Inhibition of endocytic uptake of severe acute respiratory syndrome coronavirus 2 and endo-lysosomal acidification by diphenoxylate.

Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.

Discovery of (E)-2-(hydroxyimino)-N-(2 ((4methylpentyl)amino)ethyl)acetamide (KR-27425) as a non-pyridinium oxime reactivator of paraoxon-inhibited acetylcholinesterase.

This study aimed to explore non-pyridinium oxime acetylcholinesterase (AChE) reactivators that could hold the potential to overcome the limitations of the currently available compounds used in the clinic to treat the neurologic manifestations induced by intoxication with organophosphorus agents. Fifteen compounds with various non-pyridinium oxime moieties were evaluated for AChE activity at different concentrations, including aldoximes, ketoximes, and α-ketoaldoximes. The therapeutic potential of the oxime compounds was evaluated by assessing their ability to reactivate AChE inhibited by paraoxon. Among the tested compounds, α-Ketoaldoxime derivative 13 showed the highest reactivation (%) reaching 67 % and 60 % AChE reactivation when evaluated against OP-inhibited electric eel AChE at concentrations of 1,000 and 100 µM, respectively. Compound 13 showed a comparable reactivation ability of AChE (60 %) compared to that of pralidoxime (56 %) at concentrations of 100 µM. Molecular docking simulation of the most active compounds 12 and 13 was conducted to predict the binding mode of the reactivation of electric eel AChE. As a result, a non-pyridinium oxime moiety 13, is a potential reactivator of OP-inhibited AChE and is taken as a lead compound for the development of novel AChE reactivators with enhanced capacity to freely cross the blood-brain barrier.

Radicicol Inhibits Chikungunya Virus Replication by Targeting Nonstructural Protein 2.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes a debilitating febrile illness characterized by persistent muscle and joint pain. The widespread distribution of transmission-competent vectors, Aedes species mosquitoes, indicates the potential risk of large-scale epidemics with high attack rates that can severely impact public health globally. Despite this, currently, there are no antivirals available for the treatment of CHIKV infections. Thus, we aimed to identify potential drug candidates by screening a chemical library using a cytopathic effect-based high-throughput screening assay. As a result, we identified radicicol, a heat shock protein 90 (Hsp90) inhibitor that effectively suppressed CHIKV replication by blocking the synthesis of both positive- and negative-strand viral RNA as well as expression of viral proteins. Interestingly, selection for viral drug-resistant variants and mutational studies revealed nonstructural protein 2 (nsP2) as a putative molecular target of radicicol. Moreover, coimmunoprecipitation and in silico modeling analyses determined that G641D mutation in the methyltransferase (MT)-like domain of nsP2 is essential for its interaction with cytoplasmic Hsp90ß chaperone. Our findings collectively support the potential application of radicicol as an anti-CHIKV agent. The detailed study of the underlying mechanism of action further contributes to our understanding of virus-host interactions for novel therapeutics against CHIKV infection.

Identification of non-covalent 3C-like protease inhibitors against severe acute respiratory syndrome coronavirus-2 via virtual screening of a Korean compound library.

The outbreak of coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has turned into a pandemic. The enzyme 3C-like protease (3CLpro) is essential for the maturation of viral polyproteins in SARS-CoV-2 and is therefore regarded as a key drug target for treating the disease. To identify 3CLpro inhibitors that can suppress SARS-CoV-2 replication, we performed a virtual screening of 500,282 compounds in a Korean compound bank. We then subjected the top computational hits to inhibitory assays against 3CLpro in vitro, leading to the identification of a class of non-covalent inhibitors. Among these inhibitors, compound 7 showed an EC50 of 39.89 µM against SARS-CoV-2 and CC50 of 453.5 µM. This study provides candidates for the optimization of potent 3CLpro inhibitors showing antiviral effects against SARS-CoV-2.

Comparison of Antiviral Activity of Gemcitabine with 2'-Fluoro-2'-Deoxycytidine and Combination Therapy with Remdesivir against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. The virus still spreads globally through human-to-human transmission. Nevertheless, there are no specific treatments clinically approved. This study aimed to compare antiviral activity of gemcitabine and its analogue 2'-fluoro-2'-deoxycytidine (2FdC) against SARS-CoV-2 as well as cytotoxicity in vitro. Fluorescent image-based antiviral assays revealed that gemcitabine was highly potent, with a 50% effective concentration (EC50) of 1.2 µM, more active than the well-known nucleoside monophosphate remdesivir (EC50 = 35.4 µM). In contrast, 2FdC was marginally active (EC50 = 175.2 µM). For all three compounds, the 50% cytotoxic concentration (CC50) values were over 300 µM toward Vero CCL-81 cells. Western blot and quantitative reverse-transcription polymerase chain reaction analyses verified that gemcitabine blocked viral protein expression in virus-infected cells, not only Vero CCL-81 cells but also Calu-3 human lung epithelial cells in a dose-dependent manner. It was found that gemcitabine has a synergistic effect when combined with remdesivir. This report suggests that the difluoro group of gemcitabine is critical for the antiviral activity and that its combination with other evaluated antiviral drugs, such as remdesivir, could be a desirable option to treat SARS-CoV-2 infection.

Systematic editing of synthetic RIG-I ligands to produce effective antiviral and anti-tumor RNA immunotherapies.

Retinoic acid-inducible gene I (RIG-I) recognizes double-stranded viral RNAs (dsRNAs) containing two or three 5' phosphates. A few reports of 5'-PPP-independent RIG-I agonists have emerged, but little is known about the molecular principles underlying their recognition. We recently found that the bent duplex RNA from the influenza A panhandle promoter activates RIG-I even in the absence of a 5'-triphosphate moiety. Here, we report that non-canonical synthetic RNA oligonucleotides containing G-U wobble base pairs that form a bent helix can exert RIG-I-mediated antiviral and anti-tumor effects in a sequence- and site-dependent manner. We present synthetic RNAs that have been systematically modified to enhance their efficacy and we outline the basic principles for engineering RIG-I agonists applicable to immunotherapy.

Salinomycin Inhibits Influenza Virus Infection by Disrupting Endosomal Acidification and Viral Matrix Protein 2 Function.

Screening of chemical libraries with 2,000 synthetic compounds identified salinomycin as a hit against influenza A and B viruses, with 50% effective concentrations ranging from 0.4 to 4.3 µM in cells. This compound is a carboxylic polyether ionophore that exchanges monovalent ions for protons across lipid bilayer membranes. Monitoring the time course of viral infection showed that salinomycin blocked nuclear migration of viral nuclear protein (NP), the most abundant component of the viral ribonucleoprotein (vRNP) complex. It caused cytoplasmic accumulation of NP, particularly within perinuclear endosomes, during virus entry. This was primarily associated with failure to acidify the endosomal-lysosomal compartments. Similar to the case with amantadine (AMT), proton channel activity of viral matrix protein 2 (M2) was blocked by salinomycin. Using purified retroviral Gag-based virus-like particles (VLPs) with M2, it was proved that salinomycin directly affects the kinetics of a proton influx into the particles but in a manner different from that of AMT. Notably, oral administration of salinomycin together with the neuraminidase inhibitor oseltamivir phosphate (OSV-P) led to enhanced antiviral effect over that with either compound used alone in influenza A virus-infected mouse models. These results provide a new paradigm for developing antivirals and their combination therapy that control both host and viral factors.IMPORTANCE Influenza virus is a main cause of viral respiratory infection in humans as well as animals, occasionally with high mortality. Circulation of influenza viruses resistant to the matrix protein 2 (M2) inhibitor, amantadine, is highly prevalent. Moreover, the frequency of detection of viruses resistant to the neuraminidase inhibitors, including oseltamivir phosphate (OSV-P) or zanamivir, is also increasing. These issues highlight the need for discovery of new antiviral agents with different mechanisms. Salinomycin as the monovalent cation-proton antiporter exhibited consistent inhibitory effects against influenza A and B viruses. It plays multifunctional roles by blocking endosomal acidification and by inactivating the proton transport function of M2, the key steps for influenza virus uncoating. Notably, salinomycin resulted in marked therapeutic effects in influenza virus-infected mice when combined with OSV-P, suggesting that its chemical derivatives could be developed as an adjuvant antiviral therapy to treat influenza infections resistant or less sensitive to existing drugs.

Novel tacrine-pyridinium hybrid reactivators of organophosphorus-inhibited acetylcholinesterase: Synthesis, molecular docking, and in vitro reactivation study.

First-line medical treatment against nerve agents consists of co-administration of anticholinergic agents and oxime reactivators, which reactivate inhibited AChE. Pralidoxime, a commonly used oxime reactivator, is effective against some nerve agents but not against others; thus, new oxime reactivators are needed. Novel tacrine-pyridinium hybrid reactivators in which 4-pyridinealdoxime derivatives are connected to tacrine moieties by linear carbon chains of different lengths (C2-C7) were prepared (Scheme 1, 5a-f). Their binding affinities to electric eel AChE were tested because oximes can inhibit free AChE, and the highest AChE activity (95%, 92%, and 90%) was observed at 1 µM concentrations of the oximes (5a, 5b, and 5c, respectively). Based on their inhibitory affinities towards free AChE, 1 µM concentrations of the oxime derivatives (5) were used to examine reactivation of paraoxon-inhibited AChE. Reactivation ability increased as the carbon linker chains lengthened (n = 2-5), and 5c and 5d showed remarkable reactivation ability (41%) compared to that of 2-PAM (16%) and HI-6 (4%) against paraoxon-inhibited electric eel AChE at 1 µM concentrations. Molecular docking simulation showed that the most stable binding free energy was observed in 5c at 73.79 kcal⋅mol-1, and the binding mode of 5c is acceptable for the oxygen atom of oximate to attack the phosphorus atom of paraoxon and reactivate paraoxon-inhibited eel AChE model structure.

Tunable channel drop filters consisting of a tilted Bragg grating and a mode sorting polymer waveguide.

Optical wavelength filters with large tuning range and narrow bandwidth are crucial for enhancing the capability of WDM communication systems. A polymeric tunable filter for C-band, comprising a tilted Bragg grating and a mode sorting waveguide junction is proposed in this work. For dropping a certain wavelength signal, the tilted Bragg grating reflects an odd mode into an even mode and then the reflected even mode propagates towards an output port of the asymmetric Y-junction due to the mode sorting. Consequently, the output port is separated from the input port, which is not possible in an ordinary Bragg reflector. The tilted Bragg reflector with an odd-even mode coupling efficiency of 61% exhibited a maximum reflectivity of 95% for a grating of 6 mm. A linear wavelength tuning of over 10 nm was achieved for an applied thermal power of 312 mW.

Antiviral activity of micafungin against enterovirus 71.

BACKGROUND: Enterovirus 71 (EV71) is a major causative agent of hand-foot-mouth disease (HFMD) and also causes severe neurological complications, leading to fatality in young children. However, no effective therapy is currently available for the treatment of this infection. METHODS: We identified small-molecule inhibitors of EV71 from a screen of 968 Food and Drug Administration (FDA)-approved drugs, with which clinical application for EV71-associated diseases would be more feasible, using EV71 subgenomic replicon system. Primary hits were extensively evaluated for their antiviral activities in EV71-infected cells. RESULTS: We identified micafungin, an echinocandin antifungal drug, as a novel inhibitor of EV71. Micafungin potently inhibits the proliferation of EV71 as well as the replication of EV71 replicon in cells with a low micromolar IC50 (~5 µM). The strong antiviral effect of micafungin on EV71 replicon and the result from time-of-addition experiment demonstrated a targeting of micafungin on virion-independent intracellular process(es) during EV71 infection. Moreover, an extensive analysis excluded the involvement of 2C and 3A proteins, IRES-dependent translation, and also that of polyprotein processing in the antiviral effect of micafungin. CONCLUSIONS: Our research revealed a new indication of micafungin as an effective inhibitor of EV71, which is the first case reporting antiviral activity of micafungin, an antifungal drug.

Surface relief apodized grating tunable filters produced by using a shadow mask.

To produce a compact low-cost tunable filter required for WDM optical communications, a polymeric Bragg reflection filter with an apodized grating structure is proposed. A high-contrast polymeric waveguide is incorporated in order to obtain high reflectivity from a short Bragg grating. To overcome the bandwidth broadening, an apodized grating with a gradually changing depth of surface relief grating along the propagation direction is fabricated through the dry etching with a shadow mask. The apodized polymer grating exhibits 3-dB, 20-dB bandwidths of 0.36 nm, and 0.72 nm, respectively with a 95% reflection. The reflection wavelength is tunable over 14 nm for an applied thermal power of 500 mW.

Tunable channel-drop filters consisting of polymeric Bragg reflectors and a mode sorting asymmetric X-junction.

A tunable channel-drop filter as essential component for the wavelength-division-multiplexing optical communication system has been demonstrated, which is based on polymer waveguide Bragg reflectors. For an ordinary Bragg reflector, the filtered signal is reflected toward the input waveguide. Thus an external circulator is required to separate the filtered signal from the input port, though it increases the total footprint and cost. For this purpose, we employed dual Bragg reflectors and a mode sorting asymmetric X-junction. The Bragg reflector exhibited a maximum reflectivity of 94% for a 6-mm long grating, a 3-dB bandwidth of 0.39 nm and a 20-dB bandwidth of 2.6 nm. The mode sorting crosstalk in asymmetric X-junction was less than -20 dB, and linear wavelength tuning was achieved over 10 nm at the applied thermal power of 377 mW.

Broadband silicon optical modulator using a graphene-integrated hybrid plasmonic waveguide.

Graphene is an excellent electronic and photonic material for developing electronic-photonic integrated circuits in Si-based semiconductor devices with ultra wide operational bandwidth. As an extended application, here we propose a broadband silicon optical modulator using a graphene-integrated hybrid plasmonic waveguide, and investigate the optical characteristics numerically at a wavelength of 1.55 µm. The optical device is based on the surface plasmon polariton absorption of graphene. By electrically tuning the graphene's refractive index as low as that of a noble metal, the hybrid plasmonic waveguide supports a strongly confined highly lossy hybrid long-range surface plasmon polariton strip mode, and hence light coupled from an input waveguide experiences significant power attenuation as it propagates along the waveguide. Over the entire C-band from 1.530 to 1.565 µm wavelengths, the on/off extinction ratio is larger than 13.7 dB. This modulator has the potential to play a key role in realizing graphene-Si waveguide-based integrated photonic devices.

Arrayed waveguide collimators for integrating free-space optics on polymeric waveguide devices.

Array-type optical devices are important for wavelength-division multiplexing optical communication system to achieve small footprint, mass production, and reliability. For fabricating transmitter module in an array configuration, it is difficult to achieve a passive alignment of isolator, collimating lens, and laser diode. To facilitate array isolator integration, a waveguide collimator is proposed in this work by using a low-contrast, large-core polymer waveguide. The diffraction of a guided mode propagating through a free-space region is suppressed by enlarging the guided mode. The fiber coupling loss due to the enlarged mode was overcome by incorporating an adiabatic taper structure. The excess loss of waveguide collimator including the loss through a 400-µm free-propagation region was less than 1.0 dB.

Investigation of 90° submicrometer radius bends of metal-insulator-silicon-insulator-metal waveguides.

We theoretically and experimentally investigate 90° submicrometer radius bends (SRB) of metal-insulator-silicon-insulator-metal (MISIM) waveguides that are plasmonic waveguides fabricated with standard CMOS technology. We focus on the bends of MISIM waveguides with a wide (e.g., 160-220 nm) silicon line. This study shows that the bend efficiently turns the direction of the MISIM waveguide by 90° if its radius is about 0.7 µm. Moreover, we discuss the fact that the bend may be superior to a SRB of a silicon photonic waveguide when it is used to implement a ring resonator with a high quality factor and small volume.

Correction: Highly potent and selective phosphatidylinositol 4-kinase IIIß inhibitors as broad-spectrum anti-rhinoviral agents.

[This corrects the article DOI: 10.1039/D3MD00630A.].

Highly potent and selective phosphatidylinositol 4-kinase IIIß inhibitors as broad-spectrum anti-rhinoviral agents.

Human rhinoviruses (hRVs) cause upper and lower respiratory tract infections and exacerbate asthma and chronic obstructive pulmonary disease. hRVs comprise more than 160 strains with considerable genetic variation. Their high diversity and strain-specific interactions with antisera hinder the development of anti-hRV therapeutic agents. Phosphatidylinositol-4-kinase IIIß (PI4KIIIß) is a key enzyme in the phosphoinositide signalling pathway that is crucial for the replication and survival of various viruses. We identified novel PI4KIIIß inhibitors, N-(4-methyl-5-arylthiazol)-2-amide derivatives, by generating a hit compound, 1a, from the high-throughput screening of a chemical library, followed by the optimization study of 1a. Inhibitor 7e exhibited the highest activity (EC50 = 0.008, 0.0068, and 0.0076 µM for hRV-B14, hRV-A16, and hRV-A21, respectively) and high toxicity (CC50 = 6.1 µM). Inhibitor 7f showed good activity and low toxicity and provided the highest selectivity index (SI ≥ 4638, >3116, and >2793 for hRV-B14, hRV-A16, and hRV-A21, respectively). Furthermore, 7f showed broad-spectrum activities against various hRVs, coxsackieviruses, and other enteroviruses, such as EV-A71 and EV-D68. The binding mode of the inhibitors was investigated using 7f, and the experimental results of plaque reduction, replicon and cytotoxicity, and time-of-drug-addition assays suggested that 7f acts as a PI4KIIIß inhibitor. The kinase inhibition activity of this series of compounds against PI4KIIIα and PI4KIIIß was assessed, and 7f demonstrated kinase inhibition activity with an IC50 value of 0.016 µM for PI4KIIIß, but not for PI4KIIIα (>10 µM). Therefore, 7f represents a highly potent and selective PI4KIIIß inhibitor for the further development of antiviral therapy against hRVs or other enteroviruses.

Antiviral Lipid Nanocarrier Loaded with Remdesivir Effective Against SARS-CoV-2 in vitro Model.

Introduction: The ongoing SARS-CoV-2 pandemic has affected public health, the economy, and society. This study reported a nanotechnology-based strategy to enhance the antiviral efficacy of the antiviral agent remdesivir (RDS). Results: We developed a nanosized spherical RDS-NLC in which the RDS was encapsulated in an amorphous form. The RDS-NLC significantly potentiated the antiviral efficacy of RDS against SARS-CoV-2 and its variants (alpha, beta, and delta). Our study revealed that NLC technology improved the antiviral effect of RDS against SARS-CoV-2 by enhancing the cellular uptake of RDS and reducing SARS-CoV-2 entry in cells. These improvements resulted in a 211% increase in the bioavailability of RDS. Conclusion: Thus, the application of NLC against SARS-CoV-2 may be a beneficial strategy to improve the antiviral effects of antiviral agents.

Characterizations of realized metal-insulator-silicon-insulator-metal waveguides and nanochannel fabrication via insulator removal.

We investigate experimentally metal-insulator-silicon-insulator-metal (MISIM) waveguides that are fabricated by using fully standard CMOS technology. They are hybrid plasmonic waveguides, and they have a feature that their insulator is replaceable with functional material. We explain a fabrication process for them and discuss fabrication results based on 8-inch silicon-on-insulator wafers. We measured the propagation characteristics of the MISIM waveguides that were actually fabricated to be connected to Si photonic waveguides through symmetric and asymmetric couplers. When incident light from an optical source has transverse electric (TE) polarization and its wavelength is 1318 or 1554 nm, their propagation losses are between 0.2 and 0.3 dB/µm. Excess losses due to the symmetric couplers are around 0.5 dB, which are smaller than those due to the asymmetric couplers. Additional measurement results indicate that the MISIM waveguide supports a TE-polarized hybrid plasmonic mode. Finally, we explain a process of removing the insulator without affecting the remaining MISIM structure to fabricate ~30-nm-wide nanochannels which may be filled with functional material.