Inhibition of Thiol-Mediated Uptake with Irreversible Covalent Inhibitors

Thiol-mediated uptake is emerging as method of choice to penetrate cells. This study focuses on irreversible covalent inhibitors of thiol-mediated uptake. High-content high-throughput screening of the so far largest collection of hypervalent iodine reagents affords inhibitors that are more than 250 times more active than Ellman’s reagent and rival the best dynamic covalent inhibitors. Comparison with other irreversible reagents reveals that inhibition within one series follows reactivity, whereas inhibition across series deviates from reactivity. These trends support that molecular recognition, besides dynamic covalent exchange, contributes significantly to thiol-mediated uptake. The most powerful inhibitors besides the best hypervalent iodine reagents were Fukuyama’s nosyl protecting group and super-cinnamaldehydes that have been introduced as irreversible activators of the pain receptor TRPA1. Considering that several viruses use different forms of thiol-mediated uptake to enter cells, the identification of new irreversible inhibitors of thiol-mediated uptake is of general interest for the discovery of new antivirals.     

Novel route to size-controlled Fe-MIL-88B-NH2 metal-organic framework nanocrystals

A new approach for the synthesis of uniform metal-organic framework (MOF) nanocrystals with controlled sizes and aspect ratios has been developed using simultaneously the non-ionic triblock co-polymer F127 and acetic acid as stabilizing and deprotonating agents, respectively. The alkylene oxide segments of the triblock co-polymer can coordinate with metal ions and stabilize MOF nuclei in the early stage of the formation of MOF nanocrystals. Acetic acid can control the deprotonation of carboxylic linkers during the synthesis and, thus, enables the control of the rate of nucleation, leading to the tailoring of the size and aspect ratio (length/width) of nanocrystals. Fe-MIL-88B-NH(2), as an iron-based MOF crystal, was selected as a typical example to illustrate our approach. The results reveal that this approach is used for not only the synthesis of uniform nanocrystals but also the control of the size and aspect ratio of the materials. The size and aspect ratio of nanocrystals increase with an increase in the concentration of acetic acid in the synthetic mixture. The non-ionic triblock co-polymer F127 and acetic acid can be easily removed from the Fe-MIL-88B-NH(2) nanocrystal products by washing with ethanol, and thus, their amine groups are available for practical applications. The approach is expected to synthesize various nanosized carboxylate-based MOF members, such as MIL-53, MIL-89, MIL-100, and MIL-101.     

Coherent potential approximation study of the Mott transition in optical lattice system with site-dependent interactions

We study the Mott transition in the half-filled Hubbard model with spatially alternating interactions by means of the coherent potential approximation. The phase boundary between metallic and insulating phases at zero temperature is derived and the nature of the Mott states is also considered. Our results are in good agreement with the ones recently obtained by the two-site dynamical mean-field theory.     

Microfabrication of stacks of acoustic matching layers for 15 MHz ultrasonic transducers

This paper presents a novel method used to manufacture stacks of multiple matching layers for 15 MHz piezoelectric ultrasonic transducers, using fabrication technology derived from the MEMS industry. The acoustic matching layers were made on a silicon wafer substrate using micromachining techniques, i.e., lithography and etch, to design silicon and polymer layers with the desired acoustic properties. Two matching layer configurations were tested: a double layer structure consisting of a silicon–polymer composite and polymer and a triple layer structure consisting of silicon, composite, and polymer. The composite is a biphase material of silicon and polymer in 2-2 connectivity. The matching layers were manufactured by anisotropic wet etch of a (1 1 0)-oriented Silicon-on-Insulator wafer. The wafer was etched by KOH 40 wt%, to form 83 μm deep and 4.5 mm long trenches that were subsequently filled with Spurr’s epoxy, which has acoustic impedance 2.4 MRayl. This resulted in a stack of three layers: The silicon substrate, a silicon–polymer composite intermediate layer, and a polymer layer on the top. The stacks were bonded to PZT disks to form acoustic transducers and the acoustic performance of the fabricated transducers was tested in a pulse-echo setup, where center frequency, −6 dB relative bandwidth and insertion loss were measured. The transducer with two matching layers was measured to have a relative bandwidth of 70%, two-way insertion loss 18.4 dB and pulse length 196 ns. The transducers with three matching layers had fractional bandwidths from 90% to 93%, two-way insertion loss ranging from 18.3 to 25.4 dB, and pulse lengths 326 and 446 ns. The long pulse lengths of the transducers with three matching layers were attributed to ripple in the passband.     

Soft magnetic properties and giant magneto-impedance effect of Fe73.5−xCrxSi13.5B9Nb3Au1 (x=1–5) alloys

In this paper, the effect of microstructural and surface morphological developments on the soft magnetic properties and giant magneto-impedance (GMI) effect of Fe_73.5−xCr_xSi_13.5B₉Nb₃Au₁ (x=1, 2, 3, 4, 5) alloys was investigated. It was found that the Cr addition causes slight decrease in the mean grain size of α-Fe(Si) grains. AFM results indicated a large variation of surface morphology of density and size of protrusions along the ribbon plane due to structural changes caused by thermal treatments with increasing Cr content. Ultrasoft magnetic properties such as the increase of magnetic permeability and the decrease of coercivity were observed in the samples annealed at 540 °C for 30 min. Accordingly, the GMI effect was also observed in the annealed samples.     

Methodological developments in overpressured-layer chromatography

Summary Since its introduction around 1977, OPLC has found widespread application and its methodology has been further developed. A possible development is gradient elution. Different continuous development methods (e.g. with eluent changeover, multistep gradient) and the possible system configurations for their execution are discussed. The advantages of the newly developed continuous development techniques are compared to earlier data. The most versatile method is demonstrated on lipid samples derived from human plasma, giving separation of six lipid classes and intra-class separation of the most polar classes in the same run.     

Detection of pathogenic microorganisms using biosensor based on multi-walled carbon nanotubes dispersed in DNA solution

The present paper introduces a facile and cost-effective route for the direct dispersion of multi-walled carbon nanotubes (MWCNTs) in DNA solution. Their application in detecting Escherichia coli O157:H7 using DNA biosensor was demonstrated. The dispersion state of the MWCNTs was characterized via UV–Vis spectroscopy, transmission electron microscopy, and field emission scanning electron microscopy. The interaction between DNA sequence and the MWCNTs was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy. As-obtained MWCNT solution was used in the preparation of DNA sensor. Results revealed that the developed DNA sensor can detect a DNA target as low as 1 nM in a buffer solution. The sensitivity of the DNA sensor reached approximately 0.19 nM/mV. The effect of dispersion parameters, including pH values, DNA concentration, ion strength, and sonication time, on sensor response was also studied. The DNA sensor can respond well to 120 min of sonication time, a pH value of 9, and 20 μM of DNA sequence concentration. The results of the present study showed a potential application of the DNA sensor in the detection of Escherichia coli O157:H7.     

Inhibitors of thiol-mediated uptake

Ellman''s reagent has caused substantial confusion and concern as a probe for thiol-mediated uptake because it is the only established inhibitor available but works neither efficiently nor reliably. Here we use fluorescent cyclic oligochalcogenides that enter cells by thiol-mediated uptake to systematically screen for more potent inhibitors, including epidithiodiketopiperazines, benzopolysulfanes, disulfide-bridged γ-turned peptides, heteroaromatic sulfones and cyclic thiosulfonates, thiosulfinates and disulfides. With nanomolar activity, the best inhibitors identified are more than 5000 times better than Ellman''s reagent. Different activities found with different reporters reveal thiol-mediated uptake as a complex multitarget process. Preliminary results on the inhibition of the cellular uptake of pseudo-lentivectors expressing SARS-CoV-2 spike protein do not exclude potential of efficient inhibitors of thiol-mediated uptake for the development of new antivirals.

Thiol-reactive inhibitors for the cellular entry of cyclic oligochalcogenide (COC) transporters and SARS-CoV-2 spike pseudo-lentivirus are reported.

Thiol-mediated uptake^1–10 has been developed to explain surprisingly efficient cellular uptake of substrates attached to thiol-reactive groups, most notably disulfides. The key step of this mechanism is the dynamic covalent thiol-disulfide exchange between disulfides of the substrates and exofacial thiols on cell surfaces (Fig. 1). The covalently bound substrate then enters the cell either by fusion, endocytosis, or direct translocation across the plasma membrane into the cytosol. Thiol-disulfide exchange has been confirmed to play an essential role in the cellular entry of some viruses^1,11–14 and toxins.² Indeed, diphtheria toxin and HIV were among the first to be recognized to enter cells via thiol-mediated uptake.^1,2 The involvement of cell-surface thiols in cellular uptake is most often probed by inhibition with Ellman''s reagent (DTNB). However, this test is not always reliable, in part due to the comparably poor reactivity of DTNB, and the comparably high reactivity of the disulfide obtained as a product. Thus, the importance of thiol-mediated uptake for viral entry and beyond remains, at least in part, unclear.Open in a separate windowFig. 1In thiol-mediated uptake, dynamic covalent exchange with thiols on the cell surface precedes entry through different mechanisms. Inhibition of thiol-mediated uptake by removal of exofacial thiols and disulfides could thus afford new antivirals.We became interested in thiol-mediated uptake^3–5 while studying the cytosolic delivery of substrates such as drugs, probes and also larger objects like proteins or quantum dots with cell-penetrating poly(disulfide)s.⁶ Our recent focus shifted to cyclic oligochalcogenides (COCs) to increase speed and selectivity of dynamic covalent thiol-oligochalcogenide exchange, and, most importantly, to assure reversibility, i.e., mobility during uptake, with a covalently tethered, intramolecular leaving group.⁷ With increasingly unorthodox COC chemistry, from strained disulfides^7,8 and diselenides⁹ to adaptive dynamic covalent networks produced by polysulfanes,¹⁰ uptake activities steadily increased. Their high activities suggested that the same, or complementary, COCs could also function as powerful inhibitors of thiol-mediated uptake that ultimately might perhaps lead to antivirals. In the following, this hypothesis is developed further.Fluorescently labeled COCs 1⁸ and 2¹⁰ were selected as reporters for the screening of thiol-mediated uptake inhibitors because of their high activity, their destination in the cytosol, and their different characteristics (Fig. 2). The COC in 1 is an epidithiodiketopiperazine (ETP). With a CSSC dihedral angle ∼0°, ETPs drive ring tension to the extreme.^15,16 Ring-opening thiol-disulfide exchange is ultrafast, and the released thiols are acidic enough to continue exchanging in neutral water, including ring closure.⁸ This unique exchange chemistry coincides with efficient cellular uptake and poor retention on thiol affinity columns.⁸Open in a separate windowFig. 2Structure of reporters 1 and 2 and inhibitor candidates 3–30 with their concentrations needed to inhibit by ∼15% (MIC) the uptake of 1 (1 h pre-incubation with inhibitors, 30 min incubation with reporter, filled symbols) and 2 (4 h pre-incubation, empty symbols). Red squares: ETPs; orange circles: BPSs; blue upward triangles: heteroaromatic sulfones; purple diamonds: thiosulfonates; magenta downward triangles: di- and polysulfides; brown hexagons: thiosulfinates. Symbols with upward arrows: MIC not reached at the highest concentration tested. Symbols with downward arrows indicate the lowest concentration tested already exceeds the MIC. (a) Similarly active upon co-incubation of reporters and inhibitor; (b–d) similarly (b), less (c), or more (d) active upon co-incubation in the presence of serum (mostly 6 h); (e) pre-incubation for 15 min; (f) isomerizes into cis22; (g) V-shaped DRC (see Fig. 3f); (h) pre-incubation for 30 min, co-incubation with 2; (i) mixture of regioisomers.The COC in 2 is a benzopolysulfane (BPS). Like ETPs, BPSs occur in natural products and have inspired total synthesis.¹⁷ Unlike ETPs, BPSs are not strained but evolve into adaptive networks of extreme sulfur species for cells to select from. Uptake efficiencies and retention on thiol affinity columns exceed other COCs clearly.^10,18With COCs 1 and 2 as cell-penetrating reporters, a fully automated, fluorescent microscopy image-based high-content high-throughput (HCHT)¹⁹ inhibitor screening assay was developed. HeLa cells in multiwell plates are incubated with a reporter at constant and inhibitors at varying concentrations and incubation times. Hindered reporter uptake then causes decrease of fluorescence inside of cells (Fig. 3a). Automated data analysis¹⁹ was established to extract average fluorescence intensity per cell and, at the same time, cell viability from propidium iodide negative nuclei count (Fig. 3 and S3–S6^†). Standard assay conditions consisted of pre-incubation of HeLa cells with inhibitors for different periods of time, followed by the removal of inhibitors and the addition of reporters, thus excluding possible interactions between the two in the extracellular environment. In alternative co-incubation conditions, inhibitors were not removed before the addition of reporters to allow for eventual interactions between the two.Open in a separate windowFig. 3(a) Fluorescence image of HCHT plates (4 images per well) with HeLa cells pre-incubated with 6 (30 min) followed by co-incubation with 1 (left) and 2 (right, 10 μM each) for constant 30 min. (b–f) HCHT data showing relative fluorescence intensity (filled symbols) and cell viability (empty symbols) of HeLa cells after (b) pre-incubation with 4 for 1 h, followed by washing and incubation with 1 (top), or pre-incubation with 4 for 30 min, followed by co-incubation with 4 and 2 (bottom). (c) As in (b) with 18. (d) As in (b) after incubation for 4 h with 16 followed by incubation with 2. (e) As in (b) after pre-incubation with 11 (circles), 14 (crosses), or 21 (diamonds) for 15 min, followed by washing and incubation with 1. (f) As in (b) after pre-incubation with 20 (30 min), followed by washing and incubation with 1.Among the very high number of thiol-reactive probes, compounds 3–30 were selected based on promise, experience, availability and accessibility. Main focus was on COCs offering increasingly extreme sulfur chemistry because dynamic covalent thiol-oligochalcogenide exchange with different intramolecular leaving groups promises access to different exchange cascades for the intramolecular and, perhaps, also intermolecular crosslinking of the target proteins. More hydrophilic, often anionic COCs were preferred to prevent diffusion into cells and thus minimize toxicity. The expectation was that from such a sketchy outline of an immense chemical space, leads could be identified for future, more systematic exploration. Reporters 1 and 2 and candidates 3–30 were prepared by substantial multistep synthesis (Schemes S1–S11 and Fig. S47–S93,^† commercially available: 20, 25, 30). Inhibitors were numbered in the order of efficiency against reporter 1, evaluated by their minimum inhibitory concentrations (MICs), i.e., concentrations that cause a ∼15% reduction of reporter uptake in cells (Fig. 2 and Tables S1–S37^†). We chose to use MICs because half-maximal inhibitions could not always be reached due to the onset of toxicity, formally anticooperative, or even V-shaped dose–response curves (DRCs, e.g., Fig. 3b–f, all DRCs can be found in the ESI, Fig. S7–S43^†). MICs are usually below the half-maximal cell growth inhibition concentration (GI₅₀, Tables S1–S37^†).Among the most potent inhibitors of ETP reporter 1 were ETPs 4 and 5 (Fig. 2, ​,3b).3b). This intriguing self-inhibition was even surpassed by the expanded cyclic tetrasulfide ETP₄3 (MIC < 0.1 μM), which was of interest because they are much poorer transporters.¹⁰ Further formal ring expansion leads to cyclic pentasulfides BPS₅6 as equally outstanding inhibitors (MIC ≈ 0.3 μM). This trend toward the adaptive networks, reminiscent of elemental sulfur chemistry, did not extend toward inorganic polysulfides 13 (MIC ≈ 20 μM). ETPs 4 and 5 were sensitive to modification of the carboxylate, with the cationic 12 being the worst (MIC ≈ 30 μM) and the neutral glucose hemiacetal 7 the most promising (MIC ≈ 0.5 μM).Although this study focuses on increasingly extreme dynamic covalent COC chemistry, the inclusion of one example for covalent C–S bond formation was of interest for comparison. The classical iodoacetamides⁷ and maleimides⁴ were more toxic than active (not shown). However, nucleophilic aromatic substitution of heteroaromatic sulfones,²⁰ just developed for the efficient bioorthogonal conversion of thiols into sulfides, was more promising. Weaker than dynamic covalent COCs, this irreversible inhibition was best with benzoxazole 11 (MIC ≈ 15 μM) and decreased in accordance with reactivity toward free thiols to oxadiazole 14 and benzothiazole 21 (MIC ≈ 300 μM, Fig. 3e).At constant pH, Ellman''s reagent 20 was confirmed to be erratic also in this assay. The DRC showed minor inhibition up to around 2 mM, which disappeared again at higher concentrations (Fig. 3f). Other cyclic disulfides were inactive as well (28–30). Also disappointing were oxidized disulfides, that is thiosulfinates, including allicin 25, the main odorant component of garlic,^21,22 oxidized cystine 26 and oxidized lipoic acid 27. Thiosulfinates were of interest because they should selectively target the vicinal thiols of reduced disulfides bridges, producing two disulfides.²³ The most active trans dithioerythrol (DTE) thiosulfinate 17 isomerized with time into the less active, hydrogen-bonded cis isomer 22 (Fig. S46^†).Reporter 2 was more difficult to inhibit than 1, as expected from high activity with extreme retention on thiol affinity columns.^10,18 For instance, BPS 6 was very efficient against ETP 1 but much less active against BPS 2 (Fig. 3a), although longer pre-incubation could lower the MIC down to 4 μM (Fig. 2, S41^†). The complementary ETP 4 “self-inhibited” ETP 1 but was also unable to inhibit BPS 2 as efficiently (Fig. 3b). Among the best inhibitors of BPS 2 upon co-incubation were disulfide bridged γ-turn²⁴ peptides 18 and 19 (MIC ≈ 5 μM), both less active against 1 (MIC ≈ 300 μM, Fig. 3c). Disulfide-bridged γ-turn CXC peptides consist of an 11-membered ring with significant Prelog strain. They were introduced by Wu and coworkers as transporters for efficient cytosolic delivery.⁵ The cyclic thiosulfonates 15 and 16 showed promising activities against both 1 and 2, and were tolerant toward the presence of serum (Fig. 2d, S33 and S42^†). Contrary to thiosulfinate 27, the oxidation of lipoic acid to pure thiosulfonates was not successful so far. However, weakly detectable activity of the lipoyl-glutamate conjugate oxidized to the thiosulfinate (MIC ≈ 350 μM, not shown) compared to the inactive thiosulfinate 27 implied that lipoic acid oxidized to the thiosulfonate would also be less active than the glutamate conjugate 15.The oxidized DTE 16^25–28 was particularly intriguing because it was more potent against 2 and could achieve nearly complete inhibition (MIC ∼ 20 μM, Fig. 3d). Highly selective for thiols, the cyclic thiosulfonate 16 was stable for weeks at room temperature, without precaution, in all solvents tested. The disulfides and sulfinates obtained from exchange with thiols were stable as well, and the latter can further react with disulfides²⁷ for intramolecular or eventually intermolecular crosslinking of the target proteins.The overall mismatched inhibition profiles found for reporters 1 and 2 supported that thiol-mediated uptake proceeds through a series of at least partially uncoupled parallel multitarget systems instead of a specific single protein or membrane target. From proteomics studies with cysteine-reactive irreversible probes, it is known that different probes generally target different proteins.^29b Proteomics analysis^29a for asparagusic acid derived transporters supports the involvement of many targets beyond the commonly considered protein disulfide isomerases and the confirmed transferrin receptor.^{12–14,26–30} The unusual, formally anti-cooperative (Hill coefficients < 1) DRCs further supported thiol-mediated uptake as complex multitarget systems.Despite the complexity of these systems, results did not much depend on assay conditions. Compared to the standard protocol of pre-incubation with inhibitors followed by inhibitor removal and incubation with reporters 1 or 2 for detection, the co-incubation protocol, in which pre-incubation with inhibitors is followed by co-incubation with reporters 1 or 2 without inhibitor removal, gave reasonably similar results (Fig. 2). Inhibition characteristics naturally depended on pre-incubation time, with weaker activities at shorter and longer times, reflecting incomplete exchange and cellular response or other ways of inhibitor destruction, respectively. The presence of serum also did not affect the activities much (Fig. 2b–d).Preliminary studies on antiviral activity were performed with pseudo-lentivectors³¹ that express the D614G mutant¹¹ of the SARS-CoV-2 spike protein and code for a luciferase reporter gene, which is expressed by the infected cells.¹² A549 human lung alveolar basal epithelium cell line constitutively overexpressing ACE2 and TMPRSS2 was selected to facilitate the entry of the SARS-CoV-2 spike pseudo-lentivirus. The most significant activities were found for DTE thiosulfonate 16 with an IC₅₀ around 50 μM, while toxicity was detected only at 500 μM (Fig. S44^†). The onset of inhibition could be observed for tetrasulfide ETP 3 at 50 μM, but it coincided with the appearance of cytotoxicity. Protease inhibition is less likely to be the mode of action, as similar activity was found with wild type A549 cells transduced with a standard lentivirus expressing vesicular-stomatitis virus G surface protein VSVG (Fig. S45^†).¹³ Short incubation times of cells and inhibitors before the addition of viruses disfavored contributions from changes in gene expression. More detailed studies are ongoing.The lessons learned from this study are that, firstly, thiol-mediated uptake can be inhibited efficiently by thiol-reactive reagents, confirming that thiol-mediated uptake exists and transporters like ETP 1 and BPS 2 do not simply diffuse into cells; the best inhibitors are more than 5000 times better than Ellman''s reagent. Secondly, inhibitor efficiencies vary with the transporters, supporting that thiol-mediated uptake operates as a complex multitarget system. The best inhibitors are COCs that operate with fast dynamic covalent exchange, suggesting that the reversibility provided by COCs is important. The inhibition of thiol-mediated uptake might contribute to activities of thiol-reactive antivirals such as 16, ETPs or ebselen, although they have been shown to bind to zinc fingers or inhibit proteases.^{16,25,32–34} Finally, the inhibitors reported here could also be of interest for delivery applications and might be worth investigation with regard to antiviral activity. We currently plan to focus more systematically on the most promising leads within COCs, particularly cyclic thiosulfonates, and to expand the screening campaign toward new attractive motifs.^33–35     

Characterization and antimicrobial activity of silver nanoparticles prepared by a thermal decomposition technique

Recently, there has been an increasing need of efficient synthetic protocols using eco-friendly conditions including low costs and green chemicals for production of metal nanoparticles. In this work, silver nanoparticles (silver NPs) with average particle size about 10 nm were synthesized by using a thermal decomposition technique. Unlike the colloidal chemistry method, the thermal decomposition method developed has advantages such as the high crystallinity, single-reaction synthesis, and easy dispersion ability of the synthesized NPs in organic solvents. In a modified synthesis process, we used sodium oleate as a capping agent to modify the surface of silver NPs because the oleate has a C₁₈ tail with a double bond in the middle, therefore, forming a kink which is to be effective for aggregative stability. Importantly, the as-synthesized silver NPs have demonstrated strong antimicrobial effects against various bacteria and fungi strains. Electron microscopic studies reveal physical insights into the interaction and bactericidal mechanism between the prepared silver NPs and tested bacteria in question. The observed excellent antibacterial and antifungal activity of the silver NPs make them ideal for disinfection and biomedicine applications.