
Figure 10
Adaptive dynamic covalent network produced by BPS 28, with cells expected to select the best for uptake, which then will be amplified by re-equilibration.
Figure 11
Figure 11
Thiol affinity columns as a tool to fingerprint dynamic covalent chalcogenide exchange networks: (a, b) After injection of AspA 5, a first fraction is eluted without retention (A), and a second fraction is eluted upon addition of DTT (B). (c, d) DSL 26 is poorly retained, presumably due to self-release by selenophilic ring closure.
Figure 12
Figure 12
Dynamic covalent chalcogen and pnictogen exchange networks applied to molecular walkers that walk along thiol tracks 57 on a β sheet in a transmembrane pore. (a) A disulfide hopper 56 carrying DNA to hop by disulfide exchange cascades from the first (A, B) to the second thiol (B, C) and to the other end of the track (C, D). (b) A sulfophenyl (SP) arsonodithioite walker 58 combining chalcogen and pnictogen exchange chemistry to step on the first (E, F) and the second thiol (F, G) then moves from the first (G) to the third (H, I) and then from the second toward the terminal thiol (I, J). (c) The dynamic covalent network of Ehrlich’s arsphenamine 60.
Figure 13
Figure 13
Dynamic covalent conjugate addition cascades to (a) create molecular walkers 65 that walk along polyamine tracks 66 from the first two (A–C) to the last two amines (C, D) and (b) to create molecular probes 68 that recognize neighboring thiols 67, with addition of the first thiol (E) generating the second acceptor in situ (F) for addition to the neighboring thiols (G), producing the two, possibly dynamic sulfide bridges in product 69. (c) Cyano and phenyl acceptors in 70 to enhance the reversibility of Michael addition cascades (EWG, electron-withdrawing group) and carbonyl acceptors in 71 to enhance the spiciness of cinnamaldehyde.
Figure 14
Figure 14
Ring-opening polymerization (ROP) of cyclic oligochalcogenides (COCs) attached with a cleavable linker L along a template.
Figure 15
Figure 15
Examples for ring-opening polymerization (ROP) of 1,2-dithiolanes templated in vesicles (A, B), on DNA nanoparticles (C, D), along π stacks on electrodes (E) and through macrocycles 74, compared to the practically unexplored ROP of 1,2-diselenolanes 26.
Figure 16
Figure 16
Schematic side, perspective, and top view (top to bottom) of micellar pores (or lipid ion channels, toroidal pores) next to dynamic covalent chalcogen exchange networks as outlined in preceding figures.
Figure 17
Figure 17
Hypothetical thiol-mediated uptake mechanisms with disulfide-network candidates covering (a) transferrin receptor (TFR), (b) chloride intracellular channel protein 1 (CLIC1), (c) Ca2+-activated scramblase (TMEM16F), (d) transient receptor potential cation channel (TRPA1), (e) voltage-gated calcium channel (Cav1.1), and (f) epidermal growth factor receptor (EGFR).
Figure 18
Figure 18
Thiol-mediated uptake of HIV:DTNB-inhibitable dynamic covalent disulfide exchange between PDI and gp120 (A, B) prepares for fusion (C).
See this image and copyright information in PMC

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