Inhibitors of thiol-mediated uptake.
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ABSTRACT:
SUBMITTER: Cheng Y
PROVIDER: S-EPMC8179002 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 IC50 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.
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Project description:Oligonucleotide phosphorothioates (OPS) are DNA or RNA mimics where one phosphate oxygen is replaced by a sulfur atom. They have been shown to enter mammalian cells much more efficiently than non-modified DNA. Thus, solving one of the key challenges with oligonucleotide technology, OPS became very useful in practice, with several FDA-approved drugs on the market or in late clinical trials. However, the mechanism accounting for this facile cellular uptake is unknown. Here, we show that OPS enter cells by thiol-mediated uptake. The transient adaptive network produced by dynamic covalent pseudo-disulfide exchange is characterized in action. Inhibitors with nanomolar efficiency are provided, together with activators that reduce endosomal capture for efficient delivery of OPS into the cytosol, the site of action.
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Project description:The disulfide dihedral angle in epidithiodiketopiperazines (ETPs) is near 0°. Application of this highest possible ring tension to strain-promoted thiol-mediated uptake results in efficient delivery to the cytosol and nucleus. Compared to the previous best asparagusic acid (AspA), ring-opening disulfide exchange with ETPs occurs more efficiently even with nonactivated thiols, and the resulting thiols exchange rapidly with nonactivated disulfides. ETP-mediated cellular uptake is more than 20 times more efficient compared to AspA, occurs without endosomal capture, depends on temperature, and is "unstoppable" by inhibitors of endocytosis and conventional thiol-mediated uptake, including siRNA against the transferrin receptor. These results suggest that ETP-mediated uptake not only maximizes delivery to the cytosol and nucleus but also opens the door to a new multitarget hopping mode of action.
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Project description:With a small amount of maleimide modification on the liposome surface, enhanced cellular uptake of liposomes and drug-delivery efficiency can be obtained both in vitro and in vivo. Herein, we describe the mechanisms underlying this enhanced cellular uptake. Suppression of the cellular uptake of maleimide-modified liposomes (M-GGLG, composed of 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-glutamate [GGLG]/cholesterol/poly(ethylene glycol) - 1,2-distearoyl-sn-glycero-3-phosphoethanolamine [PEG????-DSPE]/maleimide [M]-PEG????-Glu2C18 at a molar ratio of 5:5:0.03:0.03) caused by temperature block and addition of serum was alleviated compared with that of liposomes without maleimide modification (GGLG liposomes, composed of GGLG/cholesterol/PEG????-DSPE/PEG????-Glu2C?? at a molar ratio of 5:5:0.03:0.03). When 0.01 nM N-ethylmaleimide was used to pre-block cellular thiols, the cellular uptake of M-GGLG liposomes was decreased to approximately 70% in HeLa, HCC1954, MDA-MB-468, and COS-7 cell lines. Moreover, inhibition of a thiol-related reductase such as protein disulfide isomerase resulted in a 15%-45% inhibition of the cellular uptake of M-GGLG liposomes, whereas GGLG liposomes were not influenced. Further, single and mixed inhibitors of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis did not efficiently inhibit the cellular uptake of M-GGLG liposomes. Using confocal microscopy, we verified that M-GGLG liposomes were localized partially in lysosomes after inhibition of the mentioned conventional endocytic pathways. Therefore, it was hypothesized that the mechanisms underlying the enhanced cellular uptake of liposomes by maleimide modification was thiol-mediated membrane trafficking, including endocytosis and energy-independent transport.
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Project description:The cytosolic delivery of various substrates in 3D multicellular spheroids by thiol-mediated uptake is reported. This is important because most orthodox systems, including polycationic cell-penetrating peptides, fail to deliver efficiently into deep tissue. The grand principles of supramolecular chemistry, that is the pH dependence of dynamic covalent disulfide exchange with known thiols on the transferrin receptor, are proposed to account for transcytosis into deep tissue, while the known but elusive exchange cascades along the same or other partners assure cytosolic delivery in kinetic competition. For quantitative detection in the cytosol, the 2D chloroalkane penetration assay (CAPA) is translated to 3D deep tissue. The targeted delivery of quantum dots, otherwise already troublesome in 2D culture, and the controlled release of mechanophores are realized to exemplify the power of thiol-mediated uptake into spheroids. As transporters, dithiolane quartets on streptavidin templates are introduced as modular motifs. Built from two amino acids only, the varied stereochemistry and peptide sequence are shown to cover maximal functional space with minimal structural change, i.e., constitutional isomers. Reviving a classic in peptide chemistry, this templated assembly of β quartets promises to expand streptavidin biotechnology in new directions, while the discovery of general cytosolic delivery in deep tissue as an intrinsic advantage further enhances the significance and usefulness of thiol-mediated uptake.
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Project description:Dynamic covalent exchange cascades with cellular thiols are of interest to deliver substrates to the cytosol and to inhibit the entry of viruses. The best transporters and inhibitors known today are cyclic cascade exchangers (CAXs), producing a new exchanger with every exchange, mostly cyclic oligochalcogenides, particularly disulfides. The objective of this study was to expand the dynamic covalent chalcogen exchange cascades in thiol-mediated uptake by inserting pnictogen relays. A family of pnictogen-expanded cyclic disulfides covering As(III), Sb(III), and Bi(III) is introduced. Their ability to inhibit thiol-mediated cytosolic delivery is explored with fluorescently labeled CAXs as transporters. The promise of inhibiting viral entry is assessed with SARS-CoV-2 lentiviral vectors. Oxygen-bridged seven-membered 1,3,2-dithiabismepane rings are identified as privileged scaffolds. The same holds for six-membered 1,3,2-dithiarsinane rings made from asparagusic acid and para-aminophenylarsine oxide, which are inactive or toxic when used alone. These chemically complementary Bi(III) and As(III) cascade exchangers inhibit both thiol-mediated cytosolic delivery and SARS-CoV-2 lentivector uptake at concentrations of 10 μM or lower. Crystal structures, computational models, and exchange kinetics support that lentivector entry inhibition of the contracted dithiarsinane and the expanded dithiabismepane rings coincides with exchange cascades that occur without the release of the pnictogen relay and benefit from noncovalent pnictogen bonds. The identified leads open perspectives regarding drug delivery as well as unorthodox approaches toward dynamic covalent inhibition of cellular entry.
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Project description:The purpose of this study was to determine if radiation (RT)-resistant cervical cancers are dependent upon glutamine metabolism driven by activation of the PI3K pathway and test whether PI3K pathway mutation predicts radiosensitization by inhibition of glutamine metabolism. Cervical cancer cell lines with and without PI3K pathway mutations, including SiHa and SiHa PTEN-/- cells engineered by CRISPR/Cas9, were used for mechanistic studies performed in vitro in the presence and absence of glutamine starvation and the glutaminase inhibitor, telaglenastat (CB-839). These studies included cell survival, proliferation, quantification of oxidative stress parameters, metabolic tracing with stable isotope-labeled substrates, metabolic rescue, and combination studies with L-buthionine sulfoximine (BSO), auranofin (AUR), and RT. In vivo studies of telaglenastat ± RT were performed using CaSki and SiHa xenografts grown in immune-compromised mice. PI3K-activated cervical cancer cells were selectively sensitive to glutamine deprivation through a mechanism that included thiol-mediated oxidative stress. Telaglenastat treatment decreased total glutathione pools, increased the percent glutathione disulfide, and caused clonogenic cell killing that was reversed by treatment with the thiol antioxidant, N-acetylcysteine. Telaglenastat also sensitized cells to killing by glutathione depletion with BSO, thioredoxin reductase inhibition with AUR, and RT. Glutamine-dependent PI3K-activated cervical cancer xenografts were sensitive to telaglenastat monotherapy, and telaglenastat selectively radiosensitized cervical cancer cells in vitro and in vivo These novel preclinical data support the utility of telaglenastat for glutamine-dependent radioresistant cervical cancers and demonstrate that PI3K pathway mutations may be used as a predictive biomarker for telaglenastat sensitivity.
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Project description:Integrins are cell surface proteins responsible for cell motility. Inspired by the rich disulfide exchange chemistry of integrins, we show here the inhibition of cell migration by cascade exchangers (CAXs), which also enable and inhibit cell penetration by thiol-mediated uptake. Fast-moving CAXs such as reversible Michael acceptor dimers, dithiabismepanes, and bioinspired epidithiodiketopiperazines are best, much better than Ellman's reagent. The implication that integrins participate in thiol-mediated uptake is confirmed by reduced uptake in integrin-knockdown cells. Although thiol-mediated uptake is increasingly emerging as a unifying pathway to bring matter into cells, its molecular basis is essentially unknown. These results identify the integrin superfamily as experimentally validated general cellular partners in the dynamic covalent exchange cascades that are likely to account for thiol-mediated uptake. The patterns identified testify to the complexity of the dynamic covalent networks involved. This work also provides chemistry tools to explore cell motility and expands the drug discovery potential of CAXs from antiviral toward antithrombotic and antitumor perspectives.
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Project description:DNA aptamers have been widely used as biosensors for detecting a variety of targets. Despite decades of success, they have not been applied to monitor any targets in plants, even though plants are a major platform for providing oxygen, food, and sustainable products ranging from energy fuels to chemicals, and high-value products such as pharmaceuticals. A major barrier to progress is a lack of efficient methods to deliver DNA into plant cells. We herein report a thiol-mediated uptake method that more efficiently delivers DNA into Arabidopsis and tobacco leaf cells than another state-of-the-art method, DNA nanostructures. Such a method allowed efficient delivery of a glucose DNA aptamer sensor into Arabidopsis for sensing glucose. This demonstration opens a new avenue to apply DNA aptamer sensors for functional studies of various targets, including metabolites, plant hormones, metal ions, and proteins in plants for a better understanding of the biodistribution and regulation of these species and their functions.
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Project description:Chalcogen-centered cascade exchange chemistry is increasingly understood to account for thiol-mediated uptake, that is, the ability of reversibly thiol-reactive agents to penetrate cells. Here, reversible Michael acceptors are shown to enable and inhibit thiol-mediated uptake, including the cytosolic delivery of proteins. Dynamic cyano-cinnamate dimers rival the best chalcogen-centered inhibitors. Patterns generated in inhibition heatmaps reveal contributions from halogen-bonding switches that occur independent from the thyroid transporter MCT8. The uniqueness of these patterns supports that the entry of tetrel-centered exchangers into cells differs from chalcogen-centered systems. These results expand the chemical space of thiol-mediated uptake and support the existence of a universal exchange network to bring matter into cells, abiding to be decoded for drug delivery and drug discovery in the broadest sense.
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