Discovery of SARS-CoV-2-E channel inhibitors as antiviral candidates

AbstractLack of efficiency has been a major problem shared by all currently developed anti-SARS-CoV-2 therapies. Our previous study shows that SARS-CoV-2 structural envelope (2-E) protein forms a type of cation channel, and heterogeneously expression of 2-E channels causes host cell death. In this study we developed a cell-based high throughput screening (HTS) assay and used it to discover inhibitors against 2-E channels. Among 4376 compounds tested, 34 hits with cell protection activity were found. Followed by an anti-viral analysis, 15 compounds which could inhibit SARS-CoV-2 replication were identified. In electrophysiological experiments, three representatives showing inhibitory effect on 2-E channels were chosen for further characterization. Among them, proanthocyanidins directly bound to 2-E channel with binding affinity (KD) of 22.14 μM in surface plasmon resonance assay. Molecular modeling and docking analysis revealed that proanthocyanidins inserted into the pore of 2-E N-terminal vestibule acting as a channel blocker. Consistently, mutations of Glu 8 and Asn 15, two residues lining the proposed binding pocket, abolished the inhibitory effects of proanthocyanidins. The natural product proanthocyanidins are widely used as cosmetic, suggesting a potential of proanthocyanidins as disinfectant for external use. This study further demonstrates that 2-E channel is an effective antiviral drug target and provides a potential antiviral candidate against SARS-CoV-2.

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Evidence of Immunostimulating Lipoprotein Existing in the Natural Lipoteichoic Acid Fraction

Infection and Immunity ◽

10.1128/iai.02083-05 ◽

2007 ◽

Vol 75 (4) ◽

pp. 1926-1932 ◽

Cited By ~ 30

Author(s):

Masahito Hashimoto ◽

Maiko Furuyashiki ◽

Ryoko Kaseya ◽

Yuka Fukada ◽

Mai Akimaru ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cytokine Production ◽

Lipoteichoic Acid ◽

Inhibitory Effect ◽

Innate Immune ◽

Enterococcus Hirae ◽

Inhibitory Effects ◽

Active Structure ◽

Gram Positive Bacteria ◽

A Cell

ABSTRACT Lipoteichoic acid (LTA) is a cell surface glycoconjugate of gram-positive bacteria and is reported to activate the innate immune system. We previously reported that purified LTA obtained from Enterococcus hirae has no immunostimulating activity, but a subfraction (Eh-AF) in an LTA fraction possesses activity. In this study, we established a mouse monoclonal antibody neutralizing the activity of Eh-AF and investigated its inhibitory effects. Monoclonal antibody (MAbEh1) was established by the immunization of BALB/c mice with Eh-AF, followed by hybridoma screening based on its inhibitory effect for the production of interleukin-6 (IL-6) induced by Eh-AF. MAbEh1 neutralized the production of IL-6 by LTA fraction from not only E. hirae but also Staphylococcus aureus, while it failed to block that of lipopolysaccharide, suggesting that the antibody recognized a common active structure(s) in LTA fractions. Synthetic glycolipids in these LTAs did not induce cytokine production, at least in our system. Interestingly, the antibody was found to inhibit the activity of immunostimulating synthetic lipopeptides, Pam3CSK4 and FSL-1. These results suggest that MAbEh1 neutralizes the activity of lipoprotein-like compounds which is responsible for the activity of the LTA fraction of E. hirae and S. aureus.

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Inhibitory Effects of Glycyrrhetinic Acid on DNA Polymerase and Inflammatory Activities

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2012/650514 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 23

Author(s):

Tsukasa Ishida ◽

Yoshiyuki Mizushina ◽

Saori Yagi ◽

Yasuhiro Irino ◽

Shin Nishiumi ◽

...

Keyword(s):

Inhibitory Effect ◽

Peritoneal Macrophages ◽

Glycyrrhetinic Acid ◽

Inhibitory Effects ◽

Hek 293 ◽

293 Cells ◽

Mouse Macrophages ◽

Mouse Ear ◽

A Cell ◽

Acid Compound

We investigated the inhibitory effect of three glycyrrhizin derivatives, such as Glycyrrhizin (compound1), dipotassium glycyrrhizate (compound2) and glycyrrhetinic acid (compound3), on the activity of mammalian pols. Among these derivatives, compound3was the strongest inhibitor of mammalian polsα,β,κ, andλ, which belong to the B, A, Y, and X families of pols, respectively, whereas compounds1and2showed moderate inhibition. Among the these derivatives tested, compound3displayed strongest suppression of the production of tumor necrosis factor-α(TNF-α) induced by lipopolysaccharide (LPS) in a cell-culture system using mouse macrophages RAW264.7 and peritoneal macrophages derived from mice. Moreover, compound3was found to inhibit the action of nuclear factor-κB (NF-κB) in engineered human embryonic kidney (HEK) 293 cells. In addition, compound3caused greater reduction of 12-O-tetradecanoylphorbol-13-acetate-(TPA-) induced acute inflammation in mouse ear than compounds1and2. In conclusion, this study has identified compound3, which is the aglycone of compounds1and2, as a promising anti-inflammatory candidate based on mammalian pol inhibition.

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Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 cells

Viruses ◽

10.3390/v11080742 ◽

2019 ◽

Vol 11 (8) ◽

pp. 742 ◽

Cited By ~ 2

Author(s):

Hongzhuan Zhou ◽

Xia Su ◽

Lulu Lin ◽

Jin Zhang ◽

Qi Qi ◽

...

Keyword(s):

High Throughput Screening ◽

Antiviral Drug ◽

Supportive Therapy ◽

Immunofluorescence Assay ◽

Canine Parvovirus ◽

Inhibitory Effects ◽

Screening Assay ◽

Western Blot Assay ◽

High Throughput Screening Assay ◽

Approved Drugs

Canine parvovirus (CPV) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. Despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. While no specific antiviral therapy for CPV infection exists, the only treatment option for the infection is supportive therapy based on symptoms. Thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of CPV-related disease. In this study, a cytopathic effect (CPE)-based high-throughput screening assay was used to screen CPV inhibitors from a Food and Drug Administration (FDA)-approved drug library. After two rounds of screening, seven out of 1430 screened drugs were found to have >50% CPE inhibition. Three drugs—Nitazoxanide, Closantel Sodium, and Closantel—with higher anti-CPV effects were further evaluated in F81 cells by absolute PCR quantification and indirect immunofluorescence assay (IFA). The inhibitory effects of all three drugs were dose-dependent. Time of addition assay indicated that the drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection. Western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three CPV variants. In addition, antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells regardless of CPV infection, while Closantel Sodium- or Closantel-treated cells had no pro- or antiapoptotic effects. In conclusion, Nitazoxanide, Closantel Sodium, and Closantel can effectively inhibit different subspecies of CPV. Since the safety profiles of FDA-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-CPV drugs.

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High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors

Protein & Cell ◽

10.1007/s13238-021-00836-9 ◽

2021 ◽

Author(s):

Yao Zhao ◽

Xiaoyu Du ◽

Yinkai Duan ◽

Xiaoyan Pan ◽

Yifang Sun ◽

...

Keyword(s):

Clinical Trials ◽

High Throughput Screening ◽

Treatment Options ◽

Antiviral Drug ◽

Binding Pocket ◽

Binding Mode ◽

Etiologic Agent ◽

Drug Candidate ◽

Approved Drugs ◽

Pharmacologically Active

AbstractA new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 μmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three “hot” spots on PLpro, including the substrate-binding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

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Dynactin has two antagonistic regulatory domains and exerts opposing effects on dynein motility

10.1101/110775 ◽

2017 ◽

Author(s):

Takuya Kobayashi ◽

Takuya Miyashita ◽

Takashi Murayama ◽

Yoko Y. Toyoshima

Keyword(s):

Single Molecule ◽

Coiled Coil ◽

Inhibitory Effect ◽

Binding Affinities ◽

Inhibitory Effects ◽

Regulatory Domains ◽

Binding Domains ◽

Rich Domain ◽

A Cell ◽

Opposing Effects

AbstractDynactin is a dynein-regulating protein that increases the processivity of dynein movement on microtubules. Recent studies have shown that a tripartite complex of dynein–dynactin–Bicaudal D2 is essential for highly processive movement. To elucidate the regulation of dynein motility by dynactin, we focused on two isoforms (A and B) of dynactin 1 (DCTN1), the largest subunit of dynactin that contains both microtubule- and dynein-binding domains. The only difference between the primary structures of the two isoforms is that DCTN1B lacks the K-rich domain, a cluster of basic residues. We measured dynein motility by single molecule observation of recombinant dynein and dynactin. Whereas the tripartite complex containing DCTN1A exhibited highly processive movement, the complex containing DCTN1B dissociated from microtubules with no apparent processive movement. This inhibitory effect of DCTN1B was caused by reductions of the microtubule-binding affinities of both dynein and dynactin, which is attributed to the coiled-coil 1 domain of DCTN1. In DCTN1A, the K-rich domain antagonized these inhibitory effects. Therefore, dynactin has two antagonistic domains and promotes or suppresses dynein motility to accomplish correct localization and functions of dynein within a cell.

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Molecular docking and simulation investigation: effect of beta-sesquiphellandrene with ionic integration on SARS-CoV2 and SFTS viruses

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-020-00095-x ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Amit Joshi ◽

G. Sunil Krishnan ◽

Vikas Kaushik

Keyword(s):

Molecular Docking ◽

In Silico ◽

Binding Pocket ◽

Inhibitory Effect ◽

Binding Energies ◽

Spike Protein ◽

Membrane Glycoprotein ◽

Inhibitory Effects ◽

Drug Molecules ◽

Wet Lab

Abstract Background At present, viral diseases become major concern for the world. SARS-CoV2 and SFTS viruses are deadly in nature, and there is a need for developing best treatments for them. Modern in silico approaches were found to be very handy in determining putative drug molecules. In this study, we analyze interaction of beta-sesquiphellandrene (compound belongs to ginger) with spike protein (Sp) and membrane glycoprotein polyprotein (MPp). Results Our molecular docking and simulation study reveals the perfect binding pocket of Sp and MPp holding beta-sesquiphellandrene (bS). Binding energies for MPp-bS and Sp-bS were found to be − 9.5 kcal/mol and − 10.3 kcal/mol respectively. RMSD and RMSF values for docked complexes were found to be in selectable range, i.e., 1 to 3 Å and 1 to 8 Å respectively. Modern computational tools were used here to make this investigation fast and effective. Further, ADME analysis reveals the therapeutic validations for beta-sesquiphellandrene to act as a useful pharmacoactive compound. Beta-sesquiphellandrene provides not only inhibitory effect on spike protein of SARS-CoV2 but also similar inhibitory effects on membrane glycoprotein polyprotein complex of SFTS virus, which hampers the pathological initiation of the diseases caused by both the viruses, i.e., COVID-19 and severe fever with thrombocytopenia syndrome. Conclusion This method of computational analysis was found to be rapid and effective, and opens new doors in the domain of in silico drug discovery. Beta-sesquiphellandrene can be used as effective medicine to control these harmful pathogens after wet lab validations.

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Biological Control of Crown Gall of Grapevine, Rose, and Tomato by Nonpathogenic Agrobacterium vitis Strain VAR03-1

Phytopathology ◽

10.1094/phyto-98-11-1218 ◽

2008 ◽

Vol 98 (11) ◽

pp. 1218-1225 ◽

Cited By ~ 30

Author(s):

A. Kawaguchi ◽

K. Inoue ◽

Y. Ichinose

Keyword(s):

Biological Control ◽

Crown Gall ◽

Biological Control Agent ◽

Inhibitory Effect ◽

Control Agent ◽

Inhibitory Effects ◽

Agrobacterium Vitis ◽

Colony Forming Units ◽

Nonpathogenic Strain ◽

A Cell

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent for crown gall of grapevine (Vitis vinifera). When roots of grapevine, rose (Rose multiflora), and tomato (Lycopersicon esculentum) were soaked in a cell suspension of antagonists before planting in soil infested with tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens, respectively, treatment with VAR03-1 significantly reduced the number of plants with tumors and disease severity in the three plant species. The inhibitory effects of treatment with VAR03-1 and the nonpathogenic A. rhizogenes strain K84 on crown gall of rose and tomato were almost identical, and the inhibitory effect of VAR03-1 on grapevine was superior to that of K84. Moreover, VAR03-1 greatly controlled crown gall of grapevine due to tumorigenic A. vitis in the field. VAR03-1 established populations averaging 106 colony forming units (CFU)/g of root in the rhizosphere of grapevine and persisted on roots for 2 years. VAR03-1 was bacteriocinogenic, producing a halo of inhibition against those three species of Agrobacterium. This is the first report that a nonpathogenic strain, VAR03-1, can effectively control crown gall caused by tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens.

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GC-MS Analysis and Inhibitory Evaluation of Terminalia catappa Leaf Extracts on Major Enzymes Linked to Diabetes

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/6316231 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14 ◽

Cited By ~ 2

Author(s):

Franklyn Nonso Iheagwam ◽

Emmanuel Nsedu Israel ◽

Kazeem Oyindamola Kayode ◽

Opeyemi Christianah De Campos ◽

Olubanke Olujoke Ogunlana ◽

...

Keyword(s):

Amylase Activity ◽

Ethanol Extract ◽

Binding Pocket ◽

Inhibitory Effect ◽

Inhibitory Effects ◽

Autodock Vina ◽

Leaf Extracts ◽

Terminalia Catappa ◽

Southwest Nigeria

Terminalia catappa leaves are used in managing both diabetes mellitus and its complications in Southwest Nigeria. However, its inhibitory activity on enzymes implicated in diabetes is not very clear. This study investigated the in vitro inhibitory properties and mode of inhibition of T. catappa leaf extracts on enzymes associated with diabetes. The study also identified some bioactive compounds as well as their molecular interaction in the binding pocket of these enzymes. Standard enzyme inhibition and kinetics assays were performed to determine the inhibitory effects of aqueous extract (TCA) and ethanol extract (TCE) of T. catappa leaves on α-glucosidase and α-amylase activities. The phytoconstituents of TCA and TCE were determined using GC-MS. Molecular docking of the phytocompounds was performed using Autodock Vina. TCA and TCE were the most potent inhibitors of α-glucosidase (IC50 = 3.28 ± 0.47 mg/mL) and α-amylase (IC50 = 0.24 ± 0.08 mg/mL), respectively. Both extracts displayed a mixed mode of inhibition on α-amylase activity, while mixed and noncompetitive modes of inhibition were demonstrated by TCA and TCE, respectively, on α-glucosidase activity. The GC-MS analytic chromatogram revealed the presence of 24 and 22 compounds in TCE and TCA, respectively, which were identified mainly as phenolic compounds, terpenes/terpenoids, fatty acids, and other phytochemicals. The selected compounds exhibited favourable interactions with the enzymes compared with acarbose. Overall, the inhibitory effect of T. catappa on α-amylase and α-glucosidase may be ascribed to the synergistic action of its rich phenolic and terpene composition giving credence to the hypoglycaemic nature of T. catappa leaves.

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All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity

Viruses ◽

10.3390/v13081669 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1669

Author(s):

Takeshi Morita ◽

Kei Miyakawa ◽

Sundararaj Stanleyraj Jeremiah ◽

Yutaro Yamaoka ◽

Mitsuru Sada ◽

...

Keyword(s):

Retinoic Acid ◽

High Throughput Screening ◽

Antiviral Drug ◽

Antiviral Effect ◽

Inhibitory Effect ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Inhibition Concentration ◽

Natural Product Compounds

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.

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In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646570 ◽

1989 ◽

Vol 61 (02) ◽

pp. 254-258 ◽

Cited By ~ 21

Author(s):

Margaret L Rand ◽

Peter L Gross ◽

Donna M Jakowec ◽

Marian A Packham ◽

J Fraser Mustard

Keyword(s):

Cyclic Amp ◽

Inhibitory Effect ◽

Rabbit Platelet ◽

Inhibitory Effects ◽

Low Concentrations ◽

Rabbit Platelets ◽

High Concentrations ◽

In Vitro Effects ◽

Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

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