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1.
Biochem Biophys Res Commun ; 604: 76-82, 2022 May 14.
Article in English | MEDLINE | ID: covidwho-1797136

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and seriously threatened public health and safety. Despite COVID-19 vaccines being readily popularized worldwide, targeted therapeutic agents for the treatment of this disease remain very limited. Here, we studied the inhibitory activity of the scutellarein and its methylated derivatives against SARS-CoV-2 main protease (Mpro) by the fluorescence resonance energy transfer (FRET) assay. Among all the methylated derivatives we studied, 4'-O-methylscutellarein exhibited the most promising enzyme inhibitory activity in vitro, with the half-maximal inhibitory concentration value (IC50) of 0.40 ± 0.03 µM. Additionally, the mechanism of action of the hits was further characterized through enzyme kinetic studies and molecular docking. Overall, our results implied that 4'-O-methylscutellarein could be a primary lead compound with clinical potential for the development of inhibitors against the SARS-CoV-2 Mpro.


Subject(s)
Alkaloids , Coronavirus 3C Proteases , Indoles , SARS-CoV-2 , Viral Protease Inhibitors , Alkaloids/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Humans , Indoles/pharmacology , Kinetics , Molecular Docking Simulation , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Viral Protease Inhibitors/pharmacology
2.
J Virol ; 95(24): e0139921, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1691426

ABSTRACT

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.


Subject(s)
Apoptosis , Arenaviridae/drug effects , COVID-19/drug therapy , Proto-Oncogene Proteins c-bcl-2/metabolism , A549 Cells , Animals , Antiviral Agents/pharmacology , Apoptosis Regulatory Proteins/pharmacology , Biphenyl Compounds/pharmacology , COVID-19/virology , Cell Cycle , Cell Cycle Checkpoints/drug effects , Cells, Cultured/drug effects , Cells, Cultured/virology , Chlorocebus aethiops , Cyclin A2/biosynthesis , Cyclin B1/biosynthesis , G1 Phase , Humans , Indoles/pharmacology , Mice , Mice, Inbred C57BL , Nitrophenols/pharmacology , Piperazines/pharmacology , Pyrroles/pharmacology , Resting Phase, Cell Cycle , SARS-CoV-2 , Sulfonamides/pharmacology , Thymidine Kinase/biosynthesis , Vero Cells
3.
Med Sci Monit ; 28: e934102, 2022 Jan 25.
Article in English | MEDLINE | ID: covidwho-1651076

ABSTRACT

BACKGROUND Heat-clearing and detoxifying herbs (HDHs) play an important role in the prevention and treatment of coronavirus infection. However, their mechanism of action needs further study. This study aimed to explore the anti-coronavirus basis and mechanism of HDHs. MATERIAL AND METHODS Database mining was performed on 7 HDHs. Core ingredients and targets were screened according to ADME rules combined with Neighborhood, Co-occurrence, Co-expression, and other algorithms. GO enrichment and KEGG pathway analyses were performed using the R language. Finally, high-throughput molecular docking was used for verification. RESULTS HDHs mainly acts on NOS3, EGFR, IL-6, MAPK8, PTGS2, MAPK14, NFKB1, and CASP3 through quercetin, luteolin, wogonin, indirubin alkaloids, ß-sitosterol, and isolariciresinol. These targets are mainly involved in the regulation of biological processes such as inflammation, activation of MAPK activity, and positive regulation of NF-kappaB transcription factor activity. Pathway analysis further revealed that the pathways regulated by these targets mainly include: signaling pathways related to viral and bacterial infections such as tuberculosis, influenza A, Ras signaling pathways; inflammation-related pathways such as the TLR, TNF, MAPK, and HIF-1 signaling pathways; and immune-related pathways such as NOD receptor signaling pathways. These pathways play a synergistic role in inhibiting lung inflammation and regulating immunity and antiviral activity. CONCLUSIONS HDHs play a role in the treatment of coronavirus infection by regulating the body's immunity, fighting inflammation, and antiviral activities, suggesting a molecular basis and new strategies for the treatment of COVID-19 and a foundation for the screening of new antiviral drugs.


Subject(s)
COVID-19/drug therapy , Coronavirus/drug effects , Drugs, Chinese Herbal/pharmacology , SARS-CoV-2/drug effects , Alkaloids/chemistry , Alkaloids/pharmacology , Caspase 3/drug effects , Caspase 3/genetics , Coronavirus/metabolism , Coronavirus Infections/drug therapy , Cyclooxygenase 2/drug effects , Cyclooxygenase 2/genetics , Databases, Pharmaceutical , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/therapeutic use , Flavanones/chemistry , Flavanones/pharmacology , Humans , Indoles/chemistry , Indoles/pharmacology , Interleukin-6/genetics , Lignin/chemistry , Lignin/pharmacology , Luteolin/chemistry , Luteolin/pharmacology , Mitogen-Activated Protein Kinase 14/drug effects , Mitogen-Activated Protein Kinase 14/genetics , Mitogen-Activated Protein Kinase 8/drug effects , Mitogen-Activated Protein Kinase 8/genetics , Molecular Docking Simulation , NF-kappa B p50 Subunit/drug effects , NF-kappa B p50 Subunit/genetics , Naphthols/chemistry , Naphthols/pharmacology , Nitric Oxide Synthase Type III/drug effects , Nitric Oxide Synthase Type III/genetics , Protein Interaction Maps , Quercetin/chemistry , Quercetin/pharmacology , SARS-CoV-2/metabolism , Signal Transduction , Sitosterols/chemistry , Sitosterols/pharmacology , Transcriptome/drug effects , Transcriptome/genetics
4.
Emerg Microbes Infect ; 11(1): 483-497, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1606402

ABSTRACT

Coronavirus disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has set off a global pandemic. There is an urgent unmet need for safe, affordable, and effective therapeutics against COVID-19. In this regard, drug repurposing is considered as a promising approach. We assessed the compounds that affect the endosomal acidic environment by applying human angiotensin-converting enzyme 2 (hACE2)- expressing cells infected with a SARS-CoV-2 spike (S) protein-pseudotyped HIV reporter virus and identified that obatoclax resulted in the strongest inhibition of S protein-mediated virus entry. The potent antiviral activity of obatoclax at nanomolar concentrations was confirmed in different human lung and intestinal cells infected with the SARS-CoV-2 pseudotype system as well as clinical virus isolates. Furthermore, we uncovered that obatoclax executes a double-strike against SARS-CoV-2. It prevented SARS-CoV-2 entry by blocking endocytosis of virions through diminished endosomal acidification and the corresponding inhibition of the enzymatic activity of the endosomal cysteine protease cathepsin L. Additionally, obatoclax impaired the SARS-CoV-2 S-mediated membrane fusion by targeting the MCL-1 protein and reducing furin protease activity. In accordance with these overarching mechanisms, obatoclax blocked the virus entry mediated by different S proteins derived from several SARS-CoV-2 variants of concern such as, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Taken together, our results identified obatoclax as a novel effective antiviral compound that keeps SARS-CoV-2 at bay by blocking both endocytosis and membrane fusion. Our data suggested that obatoclax should be further explored as a clinical drug for the treatment of COVID-19.


Subject(s)
Cathepsins/metabolism , Furin/metabolism , Indoles/pharmacology , Pyrroles/pharmacology , SARS-CoV-2 , Virus Internalization/drug effects , COVID-19 , Humans , Hydrogen-Ion Concentration , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus
5.
Molecules ; 26(24)2021 Dec 09.
Article in English | MEDLINE | ID: covidwho-1572567

ABSTRACT

COVID-19 is the name of the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that occurred in 2019. The virus-host-specific interactions, molecular targets on host cell deaths, and the involved signaling are crucial issues, which become potential targets for treatment. Spike protein, angiotensin-converting enzyme 2 (ACE2), cathepsin L-cysteine peptidase, transmembrane protease serine 2 (TMPRSS2), nonstructural protein 1 (Nsp1), open reading frame 7a (ORF7a), viral main protease (3C-like protease (3CLpro) or Mpro), RNA dependent RNA polymerase (RdRp) (Nsp12), non-structural protein 13 (Nsp13) helicase, and papain-like proteinase (PLpro) are molecules associated with SARS-CoV infection and propagation. SARS-CoV-2 can induce host cell death via five kinds of regulated cell death, i.e., apoptosis, necroptosis, pyroptosis, autophagy, and PANoptosis. The mechanisms of these cell deaths are well established and can be disrupted by synthetic small molecules or natural products. There are a variety of compounds proven to play roles in the cell death inhibition, such as pan-caspase inhibitor (z-VAD-fmk) for apoptosis, necrostatin-1 for necroptosis, MCC950, a potent and specific inhibitor of the NLRP3 inflammasome in pyroptosis, and chloroquine/hydroxychloroquine, which can mitigate the corresponding cell death pathways. However, NF-κB signaling is another critical anti-apoptotic or survival route mediated by SARS-CoV-2. Such signaling promotes viral survival, proliferation, and inflammation by inducing the expression of apoptosis inhibitors such as Bcl-2 and XIAP, as well as cytokines, e.g., TNF. As a result, tiny natural compounds functioning as proteasome inhibitors such as celastrol and curcumin can be used to modify NF-κB signaling, providing a responsible method for treating SARS-CoV-2-infected patients. The natural constituents that aid in inhibiting viral infection, progression, and amplification of coronaviruses are also emphasized, which are in the groups of alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones. Natural constituents derived from medicinal herbs have anti-inflammatory and antiviral properties, as well as inhibitory effects, on the viral life cycle, including viral entry, replication, assembly, and release of COVID-19 virions. The phytochemicals contain a high potential for COVID-19 treatment. As a result, SARS-CoV-2-infected cell death processes and signaling might be of high efficacy for therapeutic targeting effects and yielding encouraging outcomes.


Subject(s)
COVID-19/drug therapy , Cell Death/drug effects , Drug Discovery/methods , Molecular Targeted Therapy/methods , SARS-CoV-2/drug effects , Amino Acid Chloromethyl Ketones/pharmacology , Antiviral Agents/pharmacology , Apoptosis/drug effects , Furans/pharmacology , Humans , Hydroxychloroquine/pharmacology , Imidazoles/pharmacology , Indenes/pharmacology , Indoles/pharmacology , Necroptosis/drug effects , Phytochemicals/pharmacology , Pyroptosis/drug effects , SARS-CoV-2/metabolism , Signal Transduction/drug effects , Sulfonamides/pharmacology , Viral Proteins/antagonists & inhibitors
6.
SAR QSAR Environ Res ; 32(12): 963-983, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1532255

ABSTRACT

The coronavirus helicase is an essential enzyme required for viral replication/transcription pathways. Structural studies revealed a sulphate moiety that interacts with key residues within the nucleotide-binding site of the helicase. Compounds with a sulphoxide or a sulphone moiety could interfere with these interactions and consequently inhibit the enzyme. The molecular operating environment (MOE) was used to dock 189 sulphoxide and sulphone-containing FDA-approved compounds to the nucleotide-binding site. Zafirlukast, a leukotriene receptor antagonist used to treat chronic asthma, achieved the lowest docking score at -8.75 kcals/mol. The inhibitory effect of the compounds on the SARS-CoV-2 helicase dsDNA unwinding activity was tested by a FRET-based assay. Zafirlukast was the only compound to inhibit the enzyme (IC50 = 16.3 µM). The treatment of Vero E6 cells with 25 µM zafirlukast prior to SARS-CoV-2 infection decreased the cytopathic effects of SARS-CoV-2 significantly. These results suggest that zafirlukast alleviates SARS-CoV-2 pathogenicity by inhibiting the viral helicase and impairing the viral replication/transcription pathway. Zafirlukast could be clinically developed as a new antiviral treatment for SARS-CoV-2 and other coronavirus diseases. This discovery is based on molecular modelling, in vitro inhibition of the SARS-CoV helicase activity and cell-based SARS-CoV-2 viral replication.


Subject(s)
Antiviral Agents/pharmacology , DNA Helicases/antagonists & inhibitors , Indoles/pharmacology , Phenylcarbamates/pharmacology , SARS-CoV-2/drug effects , Sulfonamides/pharmacology , Animals , COVID-19/drug therapy , Chlorocebus aethiops , Fluorescence Resonance Energy Transfer , Quantitative Structure-Activity Relationship , SARS-CoV-2/enzymology , Vero Cells , Virus Replication/drug effects
7.
Theranostics ; 11(14): 7005-7017, 2021.
Article in English | MEDLINE | ID: covidwho-1524524

ABSTRACT

The tumor suppressor protein p53 remains in a wild type but inactive form in ~50% of all human cancers. Thus, activating it becomes an attractive approach for targeted cancer therapies. In this regard, our lab has previously discovered a small molecule, Inauhzin (INZ), as a potent p53 activator with no genotoxicity. Method: To improve its efficacy and bioavailability, here we employed nanoparticle encapsulation, making INZ-C, an analog of INZ, to nanoparticle-encapsulated INZ-C (n-INZ-C). Results: This approach significantly improved p53 activation and inhibition of lung and colorectal cancer cell growth by n-INZ-C in vitro and in vivo while it displayed a minimal effect on normal human Wi38 and mouse MEF cells. The improved activity was further corroborated with the enhanced cellular uptake observed in cancer cells and minimal cellular uptake observed in normal cells. In vivo pharmacokinetic evaluation of these nanoparticles showed that the nanoparticle encapsulation prolongates the half-life of INZ-C from 2.5 h to 5 h in mice. Conclusions: These results demonstrate that we have established a nanoparticle system that could enhance the bioavailability and efficacy of INZ-C as a potential anti-cancer therapeutic.


Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Indoles/pharmacology , Lung Neoplasms/drug therapy , Nanoparticles/chemistry , Phenothiazines/pharmacology , Tumor Suppressor Protein p53/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Biological Availability , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Indoles/chemistry , Indoles/pharmacokinetics , Indoles/therapeutic use , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Nanoparticles/toxicity , Nanoparticles/ultrastructure , Phenothiazines/chemistry , Phenothiazines/pharmacokinetics , Phenothiazines/therapeutic use , Spectroscopy, Fourier Transform Infrared , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
8.
ACS Chem Biol ; 16(12): 2845-2851, 2021 12 17.
Article in English | MEDLINE | ID: covidwho-1521690

ABSTRACT

Arbidol (ARB) is a broad-spectrum antiviral drug approved in Russia and China for the treatment of influenza. ARB was tested in patients as a drug candidate for the treatment at the early onset of COVID-19 caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite promising clinical results and multiple ongoing trials, preclinical data are lacking and the molecular mechanism of action of ARB against SARS-CoV-2 remains unknown. Here, we demonstrate that ARB binds to the spike viral fusion glycoprotein of the SARS-CoV-2 Wuhan strain as well as its more virulent variants from the United Kingdom (strain B.1.1.7) and South Africa (strain B.1.351). We pinpoint the ARB binding site on the S protein to the S2 membrane fusion domain and use an infection assay with Moloney murine leukemia virus (MLV) pseudoviruses (PVs) pseudotyped with the S proteins of the Wuhan strain and the new variants to show that this interaction is sufficient for the viral cell entry inhibition by ARB. Finally, our experiments reveal that the ARB interaction leads to a significant destabilization and eventual lysosomal degradation of the S protein in cells. Collectively, our results identify ARB as the first clinically approved small molecule drug binder of the SARS-CoV-2 S protein and place ARB among the more promising drug candidates for COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Indoles/pharmacology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Animals , Antiviral Agents/metabolism , Binding Sites , Chlorocebus aethiops , HEK293 Cells , Humans , Indoles/metabolism , Lysosomes/metabolism , Mutation , Protein Domains , Proteolysis/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Internalization/drug effects
9.
Cell Res ; 31(12): 1230-1243, 2021 12.
Article in English | MEDLINE | ID: covidwho-1475291

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.


Subject(s)
COVID-19/pathology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/drug therapy , COVID-19/mortality , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cytokines/genetics , Cytokines/metabolism , Down-Regulation/drug effects , ErbB Receptors/metabolism , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Indoles/pharmacology , Indoles/therapeutic use , Lung/pathology , Lung/virology , Mice , Mice, Transgenic , Mutation , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Survival Rate , Transcriptome/drug effects , Viral Load/drug effects , Virus Internalization
10.
J Virol ; 95(24): e0139921, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1443355

ABSTRACT

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.


Subject(s)
Apoptosis , Arenaviridae/drug effects , COVID-19/drug therapy , Proto-Oncogene Proteins c-bcl-2/metabolism , A549 Cells , Animals , Antiviral Agents/pharmacology , Apoptosis Regulatory Proteins/pharmacology , Biphenyl Compounds/pharmacology , COVID-19/virology , Cell Cycle , Cell Cycle Checkpoints/drug effects , Cells, Cultured/drug effects , Cells, Cultured/virology , Chlorocebus aethiops , Cyclin A2/biosynthesis , Cyclin B1/biosynthesis , G1 Phase , Humans , Indoles/pharmacology , Mice , Mice, Inbred C57BL , Nitrophenols/pharmacology , Piperazines/pharmacology , Pyrroles/pharmacology , Resting Phase, Cell Cycle , SARS-CoV-2 , Sulfonamides/pharmacology , Thymidine Kinase/biosynthesis , Vero Cells
11.
Nat Commun ; 12(1): 668, 2021 01 28.
Article in English | MEDLINE | ID: covidwho-1387328

ABSTRACT

Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 µM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.


Subject(s)
COVID-19/drug therapy , Coronavirus Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Viral Proteases/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Humans , Indoles/pharmacology , Pyridines/pharmacology , Vero Cells , Viral Proteases/metabolism
12.
Viruses ; 13(8)2021 08 23.
Article in English | MEDLINE | ID: covidwho-1367925

ABSTRACT

An escalating pandemic of the novel SARS-CoV-2 virus is impacting global health, and effective antivirals are needed. Umifenovir (Arbidol) is an indole-derivative molecule, licensed in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It has been shown that umifenovir has broad spectrum activity against different viruses. We evaluated the sensitivity of different coronaviruses, including the novel SARS-CoV-2 virus, to umifenovir using in vitro assays. Using a plaque assay, we revealed an antiviral effect of umifenovir against seasonal HCoV-229E and HCoV-OC43 coronaviruses in Vero E6 cells, with estimated 50% effective concentrations (EC50) of 10.0 ± 0.5 µM and 9.0 ± 0.4 µM, respectively. Umifenovir at 90 µM significantly suppressed plaque formation in CMK-AH-1 cells infected with SARS-CoV. Umifenovir also inhibited the replication of SARS-CoV-2 virus, with EC50 values ranging from 15.37 ± 3.6 to 28.0 ± 1.0 µM. In addition, 21-36 µM of umifenovir significantly suppressed SARS-CoV-2 virus titers (≥2 log TCID50/mL) in the first 24 h after infection. Repurposing of antiviral drugs is very helpful in fighting COVID-19. A safe, pan-antiviral drug such as umifenovir could be extremely beneficial in combating the early stages of a viral pandemic.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 229E, Human/drug effects , Coronavirus OC43, Human/drug effects , Indoles/pharmacology , SARS Virus/drug effects , SARS-CoV-2/drug effects , Animals , Antiviral Agents/administration & dosage , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus 229E, Human/physiology , Coronavirus OC43, Human/physiology , Cytopathogenic Effect, Viral/drug effects , Humans , Indoles/administration & dosage , Microbial Sensitivity Tests , SARS Virus/physiology , SARS-CoV-2/physiology , Vero Cells , Viral Load/drug effects , Viral Plaque Assay , Virus Replication/drug effects
13.
Biochem J ; 478(13): 2481-2497, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1289949

ABSTRACT

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Exoribonucleases/antagonists & inhibitors , Methyltransferases/antagonists & inhibitors , RNA Caps/metabolism , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/chemistry , Chlorobenzenes/pharmacology , Chlorocebus aethiops , Enzyme Assays , Exoribonucleases/genetics , Exoribonucleases/isolation & purification , Exoribonucleases/metabolism , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Indazoles/pharmacology , Indenes/pharmacology , Indoles/pharmacology , Methyltransferases/genetics , Methyltransferases/isolation & purification , Methyltransferases/metabolism , Nitriles/pharmacology , Phenothiazines/pharmacology , Purines/pharmacology , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Substrate Specificity , Trifluperidol/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/isolation & purification , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/isolation & purification , Viral Regulatory and Accessory Proteins/metabolism
14.
Sci Adv ; 7(25)2021 06.
Article in English | MEDLINE | ID: covidwho-1276873

ABSTRACT

Infection by highly pathogenic coronaviruses results in substantial apoptosis. However, the physiological relevance of apoptosis in the pathogenesis of coronavirus infections is unknown. Here, with a combination of in vitro, ex vivo, and in vivo models, we demonstrated that protein kinase R-like endoplasmic reticulum kinase (PERK) signaling mediated the proapoptotic signals in Middle East respiratory syndrome coronavirus (MERS-CoV) infection, which converged in the intrinsic apoptosis pathway. Inhibiting PERK signaling or intrinsic apoptosis both alleviated MERS pathogenesis in vivo. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV induced apoptosis through distinct mechanisms but inhibition of intrinsic apoptosis similarly limited SARS-CoV-2- and SARS-CoV-induced apoptosis in vitro and markedly ameliorated the lung damage of SARS-CoV-2-inoculated human angiotensin-converting enzyme 2 (hACE2) mice. Collectively, our study provides the first evidence that virus-induced apoptosis is an important disease determinant of highly pathogenic coronaviruses and demonstrates that this process can be targeted to attenuate disease severity.


Subject(s)
Antiviral Agents/pharmacology , Apoptosis/drug effects , COVID-19/drug therapy , Coronavirus Infections/drug therapy , eIF-2 Kinase/metabolism , Adenine/analogs & derivatives , Adenine/pharmacology , Angiotensin-Converting Enzyme 2/genetics , Animals , Apoptosis/physiology , COVID-19/etiology , COVID-19/pathology , Cell Line , Coronavirus Infections/etiology , Coronavirus Infections/pathology , Dipeptidyl Peptidase 4/genetics , Epithelial Cells/virology , Female , Humans , Indoles/pharmacology , Lung/virology , Male , Mice, Transgenic , eIF-2 Kinase/antagonists & inhibitors , eIF-2 Kinase/genetics
15.
Comput Biol Med ; 135: 104568, 2021 08.
Article in English | MEDLINE | ID: covidwho-1267638

ABSTRACT

The disease outbreak of Coronavirus disease-19 (COVID-19), caused by the novel SARS-CoV-2 virus, remains a public health concern. COVID-19 is spreading rapidly with a high mortality rate due to unavailability of effective treatment or vaccine for the disease. The high rate of mutation and recombination in SARS-CoV2 makes it difficult for scientist to develop specific anti-CoV2 drugs and vaccines. SARS-CoV-2-Mpro cleaves the viral polyprotein to produce a variety of non-structural proteins, but in human host it also cleaves the nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), which suppresses the activation of the NF-κB pathway and weakens the immune response. Since the main protease (Mpro) is required for viral gene expression and replication, it is a promising target for antagonists to treat novel coronavirus disease and discovery of high resolution crystal structure of SARS-CoV-2-Mpro provide an opportunity for in silico identification of its possible inhibitors. In this study we intend to find novel and potential Mpro inhibitors from around 1830 chemically diverse and therapeutically important secondary metabolites available in the MeFSAT database by performing molecular docking against the Mpro structure of SARS-CoV-2 (PDB ID: 6LZE). After ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and binding energy calculation through MM-GBSA for top five hits, Sterenin M was proposed as a SARS-CoV2-Mpro inhibitor with validation of molecular dynamics (MD) simulation study. Sterenin M seems to have the potential to be a promising ligand against SARS-CoV-2, and thus it requires further validation by in vitro and in vivo studies.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Indoles/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Molecular Docking Simulation , Molecular Dynamics Simulation , RNA, Viral
16.
Bratisl Lek Listy ; 121(10): 705-711, 2020.
Article in English | MEDLINE | ID: covidwho-1231555

ABSTRACT

BACKGROUND: COVID-19 is not fully known and causes severe inflammation and cytokine storm. It has many symptoms, such as: fever, sore throat, headache, dyspnoea, and diarrhoea. Arbidol was used in the treatment of COVID19, which was the most critical health problem in the world. However, the desired recovery was not achieved with Arbidol. Many countries still use this drug in the treatment of COVID19. AIM: We aimed to determine whether Arbidol, the hemagglutinin esterase inhibitor used in the treatment of COVID-19, was effective against SARS Cov-2 in silico. RESULTS AND CONCLUSION: The similarity between hemagglutinin and spike proteins were reported due to the fact that inhibition properties of Arbidol and its 39 analogues were examined in detail against hemagglutinin esterase and spike glycoproteins. CID 1070884 and CID 1207786 were found to be more active against hemagglutinin esterase than in Arbidol, while these compounds were inactive against spike glycoproteins. The interaction mechanism was clarified between arbidol and spike proteins. Phenylalanine, tyrosine, glycine, lysine, and aspartic acid were found to be the headliner amino acids in the interactions between Arbidol and binding domains of spike glycoproteins in the SARS-CoV2 (Tab. 3, Fig. 8, Ref. 28).


Subject(s)
Betacoronavirus/drug effects , Indoles/pharmacology , Binding Sites , COVID-19 , Coronavirus Infections , Humans , Pandemics , Pneumonia, Viral , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/antagonists & inhibitors
17.
Viruses ; 13(4)2021 04 09.
Article in English | MEDLINE | ID: covidwho-1178430

ABSTRACT

Photodynamic inactivation of pathogenic microorganisms can be successfully used to eradicate pathogens in localized lesions, infected liquid media, and on various surfaces. This technique utilizes the photosensitizer (PS), light, and molecular oxygen to produce reactive oxygen species that kill pathogens. Here, we used the PS, water soluble octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+), to inactivate an initial 4.75-5.00 IgTCID50/mL titer of SARS-CoV-2, thereby preventing viral infection when tested in Vero E6 cell cultures. Zn-PcChol8+ in a minimally studied concentration, 1 µM and LED 3.75 J/cm2, completely destroyed the infectivity of SARS-CoV-2. To detect possible PS binding sites on the envelope of SARS-CoV-2, we analyzed electrostatic potential and simulated binding of Zn-PcChol8+ to the spike protein of this coronavirus by means of Brownian dynamics software, ProKSim (Protein Kinetics Simulator). Most of the Zn-PcChol8+ molecules formed clusters at the upper half of the stalk within a vast area of negative electrostatic potential. Positioning of the PS on the surface of the spike protein at a distance of no more than 10 nm from the viral membrane may be favorable for the oxidative damage. The high sensitivity of SARS-CoV-2 to photodynamic inactivation by Zn-PcChol8+ is discussed with respect to the application of this PS to control the spread of COVID-19.


Subject(s)
Indoles/pharmacology , Organometallic Compounds/pharmacology , Photosensitizing Agents/pharmacology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Virus Inactivation/drug effects , Animals , COVID-19/prevention & control , Chlorocebus aethiops , Indoles/chemistry , Isoindoles , Light , Molecular Dynamics Simulation , Organometallic Compounds/chemistry , Photosensitizing Agents/chemistry , Vero Cells , Zinc Compounds
18.
Cell Death Dis ; 12(4): 310, 2021 03 24.
Article in English | MEDLINE | ID: covidwho-1149708

ABSTRACT

SARS-CoV-2 is responsible for the ongoing world-wide pandemic which has already taken more than two million lives. Effective treatments are urgently needed. The enzymatic activity of the HECT-E3 ligase family members has been implicated in the cell egression phase of deadly RNA viruses such as Ebola through direct interaction of its VP40 Protein. Here we report that HECT-E3 ligase family members such as NEDD4 and WWP1 interact with and ubiquitylate the SARS-CoV-2 Spike protein. Furthermore, we find that HECT family members are overexpressed in primary samples derived from COVID-19 infected patients and COVID-19 mouse models. Importantly, rare germline activating variants in the NEDD4 and WWP1 genes are associated with severe COVID-19 cases. Critically, I3C, a natural NEDD4 and WWP1 inhibitor from Brassicaceae, displays potent antiviral effects and inhibits viral egression. In conclusion, we identify the HECT family members of E3 ligases as likely novel biomarkers for COVID-19, as well as new potential targets of therapeutic strategy easily testable in clinical trials in view of the established well-tolerated nature of the Brassicaceae natural compounds.


Subject(s)
COVID-19/drug therapy , COVID-19/enzymology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/metabolism , Adult , Aged , Animals , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/metabolism , Chlorocebus aethiops , Endosomal Sorting Complexes Required for Transport/metabolism , Female , Humans , Indoles/pharmacology , Male , Mice , Mice, Inbred BALB C , Middle Aged , Nedd4 Ubiquitin Protein Ligases/genetics , Nedd4 Ubiquitin Protein Ligases/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Vero Cells
19.
Molecules ; 26(6)2021 Mar 17.
Article in English | MEDLINE | ID: covidwho-1138745

ABSTRACT

The COVID-19 outbreak continues to spread worldwide at a rapid rate. Currently, the absence of any effective antiviral treatment is the major concern for the global population. The reports of the occurrence of various point mutations within the important therapeutic target protein of SARS-CoV-2 has elevated the problem. The SARS-CoV-2 main protease (Mpro) is a major therapeutic target for new antiviral designs. In this study, the efficacy of PF-00835231 was investigated (a Mpro inhibitor under clinical trials) against the Mpro and their reported mutants. Various in silico approaches were used to investigate and compare the efficacy of PF-00835231 and five drugs previously documented to inhibit the Mpro. Our study shows that PF-00835231 is not only effective against the wild type but demonstrates a high affinity against the studied mutants as well.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Indoles/chemistry , Indoles/pharmacology , Leucine/chemistry , Leucine/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Binding Sites , COVID-19/drug therapy , Computer Simulation , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Databases, Protein , Diarylquinolines/chemistry , Diarylquinolines/pharmacology , Dihydropyridines/chemistry , Dihydropyridines/pharmacology , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Nitrobenzenes/chemistry , Nitrobenzenes/pharmacology , Nitrophenols/chemistry , Nitrophenols/pharmacology , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Proline/analogs & derivatives , Proline/chemistry , Proline/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics
20.
Nat Commun ; 12(1): 1517, 2021 03 09.
Article in English | MEDLINE | ID: covidwho-1125914

ABSTRACT

Up to date, effective antivirals have not been widely available for treating COVID-19. In this study, we identify a dual-functional cross-linking peptide 8P9R which can inhibit the two entry pathways (endocytic pathway and TMPRSS2-mediated surface pathway) of SARS-CoV-2 in cells. The endosomal acidification inhibitors (8P9R and chloroquine) can synergistically enhance the activity of arbidol, a spike-ACE2 fusion inhibitor, against SARS-CoV-2 and SARS-CoV in cells. In vivo studies indicate that 8P9R or the combination of repurposed drugs (umifenovir also known as arbidol, chloroquine and camostat which is a TMPRSS2 inhibitor), simultaneously interfering with the two entry pathways of coronaviruses, can significantly suppress SARS-CoV-2 replication in hamsters and SARS-CoV in mice. Here, we use drug combination (arbidol, chloroquine, and camostat) and a dual-functional 8P9R to demonstrate that blocking the two entry pathways of coronavirus can be a promising and achievable approach for inhibiting SARS-CoV-2 replication in vivo. Cocktail therapy of these drug combinations should be considered in treatment trials for COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning , Peptides/pharmacology , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Animals , COVID-19/virology , Chlorocebus aethiops , Chloroquine/pharmacology , Drug Discovery , Female , HEK293 Cells , Humans , Indoles/pharmacology , Mice , Mice, Inbred BALB C , Serine Endopeptidases/metabolism , Vero Cells
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