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1.
Antiviral Res ; 202: 105311, 2022 06.
Article in English | MEDLINE | ID: covidwho-1773103

ABSTRACT

Nelfinavir is an HIV protease inhibitor that has been widely prescribed as a component of highly active antiretroviral therapy, and has been reported to exert in vitro antiviral activity against SARS-CoV-2. We here assessed the effect of Nelfinavir in a SARS-CoV-2 infection model in hamsters. Despite the fact that Nelfinavir, [50 mg/kg twice daily (BID) for four consecutive days], did not reduce viral RNA load and infectious virus titres in the lung of infected animals, treatment resulted in a substantial improvement of SARS-CoV-2-induced lung pathology. This was accompanied by a dense infiltration of neutrophils in the lung interstitium which was similarly observed in non-infected hamsters. Nelfinavir resulted also in a marked increase in activated neutrophils in the blood, as observed in non-infected animals. Although Nelfinavir treatment did not alter the expression of chemoattractant receptors or adhesion molecules on human neutrophils, in vitro migration of human neutrophils to the major human neutrophil attractant CXCL8 was augmented by this protease inhibitor. Nelfinavir appears to induce an immunomodulatory effect associated with increasing neutrophil number and functionality, which may be linked to the marked improvement in SARS-CoV-2 lung pathology independent of its lack of antiviral activity. Since Nelfinavir is no longer used for the treatment of HIV, we studied the effect of two other HIV protease inhibitors, namely the combination Lopinavir/Ritonavir (Kaletra™) in this model. This combination resulted in a similar protective effect as Nelfinavir against SARS-CoV2 induced lung pathology in hamsters.


Subject(s)
COVID-19 , HIV Infections , HIV Protease Inhibitors , Animals , COVID-19/drug therapy , Cricetinae , HIV Infections/drug therapy , HIV Protease Inhibitors/pharmacology , HIV Protease Inhibitors/therapeutic use , Lopinavir/pharmacology , Lopinavir/therapeutic use , Lung , Mesocricetus , Nelfinavir/pharmacology , Nelfinavir/therapeutic use , RNA, Viral , Ritonavir/therapeutic use , SARS-CoV-2
3.
IUBMB Life ; 74(1): 93-100, 2022 01.
Article in English | MEDLINE | ID: covidwho-1353459

ABSTRACT

Unfolded protein response (UPR) and endoplasmic reticulum (ER) stress are aspects of SARS-CoV-2-host cell interaction with proposed role in the cytopathic and inflammatory pathogenesis of this viral infection. The role of the NF-kB pathway in these cellular processes remains poorly characterized. When investigated in VERO-E6 cells, SARS-CoV-2 infection was found to markedly stimulate NF-kB protein expression and activity. NF-kB activation occurs early in the infection process (6 hpi) and it is associated with increased MAPK signaling and expression of the UPR inducer IRE-1α. These signal transduction processes characterize the cellular stress response to the virus promoting a pro-inflammatory environment and caspase activation in the host cell. Inhibition of viral replication by the viral protease inhibitor Nelfinavir reverts all these molecular changes also stimulating c-Jun expression, a key component of the JNK/AP-1 pathway with important role in the IRE-1α-mediated transcriptional regulation of stress response genes with anti-inflammatory and cytoprotection function. The present study demonstrates that UPR signaling and its interaction with cellular MAPKs and the NF-kB activity are important aspects of SARS-CoV-2-host cell interaction that deserve further investigation to identify more efficient therapies for this viral infection.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/metabolism , Endoplasmic Reticulum Stress/drug effects , NF-kappa B/metabolism , SARS-CoV-2 , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , COVID-19/virology , Caspase 9/metabolism , Chlorocebus aethiops , Cytopathogenic Effect, Viral/drug effects , Humans , MAP Kinase Signaling System/drug effects , Models, Biological , Nelfinavir/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Unfolded Protein Response/drug effects , Vero Cells
4.
Nat Commun ; 12(1): 3309, 2021 06 03.
Article in English | MEDLINE | ID: covidwho-1260940

ABSTRACT

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning/methods , Pandemics , SARS-CoV-2 , Animals , COVID-19/prevention & control , COVID-19/virology , Cell Line , Cytidine/administration & dosage , Cytidine/analogs & derivatives , Cytidine/pharmacology , Databases, Pharmaceutical , Drug Discovery/methods , Drug Evaluation, Preclinical/methods , HeLa Cells , High-Throughput Screening Assays/methods , Humans , Hydroxylamines/administration & dosage , Hydroxylamines/pharmacology , Mesocricetus , Nelfinavir/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects
5.
ACS Nano ; 15(1): 857-872, 2021 01 26.
Article in English | MEDLINE | ID: covidwho-997793

ABSTRACT

The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease-ligand complex structures from ∼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 "PPC consensus anchors" (a distinct set observed in each PPC) were observed, of which three "PPC core anchors" EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived "PPC scoring S" to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 µM and an antiviral activity with EC50 of 49.89 µM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 µM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 µM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.


Subject(s)
COVID-19/drug therapy , Coronavirus 3C Proteases/chemistry , Drug Repositioning , SARS-CoV-2/enzymology , Animals , Antiviral Agents/pharmacology , Catalytic Domain , Chlorocebus aethiops , Drug Evaluation, Preclinical , Humans , Inhibitory Concentration 50 , Nelfinavir/pharmacology , Oligopeptides/pharmacology , Protease Inhibitors/pharmacology , Protein Conformation , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
6.
Sci Rep ; 10(1): 20927, 2020 12 01.
Article in English | MEDLINE | ID: covidwho-954796

ABSTRACT

Coronavirus SARS-CoV-2 is a recently discovered single-stranded RNA betacoronavirus, responsible for a severe respiratory disease known as coronavirus disease 2019, which is rapidly spreading. Chinese health authorities, as a response to the lack of an effective therapeutic strategy, started to investigate the use of lopinavir and ritonavir, previously optimized for the treatment and prevention of HIV/AIDS viral infection. Despite the clinical use of these two drugs, no information regarding their possible mechanism of action at the molecular level is still known for SARS-CoV-2. Very recently, the crystallographic structure of the SARS-CoV-2 main protease (Mpro), also known as C30 Endopeptidase, was published. Starting from this essential structural information, in the present work we have exploited supervised molecular dynamics, an emerging computational technique that allows investigating at an atomic level the recognition process of a ligand from its unbound to the final bound state. In this research, we provided molecular insight on the whole recognition pathway of Lopinavir, Ritonavir, and Nelfinavir, three potential C30 Endopeptidase inhibitors, with the last one taken into consideration due to the promising in-vitro activity shown against the structurally related SARS-CoV protease.


Subject(s)
COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Lopinavir/pharmacology , Nelfinavir/pharmacology , Protease Inhibitors/pharmacology , Ritonavir/pharmacology , SARS-CoV-2/drug effects , Antiviral Agents/pharmacology , Drug Combinations , Drug Discovery , Drug Repositioning , Humans , Molecular Dynamics Simulation
7.
J Diabetes ; 13(3): 243-252, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-933955

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is currently posing significant threats to public health worldwide. It is notable that a substantial proportion of patients with sever COVID-19 have coexisting diabetic conditions, indicating the progression and outcome of COVID-19 may relate to diabetes. However, it is still unclear whether diabetic treatment principles can be used for the treatment of COVID-19. METHODS: We conducted a computational approach to screen all commonly used clinical oral hypoglycemic drugs to identify the potential inhibitors for the main protease (Mpro ) of SARS-CoV-2, which is one of the key drug targets for anti-COVID-19 drug discovery. RESULTS: Six antidiabetic drugs with docking scores higher than 8.0 (cutoff value), including repaglinide, canagliflozin, glipizide, gliquidone, glimepiride, and linagliptin, were predicted as the promising inhibitors of Mpro . Interestingly, repaglinide, one of the six antidiabetic drugs with the highest docking score for Mpro , was similar to a previously predicted active molecule nelfinavir, which is a potential anti-HIV and anti-COVID-19 drug. Moreover, we found repaglinide shared similar docking pose and pharmacophores with a reported ligand (N3 inhibitor) and nelfinavir, demonstrating that repaglinide would interact with Mpro in a similar way. CONCLUSION: These results indicated that these six antidiabetic drugs may have an extra effect on the treatment of COVID-19, although further studies are necessary to confirm these findings.


Subject(s)
COVID-19/drug therapy , Hypoglycemic Agents/pharmacology , Viral Matrix Proteins/antagonists & inhibitors , A549 Cells , Antiviral Agents/pharmacology , Binding Sites , Drug Discovery , Humans , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Nelfinavir/pharmacology , Protease Inhibitors/pharmacology
8.
Pharmacol Rep ; 72(6): 1553-1561, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-871625

ABSTRACT

BACKGROUND: A large body of research has focused on fluoroquinolones. It was shown that this class of synthetic antibiotics could possess antiviral activity as a broad range of anti-infective activities. Based on these findings, we have undertaken in silico molecular docking study to demonstrate, for the first time, the principle for the potential evidence pointing ciprofloxacin and moxifloxacin ability to interact with COVID-19 Main Protease. METHODS: In silico molecular docking and molecular dynamics techniques were applied to assess the potential for ciprofloxacin and moxifloxacin interaction with COVID-19 Main Protease (Mpro). Chloroquine and nelfinavir were used as positive controls. RESULTS: We revealed that the tested antibiotics exert strong capacity for binding to COVID-19 Main Protease (Mpro). According to the results obtained from the GOLD docking program, ciprofloxacin and moxifloxacin bind to the protein active site more strongly than the native ligand. When comparing with positive controls, a detailed analysis of the ligand-protein interactions shows that the tested fluoroquinolones exert a greater number of protein interactions than chloroquine and nelfinavir. Moreover, lower binding energy values obtained from KDEEP program were stated when compared to nelfinavir. CONCLUSIONS: Here, we have demonstrated for the first time that ciprofloxacin and moxifloxacin may interact with COVID-19 Main Protease (Mpro).


Subject(s)
COVID-19/drug therapy , Ciprofloxacin/pharmacology , Coronavirus 3C Proteases/drug effects , Moxifloxacin/pharmacology , Antiviral Agents/pharmacology , Binding Sites , COVID-19/virology , Chloroquine/pharmacology , Coronavirus 3C Proteases/metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Nelfinavir/pharmacology , Protein Binding , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
9.
Biomed Res Int ; 2020: 6237160, 2020.
Article in English | MEDLINE | ID: covidwho-868383

ABSTRACT

Coronaviruses have been reported previously due to their association with the severe acute respiratory syndrome (SARS). After SARS, these viruses were known to be causing Middle East respiratory syndrome (MERS) and caused 35% evanescence amid victims pursuing remedial care. Nowadays, beta coronaviruses, members of Coronaviridae, family order Nidovirales, have become subjects of great importance due to their latest pandemic originating from Wuhan, China. The virus named as human-SARS-like coronavirus-2 contains four structural as well as sixteen nonstructural proteins encoded by single-stranded ribonucleic acid of positive polarity. As there is no vaccine available to treat the infection caused by these viruses, there is a dire need for taking necessary steps against this virus. Herein, we have targeted two nonstructural proteins of SARS-CoV-2, namely, methyltransferase (nsp16) and helicase (nsp13), respectively, due to their substantial activity in viral pathogenesis. A total of 2035 compounds were analyzed for their pharmacokinetics and pharmacological properties. The screened 108 compounds were docked against both targeted proteins and were compared with previously reported known compounds. Compounds with high binding affinity were analyzed for their reactivity through DFT analysis, and binding was analyzed using molecular dynamics simulations. Through the analyses performed in this study, it is concluded that EryvarinM, Silydianin, Osajin, and Raddeanine can be considered potential inhibitors for MTase, while TomentodiplaconeB, Osajin, Sesquiterpene Glycoside, Rhamnetin, and Silydianin for helicase after these compounds are validated thoroughly using in vitro and in vivo protocols.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Phytochemicals/chemistry , Phytochemicals/pharmacology , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Antimetabolites/chemistry , Antimetabolites/pharmacology , Antiviral Agents/chemistry , COVID-19/epidemiology , COVID-19/virology , China/epidemiology , Dioxolanes/chemistry , Dioxolanes/pharmacology , Fluoroquinolones/chemistry , Fluoroquinolones/pharmacology , Humans , Methyltransferases/drug effects , Molecular Docking Simulation , Nelfinavir/chemistry , Nelfinavir/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Protein Conformation , RNA Helicases/drug effects , SARS-CoV-2/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
10.
Sci Rep ; 10(1): 16986, 2020 10 12.
Article in English | MEDLINE | ID: covidwho-851312

ABSTRACT

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein-ligand complexes and suggest the possibilities of further drug optimisations.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Cysteine Endopeptidases/metabolism , Drug Repositioning/methods , HIV Protease Inhibitors/pharmacology , Pneumonia, Viral/drug therapy , Viral Nonstructural Proteins/metabolism , Betacoronavirus/metabolism , Binding Sites/drug effects , Biophysical Phenomena , COVID-19 , Catalytic Domain/drug effects , Computational Biology , Coronavirus 3C Proteases , Darunavir/metabolism , Darunavir/pharmacology , HIV Protease Inhibitors/metabolism , Humans , Indinavir/metabolism , Indinavir/pharmacology , Lopinavir/metabolism , Lopinavir/pharmacology , Molecular Dynamics Simulation , Nelfinavir/metabolism , Nelfinavir/pharmacology , Pandemics , Ritonavir/metabolism , Ritonavir/pharmacology , SARS-CoV-2 , Saquinavir/metabolism , Saquinavir/pharmacology
11.
J Med Virol ; 92(10): 2087-2095, 2020 10.
Article in English | MEDLINE | ID: covidwho-763177

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is the causative agent of the coronavirus disease-2019 (COVID-19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV-2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID-19. These results suggest that SARS CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA-approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n- and S-o-mediated cell fusion with complete inhibition at a 10-µM concentration. In-silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n- and S-o-mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV-2 symptoms appear.


Subject(s)
Anti-HIV Agents/pharmacology , Membrane Fusion/drug effects , Nelfinavir/pharmacology , SARS Virus/drug effects , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Animals , Anti-HIV Agents/chemistry , Binding Sites , COVID-19/drug therapy , Cell Fusion , Chlorocebus aethiops , Giant Cells/drug effects , Giant Cells/pathology , Giant Cells/virology , Humans , Molecular Docking Simulation , Nelfinavir/chemistry , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS Virus/pathogenicity , SARS Virus/physiology , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virion/drug effects , Virion/pathogenicity , Virion/physiology
12.
Viruses ; 12(6)2020 06 13.
Article in English | MEDLINE | ID: covidwho-602214

ABSTRACT

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Neutralization Tests/methods , Pneumonia, Viral/drug therapy , Amodiaquine/pharmacology , Animals , COVID-19 , Caco-2 Cells , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Infections/therapy , Drug Therapy, Combination , Emetine/pharmacology , HEK293 Cells , HT29 Cells , Homoharringtonine/pharmacology , Humans , Immune Sera/immunology , Immunization, Passive/methods , Indoles , Nelfinavir/pharmacology , Pandemics , Pyrans/pharmacology , Pyrroles/pharmacology , SARS-CoV-2 , Vero Cells
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