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1.
Biomolecules ; 12(11)2022 Nov 11.
Article in English | MEDLINE | ID: covidwho-2109923

ABSTRACT

BACKGROUND: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants. HYPOTHESIS: We hypothesized that in SARS-CoV-2, two highly conserved Orf3a and E channels directly related to the virus replication were a target for the detection and inhibition of the viral replication, independent of the variant, using FDA-approved ion channel modulators. METHODS: A combination of a fluorescence potassium ion assay with channel modulators was developed to detect SARS-CoV-2 Orf3a/E channel activity. Two FDA-approved drugs, amantadine (an antiviral) and amitriptyline (an antidepressant), which are ion channel blockers, were tested as to whether they inhibited Orf3a/E channel activity in isolated virus variants and in nasal swab samples from COVID-19 patients. The variants were confirmed by PCR sequencing. RESULTS: In isolated SARS-CoV-2 Alpha, Beta, and Delta variants, the channel activity of Orf3a/E was detected and inhibited by emodin and gliclazide (IC50 = 0.42 mM). In the Delta swab samples, amitriptyline and amantadine inhibited the channel activity of viral proteins, with IC50 values of 0.73 mM and 1.11 mM, respectively. In the Omicron swab samples, amitriptyline inhibited the channel activity, with an IC50 of 0.76 mM. CONCLUSIONS: We developed an efficient method to screen FDA-approved ion channel modulators that could be repurposed to detect and inhibit SARS-CoV-2 viral replication, independent of variants.


Subject(s)
COVID-19 , Ion Channels , SARS-CoV-2 , Humans , Amantadine/pharmacology , Amitriptyline/pharmacology , COVID-19/drug therapy , Ion Channels/antagonists & inhibitors , SARS-CoV-2/drug effects , Drug Evaluation, Preclinical , Drug Repositioning
2.
Viruses ; 14(11)2022 Nov 06.
Article in English | MEDLINE | ID: covidwho-2099866

ABSTRACT

New variants of SARS-CoV-2 continue to evolve. The novel SARS-CoV-2 variant of concern (VOC) B.1.1.529 (Omicron) was particularly menacing due to the presence of numerous consequential mutations. In this study, we reviewed about 12 million SARS-CoV-2 genomic and associated metadata using extensive bioinformatic approaches to understand how evolutionary and mutational changes affect Omicron variant properties. Subsampled global data based analysis of molecular clock in the phylogenetic tree showed 29.56 substitutions per year as the evolutionary rate of five VOCs. We observed extensive mutational changes in the spike structural protein of the Omicron variant. A total of 20% of 7230 amino acid and structural changes exclusive to Omicron's spike protein were detected in the receptor binding domain (RBD), suggesting differential selection pressures exerted during evolution. Analyzing key drug targets revealed mutation-derived differential binding affinities between Delta and Omicron variants. Nine single-RBD substitutions were detected within the binding site of approved therapeutic monoclonal antibodies. T-cell epitope prediction revealed eight immunologically important functional hotspots in three conserved non-structural proteins. A universal vaccine based on these regions may likely protect against all these SARS-CoV-2 variants. We observed key structural changes in the spike protein, which decreased binding affinities, indicating that these changes may help the virus escape host cellular immunity. These findings emphasize the need for continuous genomic surveillance of SARS-CoV-2 to better understand how novel mutations may impact viral spread and disease outcome.


Subject(s)
Antiviral Agents , COVID-19 , Immune Evasion , SARS-CoV-2 , Humans , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/immunology , COVID-19/virology , Mutation , Phylogeny , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Envelope Proteins/genetics
3.
Biochemistry ; 61(22): 2495-2505, 2022 Nov 15.
Article in English | MEDLINE | ID: covidwho-2096611

ABSTRACT

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and has been the focus of many drug discovery efforts since the start of the COVID-19 pandemic. Nirmatrelvir (NTV) is an inhibitor of SARS-CoV-2 Mpro that is used in the combination drug Paxlovid for the treatment of mild to moderate COVID-19. However, with increased use of NTV across the globe, there is a possibility that future SARS-CoV-2 lineages will evolve resistance to NTV. Early prediction and monitoring of resistance mutations could allow for measures to slow the spread of resistance and for the development of new compounds with activity against resistant strains. In this work, we have used in silico mutational scanning and inhibitor docking of Mpro to identify potential resistance mutations. Subsequent in vitro experiments revealed five mutations (N142L, E166M, Q189E, Q189I, and Q192T) that reduce the potency of NTV and of a previously identified non-covalent cyclic peptide inhibitor of Mpro. The E166M mutation reduced the half-maximal inhibitory concentration (IC50) of NTV 24-fold and 118-fold for the non-covalent peptide inhibitor. Our findings inform the ongoing genomic surveillance of emerging SARS-CoV-2 lineages.


Subject(s)
Antiviral Agents , COVID-19 , Coronavirus 3C Proteases , Drug Resistance, Viral , Protease Inhibitors , SARS-CoV-2 , Humans , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/virology , Molecular Docking Simulation , Mutation , Pandemics , Protease Inhibitors/pharmacology , Protease Inhibitors/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Drug Resistance, Viral/genetics , Coronavirus 3C Proteases/antagonists & inhibitors
4.
Antiviral Res ; 208: 105428, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-2068666

ABSTRACT

The continuous emergence of SARS-CoV-2 variants prolongs COVID-19 pandemic. Although SARS-CoV-2 vaccines and therapeutics are currently available, there is still a need for development of safe and effective drugs against SARS-CoV-2 and also for preparedness for the next pandemic. Here, we discover that astersaponin I (AI), a triterpenoid saponin in Aster koraiensis inhibits SARS-CoV-2 entry pathways at the plasma membrane and within the endosomal compartments mainly by increasing cholesterol content in the plasma membrane and interfering with the fusion of SARS-CoV-2 envelope with the host cell membrane. Moreover, we find that this functional property of AI as a fusion blocker enables it to inhibit the infection with SARS-CoV-2 variants including the Alpha, Beta, Delta, and Omicron with a similar efficacy, and the formation of syncytium, a multinucleated cells driven by SARS-CoV-2 spike protein-mediated cell-to-cell fusion. Finally, we claim that the triterpene backbone as well as the attached hydrophilic sugar moieties of AI are structurally important for its inhibitory activity against the membrane fusion event. Overall, this study demonstrates that AI is a natural viral fusion inhibitor and proposes that it can be a broad-spectrum antiviral agent against current COVID-19 pandemic and future outbreaks of novel viral pathogens.


Subject(s)
COVID-19 , SARS-CoV-2 , Saponins , Humans , COVID-19/drug therapy , COVID-19 Vaccines , Giant Cells , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Asteraceae/chemistry , Saponins/pharmacology
5.
J Nat Prod ; 85(11): 2583-2591, 2022 Nov 25.
Article in English | MEDLINE | ID: covidwho-2062146

ABSTRACT

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 µM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 µM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.


Subject(s)
Antiviral Agents , Azo Compounds , COVID-19 , Influenza A Virus, H1N1 Subtype , SARS-CoV-2 , Streptomyces , Humans , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Azo Compounds/chemistry , Azo Compounds/metabolism , Azo Compounds/pharmacology , Heat-Shock Response , HEK293 Cells , Influenza A Virus, H1N1 Subtype/drug effects , Influenza, Human/drug therapy , Madin Darby Canine Kidney Cells , Orthomyxoviridae Infections/drug therapy , SARS-CoV-2/drug effects , Streptomyces/chemistry , Streptomyces/metabolism , Vero Cells , Chlorocebus aethiops , Dogs
6.
Bioorg Chem ; 129: 106185, 2022 12.
Article in English | MEDLINE | ID: covidwho-2060449

ABSTRACT

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. Mpro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of Mpro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against Mpro with IC50 of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 Mpro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of Mpro, and ACF has been previously reported as a PLpro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.


Subject(s)
Acriflavine , COVID-19 , Coronavirus 3C Proteases , Cysteine Proteinase Inhibitors , Proflavine , SARS-CoV-2 , Viral Protease Inhibitors , Acriflavine/pharmacology , COVID-19/drug therapy , Proflavine/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Viral Protease Inhibitors/pharmacology , Mesocricetus , Animals , Cricetinae , Cell Line , Virus Replication/drug effects
7.
Food Chem Toxicol ; 169: 113438, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2049210

ABSTRACT

High infection caused by mutations of SARS-CoV-2 calls for new prevention strategy. Ganoderma lucidum known as a superior immunoenhancer exhibits various antiviral effects, whether it can resist SARS-CoV-2 remains unclear. Herein, virtual screening combined with in vitro hACE2 inhibition assays were used to investigate its anti SARS-CoV-2 effect. Potential 54 active components, 80 core targets and 20 crucial pathways were identified by the component-target-pathway network. The binding characters of these components to hACE2 and its complexes with spike protein including omicron variant was analyzed by molecular docking. Lucidenic acid A was selected as the top molecule with high affinity to all receptors by forming hydrogen bonds. Molecular dynamics simulation showed it had good binding stability with the receptor proteins. Finally, in vitro FRET test demonstrated it inhibited the hACE2 activity with IC50 2 µmol/mL. Therefore, lucidenic acid A can prevent the virus invasion by blocking hACE2 binding with SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2 , Antiviral Agents , COVID-19 , Cholic Acids , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Virus Internalization , Humans , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Cholic Acids/pharmacology , COVID-19/prevention & control , Molecular Docking Simulation , Protein Binding , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , Reishi/chemistry
9.
Biomolecules ; 12(9)2022 09 15.
Article in English | MEDLINE | ID: covidwho-2043569

ABSTRACT

Novel and efficient strategies need to be developed to interfere with the SARS-CoV-2 virus. One of the most promising pharmaceutical targets is the nucleocapsid protein (N), responsible for genomic RNA packaging. N is composed of two folded domains and three intrinsically disordered regions (IDRs). The globular RNA binding domain (NTD) and the tethered IDRs are rich in positively charged residues. The study of the interaction of N with polyanions can thus help to elucidate one of the key driving forces responsible for its function, i.e., electrostatics. Heparin, one of the most negatively charged natural polyanions, has been used to contrast serious cases of COVID-19 infection, and we decided to study its interaction with N at the molecular level. We focused on the NTR construct, which comprises the NTD and two flanking IDRs, and on the NTD construct in isolation. We characterized this interaction using different nuclear magnetic resonance approaches and isothermal titration calorimetry. With these tools, we were able to identify an extended surface of NTD involved in the interaction. Moreover, we assessed the importance of the IDRs in increasing the affinity for heparin, highlighting how different tracts of these flexible regions modulate the interaction.


Subject(s)
Enoxaparin , Nucleocapsid Proteins , SARS-CoV-2 , COVID-19 , Enoxaparin/pharmacology , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Polyelectrolytes , RNA , SARS-CoV-2/drug effects
10.
Mar Drugs ; 20(9)2022 Sep 19.
Article in English | MEDLINE | ID: covidwho-2039913

ABSTRACT

SARS-CoV-2 is the causative agent of the COVID-19 pandemic. This in silico study aimed to elucidate therapeutic efficacies against SARS-CoV-2 of phyco-compounds from the seaweed, Ulva fasciata. Twelve phyco-compounds were isolated and toxicity was analyzed by VEGA QSAR. Five compounds were found to be nonmutagenic, noncarcinogenic and nontoxic. Moreover, antiviral activity was evaluated by PASS. Binding affinities of five of these therapeutic compounds were predicted to possess probable biological activity. Fifteen SARS-CoV-2 target proteins were analyzed by the AutoDock Vina program for molecular docking binding energy analysis and the 6Y84 protein was determined to possess optimal binding affinities. The Desmond program from Schrödinger's suite was used to study high performance molecular dynamic simulation properties for 3,7,11,15-Tetramethyl-2-hexadecen-1-ol-6Y84 for better drug evaluation. The ligand with 6Y84 had stronger binding affinities (-5.9 kcal/mol) over two standard drugs, Chloroquine (-5.6 kcal/mol) and Interferon α-2b (-3.8 kcal/mol). Swiss ADME calculated physicochemical/lipophilicity/water solubility/pharmacokinetic properties for 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, showing that this therapeutic agent may be effective against SARS-CoV-2.


Subject(s)
Antiviral Agents , SARS-CoV-2 , Ulva , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Chloroquine , Fatty Alcohols/chemistry , Fatty Alcohols/pharmacology , Humans , Interferon-alpha , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , SARS-CoV-2/drug effects , Terpenes/chemistry , Terpenes/pharmacology , Ulva/chemistry
11.
Proc Natl Acad Sci U S A ; 119(40): e2210990119, 2022 10 04.
Article in English | MEDLINE | ID: covidwho-2037061

ABSTRACT

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Peptides/chemistry , Peptides/pharmacology , SARS-CoV-2/drug effects
12.
Phys Chem Chem Phys ; 24(37): 22898-22904, 2022 Sep 28.
Article in English | MEDLINE | ID: covidwho-2036937

ABSTRACT

Coronavirus 3C-like protease (3CLpro) is found in SARS-CoV-2 virus, which causes COVID-19. 3CLpro controls virus replication and is a major target for target-based antiviral discovery. As reported by Pfizer, Nirmatrelvir (PF-07321332) is a competitive protein inhibitor and a clinical candidate for orally delivered medication. However, the binding mechanisms between Nirmatrelvir and 3CLpro complex structures remain unknown. This study incorporated ligand Gaussian accelerated molecular dynamics, the one-dimensional and two-dimensional potential of mean force, normal molecular dynamics, and Kramers' rate theory to determine the binding and dissociation rate constants (koff and kon) associated with the binding of the 3CLpro protein to the Nirmatrelvir inhibitor. The proposed approach addresses the challenges in designing small-molecule antiviral drugs.


Subject(s)
Antiviral Agents , Coronavirus 3C Proteases , SARS-CoV-2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Endopeptidases/metabolism , Lactams , Leucine , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Nitriles , Peptide Hydrolases/metabolism , Proline , SARS-CoV-2/drug effects
14.
J Nanobiotechnology ; 20(1): 411, 2022 Sep 15.
Article in English | MEDLINE | ID: covidwho-2029712

ABSTRACT

The major challenge to controlling the COVID pandemic is the rapid mutation rate of the SARS-CoV-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is essential to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-CoV-2 variants. Here, we report a synthetic nanobody (named C5G2) obtained by phage display and subsequent antibody engineering. C5G2 has a single-digit nanomolar binding affinity to the RBD domain and inhibits its binding to ACE2 with an IC50 of 3.7 nM. Pseudovirus assays indicated that monovalent C5G2 could protect the cells from infection with SARS-CoV-2 wild-type virus and most of the viruses of concern, i.e., Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against Omicron BA.1 among all the variants, with an IC50 of 4.9 ng/mL. The cryo-EM structure of C5G2 in complex with the spike trimer showed that C5G2 binds to RBD mainly through its CDR3 at a conserved region that does not overlap with the ACE2 binding surface. Additionally, C5G2 binds simultaneously to the neighboring NTD domain of the spike trimer through the same CDR3 loop, which may further increase its potency against viral infection. Third, the steric hindrance caused by FR2 of C5G2 could inhibit the binding of ACE2 to RBD as well. Thus, this triple-function nanobody may serve as an effective drug for prophylaxis and therapy against Omicron as well as future variants.


Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , SARS-CoV-2 , Single-Domain Antibodies , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , COVID-19 , SARS-CoV-2/drug effects , Single-Domain Antibodies/pharmacology , Spike Glycoprotein, Coronavirus
15.
J Mol Biol ; 434(16): 167720, 2022 08 30.
Article in English | MEDLINE | ID: covidwho-2028233

ABSTRACT

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.


Subject(s)
ADP-Ribosylation , Adenosine/analogs & derivatives , Coronavirus Protease Inhibitors , Poly(ADP-ribose) Polymerases , SARS-CoV-2 , ADP-Ribosylation/drug effects , Adenosine/chemistry , Adenosine/pharmacology , Adenosine Diphosphate Ribose/chemistry , Coronavirus Protease Inhibitors/chemistry , Coronavirus Protease Inhibitors/pharmacology , Humans , Poly(ADP-ribose) Polymerases/chemistry , Protein Binding , Protein Domains , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
16.
Biochemistry ; 61(18): 1966-1973, 2022 09 20.
Article in English | MEDLINE | ID: covidwho-2016509

ABSTRACT

Remdesivir is an adenosine analogue that has a cyano substitution in the C1' position of the ribosyl moiety and a modified base structure to stabilize the linkage of the base to the C1' atom with its strong electron-withdrawing cyano group. Within the replication-transcription complex (RTC) of SARS-CoV-2, the RNA-dependent RNA polymerase nsp12 selects remdesivir monophosphate (RMP) over adenosine monophosphate (AMP) for nucleotide incorporation but noticeably slows primer extension after the added RMP of the RNA duplex product is translocated by three base pairs. Cryo-EM structures have been determined for the RTC with RMP at the nucleotide-insertion (i) site or at the i + 1, i + 2, or i + 3 sites after product translocation to provide a structural basis for a delayed-inhibition mechanism by remdesivir. In this study, we applied molecular dynamics (MD) simulations to extend the resolution of structures to the measurable maximum that is intrinsically limited by MD properties of these complexes. Our MD simulations provide (i) a structural basis for nucleotide selectivity of the incoming substrates of remdesivir triphosphate over adenosine triphosphate and of ribonucleotide over deoxyribonucleotide, (ii) new detailed information on hydrogen atoms involved in H-bonding interactions between the enzyme and remdesivir, and (iii) direct information on the catalytically active complex that is not easily captured by experimental methods. Our improved resolution of interatomic interactions at the nucleotide-binding pocket between remedesivir and the polymerase could help to design a new class of anti-SARS-CoV-2 inhibitors.


Subject(s)
Adenosine Triphosphate , Antiviral Agents , SARS-CoV-2 , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Alanine/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase , Deoxyribonucleotides , Hydrogen , Nucleotides , RNA, Viral/genetics , Ribonucleotides , SARS-CoV-2/drug effects
17.
J Mol Model ; 28(9): 279, 2022 Aug 29.
Article in English | MEDLINE | ID: covidwho-2014173

ABSTRACT

Main protease (Mpro) plays a key role in replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study was designed for finding natural inhibitors of SARS-CoV-2 Mpro by in silico methods. To this end, the co-crystal structure of Mpro with telaprevir was explored and receptor-ligand pharmacophore models were developed and validated using pharmit. The database of "ZINC Natural Products" was screened, and 288 compounds were filtered according to pharmacophore features. In the next step, Lipinski's rule of five was applied and absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the filtered compounds were calculated using in silico methods. The resulted 15 compounds were docked into the active site of Mpro and those with the highest binding scores and better interaction including ZINC61991204, ZINC67910260, ZINC61991203, and ZINC08790293 were selected. Further analysis by molecular dynamic simulation studies showed that ZINC61991203 and ZINC08790293 dissociated from Mpro active site, while ZINC426421106 and ZINC5481346 were stable. Root mean square deviation (RMSD), radius of gyration (Rg), number of hydrogen bonds between ligand and protein during the time of simulation, and root mean square fluctuations (RMSF) of protein and ligands were calculated, and components of binding free energy were calculated using the molecular mechanic/Poisson-Boltzmann surface area (MM/PBSA) method. The result of all the analysis indicated that ZINC61991204 and ZINC67910260 are drug-like and nontoxic and have a high potential for inhibiting Mpro.


Subject(s)
Coronavirus 3C Proteases , Protease Inhibitors , SARS-CoV-2 , COVID-19 , Coronavirus 3C Proteases/antagonists & inhibitors , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects
18.
Front Cell Infect Microbiol ; 12: 965273, 2022.
Article in English | MEDLINE | ID: covidwho-2005850

ABSTRACT

Purpose: The Corona Virus Disease 2019 (COVID-19) pandemic has become a challenge of world. The latest research has proved that Xuanfei Baidu granule (XFBD) significantly improved patient's clinical symptoms, the compound drug improves immunity by increasing the number of white blood cells and lymphocytes, and exerts anti-inflammatory effects. However, the analysis of the effective monomer components of XFBD and its mechanism of action in the treatment of COVID-19 is currently lacking. Therefore, this study used computer simulation to study the effective monomer components of XFBD and its therapeutic mechanism. Methods: We screened out the key active ingredients in XFBD through TCMSP database. Besides GeneCards database was used to search disease gene targets and screen intersection gene targets. The intersection gene targets were analyzed by GO and KEGG. The disease-core gene target-drug network was analyzed and molecular docking was used for verification. Molecular dynamics simulation verification was carried out to combine the active ingredient and the target with a stable combination. The supercomputer platform was used to measure and analyze the number of hydrogen bonds, the binding free energy, the stability of protein target at the residue level, the solvent accessible surface area, and the radius of gyration. Results: XFBD had 1308 gene targets, COVID-19 had 4600 gene targets, the intersection gene targets were 548. GO and KEGG analysis showed that XFBD played a vital role by the signaling pathways of immune response and inflammation. Molecular docking showed that I-SPD, Pachypodol and Vestitol in XFBD played a role in treating COVID-19 by acting on NLRP3, CSF2, and relieve the clinical symptoms of SARS-CoV-2 infection. Molecular dynamics was used to prove the binding stability of active ingredients and protein targets, CSF2/I-SPD combination has the strongest binding energy. Conclusion: For the first time, it was found that the important active chemical components in XFBD, such as I-SPD, Pachypodol and Vestitol, reduce inflammatory response and apoptosis by inhibiting the activation of NLRP3, and reduce the production of inflammatory factors and chemotaxis of inflammatory cells by inhibiting the activation of CSF2. Therefore, XFBD can effectively alleviate the clinical symptoms of COVID-19 through NLRP3 and CSF2.


Subject(s)
COVID-19 , Drugs, Chinese Herbal , SARS-CoV-2 , COVID-19/drug therapy , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Humans , Medicine, Chinese Traditional , Molecular Docking Simulation , Molecular Dynamics Simulation , NLR Family, Pyrin Domain-Containing 3 Protein , SARS-CoV-2/drug effects
19.
Microbiol Spectr ; 10(5): e0105622, 2022 Oct 26.
Article in English | MEDLINE | ID: covidwho-2001788

ABSTRACT

Infection by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has posed a severe threat to global public health. The current study revealed that several inhibitors of protein kinases C (PKCs) possess protective activity against SARS-CoV-2 infection. Four pan-PKC inhibitors, Go 6983, bisindolylmaleimide I, enzastaurin, and sotrastaurin, reduced the replication of a SARS-CoV-2 replicon in both BHK-21 and Huh7 cells. A PKCδ-specific inhibitor, rottlerin, was also effective in reducing viral infection. The PKC inhibitors acted at an early step of SARS-CoV-2 infection. Finally, PKC inhibitors blocked the replication of wild-type SARS-CoV-2 in ACE2-expressing A549 cells. Our work highlights the importance of the PKC signaling pathway in infection by SARS-CoV-2 and provides evidence that PKC-specific inhibitors are potential therapeutic agents against SARS-CoV-2. IMPORTANCE There is an urgent need for effective therapeutic drugs to control the pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We found that several inhibitors of protein kinases C (PKCs) dramatically decrease the replication of SARS-CoV-2 in cultured cells. These PKC inhibitors interfere with an early step of viral infection. Therefore, the rapid and prominent antiviral effect of PKC inhibitors underscores that they are promising antiviral agents and suggests that PKCs are important host factors involved in infection by SARS-CoV-2.


Subject(s)
Antiviral Agents , Protein Kinase C , SARS-CoV-2 , Humans , Angiotensin-Converting Enzyme 2 , Antiviral Agents/pharmacology , Cells, Cultured , COVID-19/drug therapy , Protein Kinase C/pharmacology , SARS-CoV-2/drug effects
20.
Sci Rep ; 12(1): 14438, 2022 08 24.
Article in English | MEDLINE | ID: covidwho-2000930

ABSTRACT

The lack of therapeutic options to fight Covid-19 has contributed to the current global pandemic. Despite the emergence of effective vaccines, development of broad-spectrum antiviral treatment remains a significant challenge, in which antimicrobial photodynamic therapy (aPDT) may play a role, especially at early stages of infection. aPDT of the nares with methylene blue (MB) and non-thermal light has been successfully utilized to inactivate both bacterial and viral pathogens in the perioperative setting. Here, we investigated the effect of MB-aPDT to inactivate human betacoronavirus OC43 and SARS-CoV-2 in vitro and in a proof-of-principle COVID-19 clinical trial to test, in a variety of settings, the practicality, technical feasibility, and short-term efficacy of the method. aPDT yielded inactivation of up to 6-Logs in vitro, as measured by RT-qPCR and infectivity assay. From a photo-physics perspective, the in vitro results suggest that the response is not dependent on the virus itself, motivating potential use of aPDT for local destruction of SARS-CoV-2 and its variants. In the clinical trial we observed variable effects on viral RNA in nasal-swab samples as assessed by RT-qPCR attributed to aPDT-induced RNA fragmentation causing falsely-elevated counts. However, the viral infectivity in clinical nares swabs was reduced in 90% of samples and undetectable in 70% of samples. This is the first demonstration based on quantitative clinical viral infectivity measurements that MB-aPDT is a safe, easily delivered and effective front-line technique that can reduce local SARS-CoV-2 viral load.


Subject(s)
COVID-19 , Disinfection , Nose , Photochemotherapy , Anti-Infective Agents/adverse effects , Anti-Infective Agents/pharmacology , COVID-19/drug therapy , Disinfection/methods , Feasibility Studies , Humans , Methylene Blue/adverse effects , Methylene Blue/pharmacology , Nose/virology , Pandemics , RNA, Viral/analysis , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Treatment Outcome , Viral Load/drug effects
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