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1.
Biomedicines ; 10(5):1170, 2022.
Article in English | MDPI | ID: covidwho-1857309

ABSTRACT

The natural plant dietary polyphenols 1,2,3,4,6-O-Pentagalloylglucose (PGG) and proanthocyanidin (PAC) have potent antioxidant activity and a variety of pharmacological activities, including antiviral activity. In this study, we examined the inhibitory effect of PGG and PAC on SARS-CoV-2 virus infection, and elucidated its mode of action. PGG and PAC have dose-dependent inhibitory activity against SARS-CoV-2 infection in Vero cells. PGG has a lower IC50 (15.02 ±0.75 μM) than PAC (25.90 ±0.81 μM), suggesting that PGG has better inhibitory activity against SARS-CoV-2 than PAC. The PGG and PAC inhibit similar Mpro activities in a protease activity assay, with IC50 values of 25–26 μM. The effects of PGG and PAC on the activity of the other essential SARS-CoV-2 viral protein, RdRp, were analyzed using a cell-based activity assay system. The activity of RdRp is inhibited by PGG and PAC, and PGG has a lower IC50 (5.098 ±1.089 μM) than PAC (21.022 ±1.202 μM), which is consistent with their inhibitory capacity of SARS-CoV-2 infection. PGG and PAC also inhibit infection by SARS-CoV and MERS-CoV. These data indicate that PGG and PAC may be candidate broad-spectrum anticoronaviral therapeutic agents, simultaneously targeting the Mpro and RdRp proteins of SARS-CoV-2.

2.
Angewandte Chemie ; 134(11):1-6, 2022.
Article in English | Academic Search Complete | ID: covidwho-1718238

ABSTRACT

The emergence of SARS‐CoV‐2 variants is a significant concern in developing effective therapeutics and vaccines in the middle of the ongoing COVID‐19 pandemic. Here, we have identified a novel small molecule that inhibited the interactions between SARS‐CoV‐2 spike RBDs and ACE2 by modulating ACE2 without impairing its enzymatic activity necessary for normal physiological functions. Furthermore, the identified compounds suppressed viral infection in cultured cells by inhibiting the entry of ancestral and variant SARS‐CoV‐2. Our study suggests that targeting ACE2 could be a novel therapeutic strategy to inhibit SARS‐CoV‐2 entry into host cells and prevent the development of COVID‐19. [ FROM AUTHOR] Copyright of Angewandte Chemie is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

3.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-322248

ABSTRACT

Epidemiological studies suggest that men exhibit a higher mortality rate to COVID-19 than women, yet the underlying biology is largely unknown. Here, we seek to delineate sex differences in the gene expression of viral entry proteins ACE2 and TMPRSS2, and host transcriptional responses to SARS-CoV-2 through large-scale analysis of genomic and clinical data. We first compiled 220,000 human gene expression profiles from three databases and completed the meta-information through machine learning and manual annotation. Large scale analysis of these profiles indicated that male samples show higher expression levels of ACE2 and TMPRSS2 than female samples, especially in the older group (>60 years) and in the kidney. Subsequent analysis of 6,031 COVID-19 patients at Mount Sinai Health System revealed that men have significantly higher creatinine levels, an indicator of impaired kidney function. Further analysis of 782 COVID-19 patient gene expression profiles taken from upper airway and blood suggested men and women present distinct expression changes. Computational deconvolution analysis of these profiles revealed male COVID-19 patients have enriched kidney-specific mesangial cells in blood compared to healthy patients. Together, this study suggests biological differences in the kidney between sexes may contribute to sex disparity in COVID-19.

4.
Arch Virol ; 167(3): 871-879, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1680885

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Other coronaviruses (CoVs) can also infect humans, although the majority cause only mild respiratory symptoms. Because early diagnosis of SARS-CoV-2 is critical for preventing further transmission events and improving clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other SARS-related CoVs in respiratory samples. Therefore, we developed and evaluated a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the genes encoding the spike (S) and membrane (M) proteins to enable the rapid identification of SARS-CoV-2, including several new circulating variants and other emerging SARS-like CoVs. By analysis of in vitro-transcribed mRNA, we established multiplex RT-qPCR assays capable of detecting 5 × 10° copies/reaction. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays had a limit of detection of 1 × 10° TCID50/mL and showed no cross-reaction with human CoVs or other respiratory viruses. We also validated our method using human clinical samples from patients with COVID-19 and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic CoVs, including SARS-CoV-2, and may be useful for the identification of other SARS-like CoVs of zoonotic origin.


Subject(s)
COVID-19 , COVID-19/diagnosis , Clinical Laboratory Techniques , Feasibility Studies , Humans , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity
5.
Pharmaceutics ; 14(2)2022 Feb 08.
Article in English | MEDLINE | ID: covidwho-1674760

ABSTRACT

The rhizome of Dryopteris crassirhizoma Nakai. (Dryopteridaceae) has been used in traditional medicine in East Asia and has recently been reported to have anticancer, anti-inflammation, and antibacterial activity as well as antiviral activity. Natural phloroglucinols from D. crassirhizoma, dryocrassin ABBA and filixic acid ABA were reported to inhibit influenza virus infection with an inhibitory activity on neuraminidase. In this study, we found that dryocrassin ABBA and filixic acid ABA have an inhibitory activity against the main protease of SARS-CoV-2. Therefore, dryocrassin ABBA and filixic acid ABA exhibited inhibitory activity against SARS-CoV-2 infection in Vero cells dose-dependently using the immunofluorescence-based antiviral assays. Moreover, these compounds inhibited SARS-CoV and MERS-CoV infection, suggesting their broad-spectrum anticoronaviral activity. In addition, a 5-day repeated-dose toxicity study of dryocrassin ABBA and filixic acid ABA suggested that an approximately lethal dose of these compounds in mice was >10 mg/kg. Pharmacokinetic studies of dryocrassin ABBA showed good microsomal stability, low hERG inhibition, and low CYP450 inhibition. In vivo pharmacokinetic properties of dryocrassin ABBA showed a long half-life (5.5-12.6 h) and high plasma exposure (AUC 19.3-65 µg·h/mL). Therefore, dryocrassin ABBA has therapeutic potential against emerging coronavirus infections, including COVID-19.

6.
Bull Korean Chem Soc ; 43(3): 412-416, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1650887

ABSTRACT

Despite the continuing global crisis caused by coronavirus disease 2019 (COVID-19), there is still no effective treatment. Therefore, we designed and synthesized a novel series of 2-benzylaminoquinazolin-4(3H)-one derivatives and demonstrated that they are effective against SARS-CoV-2. Among the synthesized derivatives, 7-chloro-2-(((4-chlorophenyl)(phenyl)methyl)amino)quinazolin-4(3H)-one (Compound 39) showed highest anti-SARS-CoV-2 activity, with a half-maximal inhibitory concentration value greater than that of remdesivir (IC50 = 4.2 µM vs. 7.6 µM, respectively), which gained urgent approval from the U.S. Food and Drug Administration. In addition, Compound 39 showed good results in various assays measuring metabolic stability, human ether a-go-go, Cytochromes P450 (CYPs) inhibition, and plasma protein binding (PPB), and showed better solubility and pharmacokinetics than our previous work.

7.
J Microbiol Biotechnol ; 32(1): 1-5, 2022 Jan 28.
Article in English | MEDLINE | ID: covidwho-1555623

ABSTRACT

Diagnostics, vaccines, and drugs are indispensable tools and control measures employed to overcome infectious diseases such as coronavirus disease 2019 (COVID-19). Diagnostic tools based on RT-PCR were developed early in the COVID-19 pandemic and were urgently required for quarantine (testing, tracing and isolation). Vaccines such as mRNA vaccines and virus-vectored vaccines were also successfully developed using new platform technologies within one year after identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of COVID-19. Drug development has been conducted in various ways including drug repurposing, convalescent plasma therapy, and monoclonal antibody development. Among the above efforts, this review examines COVID-19 drug development along with the related and upcoming challenges.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/therapy , Drug Development , Drug Repositioning/methods , SARS-CoV-2/drug effects , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/blood , Antiviral Agents/chemistry , COVID-19/prevention & control , Humans , Immunization, Passive , SARS-CoV-2/genetics
8.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-292354

ABSTRACT

The pandemic coronavirus disease 2019 (COVID-19) is characterized as an acute respiratory infection in the majority of cases and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other coronaviruses (CoVs) can infect humans, although the majority only cause mild respiratory symptoms. As early diagnosis of SARS-CoV-2 is critical to prevent further transmission events and to improve clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other CoVs in respiratory samples. Therefore, we developed and evaluated a novel quantitative reverse transcription PCR (RT-qPCR) assay, targeting the spike (S) and membrane (M) genes, to enable the rapid identification of SARS-CoV-2 including several new circulating variants, and other emerging pan-SARS-like CoVs. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays confirmed a limit of detection of 1 × 10 0 TCID 50 /ml and no cross-reaction with human coronaviruses or other respiratory viruses. We also validated our method using human clinical samples from COVID-19 patients and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic coronaviruses, including SARS-CoV-2, and may be useful for the identification of other pan-SARS-like CoVs of zoonotic origin.

9.
Pharmaceutics ; 13(11)2021 Nov 02.
Article in English | MEDLINE | ID: covidwho-1502487

ABSTRACT

Cardiotonic steroids are steroid-like natural compounds known to inhibit Na+/K+-ATPase pumps. To develop a broad-spectrum antiviral drug against the emerging coronavirus infection, this study assessed the antiviral properties of these compounds. The activity of seven types of cardiotonic steroids against the MERS-CoV, SARS-CoV, and SARS-CoV-2 coronavirus varieties was analyzed using immunofluorescence antiviral assay in virus-infected cells. Bufalin, cinobufagin, and telocinobufagin showed high anti-MERS-CoV activities (IC50, 0.017~0.027 µM); bufalin showed the most potent anti-SARS-CoV and SARS-CoV-2 activity (IC50, 0.016~0.019 µM); cinobufotalin and resibufogenin showed comparatively low anti-coronavirus activity (IC50, 0.231~1.612 µM). Differentially expressed genes in Calu3 cells treated with cinobufagin, telocinobufagin, or bufalin, which had high antiviral activity during MERS-CoV infection were analyzed using QuantSeq 3' mRNA-Seq analysis and data showed similar gene expression patterns. Furthermore, the intraperitoneal administration of 10 mg/kg/day bufalin, cinobufagin, or digitoxin induced 100% death after 1, 2, and 4 days in 5-day repeated dose toxicity studies and it indicated that bufalin had the strongest toxicity. Pharmacokinetic studies suggested that telocinobufagin, which had high anti-coronavirus activity and low toxicity, had better microsomal stability, lower CYP inhibition, and better oral bioavailability than cinobufagin. Therefore, telocinobufagin might be the most promising cardiotonic steroid as a therapeutic for emerging coronavirus infections, including COVID-19.

10.
Chem Commun (Camb) ; 57(93): 12476-12479, 2021 Nov 23.
Article in English | MEDLINE | ID: covidwho-1500757

ABSTRACT

We identified small-molecule enhancers of cellular stress granules by observing molecular crowding of proteins and RNAs in a time-dependent manner. Hit molecules sensitized the IRF3-mediated antiviral mechanism in the presence of poly(I:C) and inhibited the replication of SARS-CoV-2 by inducing stress granule formation. Thus, modulating multimolecular crowding can be a promising strategy against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Benzopyrans/pharmacology , Cytoplasmic Granules/drug effects , Pyrazoles/pharmacology , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Benzopyrans/chemistry , Cell Line, Tumor , Chlorocebus aethiops , Cytoplasmic Granules/metabolism , Dose-Response Relationship, Drug , Drug Combinations , Humans , Interferon Regulatory Factor-3/metabolism , Lopinavir/pharmacology , Microbial Sensitivity Tests , Molecular Structure , Poly I-C/pharmacology , Pyrazoles/chemistry , Structure-Activity Relationship , Vero Cells
11.
J Med Chem ; 64(20): 14955-14967, 2021 10 28.
Article in English | MEDLINE | ID: covidwho-1461960

ABSTRACT

Blocking the association between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) is an attractive therapeutic approach to prevent the virus from entering human cells. While antibodies and other modalities have been developed to this end, d-amino acid peptides offer unique advantages, including serum stability, low immunogenicity, and low cost of production. Here, we designed potent novel D-peptide inhibitors that mimic the ACE2 α1-binding helix by searching a mirror-image version of the PDB. The two best designs bound the RBD with affinities of 29 and 31 nM and blocked the infection of Vero cells by SARS-CoV-2 with IC50 values of 5.76 and 6.56 µM, respectively. Notably, both D-peptides neutralized with a similar potency the infection of two variants of concern: B.1.1.7 and B.1.351 in vitro. These potent D-peptide inhibitors are promising lead candidates for developing SARS-CoV-2 prophylactic or therapeutic treatments.


Subject(s)
Peptides , SARS-CoV-2 , Animals , Chlorocebus aethiops , Molecular Docking Simulation , Vero Cells
13.
Microbiol Spectr ; 9(1): e0047221, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1352541

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent of the coronavirus disease 2019 (COVID-19) pandemic, and the development of therapeutic interventions is urgently needed. So far, monoclonal antibodies and drug repositioning are the main methods for drug development, and this effort was partially successful. Since the beginning of the COVID-19 pandemic, the emergence of SARS-CoV-2 variants has been reported in many parts of the world, and the main concern is whether the current vaccines and therapeutics are still effective against these variant viruses. Viral entry and viral RNA-dependent RNA polymerase (RdRp) are the main targets of current drug development; therefore, the inhibitory effects of transmembrane serine protease 2 (TMPRSS2) and RdRp inhibitors were compared among the early SARS-CoV-2 isolate (lineage A) and the two recent variants (lineage B.1.1.7 and lineage B.1.351) identified in the United Kingdom and South Africa, respectively. Our in vitro analysis of viral replication showed that the drugs targeting TMPRSS2 and RdRp are equally effective against the two variants of concern. IMPORTANCE The COVID-19 pandemic is causing unprecedented global problems in both public health and human society. While some vaccines and monoclonal antibodies were successfully developed very quickly and are currently being used, numerous variants of the causative SARS-CoV-2 are emerging and threatening the efficacy of vaccines and monoclonal antibodies. In order to respond to this challenge, we assessed antiviral efficacy of small-molecule inhibitors that are being developed for treatment of COVID-19 and found that they are still very effective against the SARS-CoV-2 variants. Since most small-molecule inhibitors target viral or host factors other than the mutated sequence of the viral spike protein, they are expected to be potent control measures against the COVID-19 pandemic.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , RNA-Dependent RNA Polymerase/drug effects , SARS-CoV-2/drug effects , Serine Endopeptidases/drug effects , Animals , Antiviral Agents/therapeutic use , Chlorocebus aethiops , Humans , South Africa , United Kingdom , Vero Cells , Virus Internalization/drug effects , Virus Replication/drug effects
14.
Brief Bioinform ; 22(6)2021 11 05.
Article in English | MEDLINE | ID: covidwho-1303894

ABSTRACT

The global efforts in the past year have led to the discovery of nearly 200 drug repurposing candidates for COVID-19. Gaining more insights into their mechanisms of action could facilitate a better understanding of infection and the development of therapeutics. Leveraging large-scale drug-induced gene expression profiles, we found 36% of the active compounds regulate genes related to cholesterol homeostasis and microtubule cytoskeleton organization. Following bioinformatics analyses revealed that the expression of these genes is associated with COVID-19 patient severity and has predictive power on anti-SARS-CoV-2 efficacy in vitro. Monensin, a top new compound that regulates these genes, was further confirmed as an inhibitor of SARS-CoV-2 replication in Vero-E6 cells. Interestingly, drugs co-targeting cholesterol homeostasis and microtubule cytoskeleton organization processes more likely present a synergistic effect with antivirals. Therefore, potential therapeutics could be centered around combinations of targeting these processes and viral proteins.

15.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Article in English | MEDLINE | ID: covidwho-1301228

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 is an unprecedentedly significant health threat, prompting the need for rapidly developing antiviral drugs for the treatment. Drug repurposing is currently one of the most tangible options for rapidly developing drugs for emerging and reemerging viruses. In general, drug repurposing starts with virtual screening of approved drugs employing various computational methods. However, the actual hit rate of virtual screening is very low, and most of the predicted compounds are false positives. Here, we developed a strategy for virtual screening with much reduced false positives through incorporating predocking filtering based on shape similarity and postdocking filtering based on interaction similarity. We applied this advanced virtual screening approach to repurpose 6,218 approved and clinical trial drugs for COVID-19. All 6,218 compounds were screened against main protease and RNA-dependent RNA polymerase of SARS-CoV-2, resulting in 15 and 23 potential repurposed drugs, respectively. Among them, seven compounds can inhibit SARS-CoV-2 replication in Vero cells. Three of these drugs, emodin, omipalisib, and tipifarnib, show anti-SARS-CoV-2 activities in human lung cells, Calu-3. Notably, the activity of omipalisib is 200-fold higher than that of remdesivir in Calu-3. Furthermore, three drug combinations, omipalisib/remdesivir, tipifarnib/omipalisib, and tipifarnib/remdesivir, show strong synergistic effects in inhibiting SARS-CoV-2. Such drug combination therapy improves antiviral efficacy in SARS-CoV-2 infection and reduces the risk of each drug's toxicity. The drug repurposing strategy reported here will be useful for rapidly developing drugs for treating COVID-19 and other viruses.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Repositioning , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/therapeutic use , Animals , Chlorocebus aethiops , Drug Evaluation, Preclinical , Drug Synergism , Humans , User-Computer Interface , Vero Cells
16.
Exp Mol Med ; 53(5): 956-972, 2021 05.
Article in English | MEDLINE | ID: covidwho-1243283

ABSTRACT

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann-Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Saponins/pharmacology , Serine Endopeptidases/metabolism , Triterpenes/pharmacology , Virus Internalization/drug effects , Antiviral Agents/chemistry , COVID-19/metabolism , Cell Line , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Models, Molecular , Platycodon/chemistry , SARS-CoV-2/physiology , Saponins/chemistry , Triterpenes/chemistry
17.
J Med Virol ; 93(3): 1403-1408, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196439

ABSTRACT

Drug repositioning represents an effective way to control the current COVID-19 pandemic. Previously, we identified 24 FDA-approved drugs which exhibited substantial antiviral effect against severe acute respiratory syndrome coronavirus 2 in Vero cells. Since antiviral efficacy could be altered in different cell lines, we developed an antiviral screening assay with human lung cells, which is more appropriate than Vero cell. The comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (IC50 = 0.0022 µM).


Subject(s)
Antiviral Agents/pharmacology , Guanidines/pharmacology , SARS-CoV-2/drug effects , Animals , Benzamidines , COVID-19/drug therapy , Cell Line, Tumor , Chlorocebus aethiops , Dose-Response Relationship, Drug , Drug Approval , Drug Repositioning , Humans , Inhibitory Concentration 50 , Lung , Microbial Sensitivity Tests , SARS-CoV-2/physiology , United States , United States Food and Drug Administration , Vero Cells
18.
Bioorg Med Chem Lett ; 39: 127885, 2021 05 01.
Article in English | MEDLINE | ID: covidwho-1116317

ABSTRACT

Despite the rising threat of fatal coronaviruses, there are no general proven effective antivirals to treat them. 2-Aminoquinazolin-4(3H)-one derivatives were newly designed, synthesized, and investigated to show the inhibitory effects on SARS-CoV-2 and MERS-CoV. Among the synthesized derivatives, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (9g) and 2-((3,5-dichlorophenyl)amino)-5-hydroxyquinazolin-4 (3H)-one (11e) showed the most potent anti-SARS-CoV-2 activities (IC50 < 0.25 µM) and anti-MERS-CoV activities (IC50 < 1.1 µM) with no cytotoxicity (CC50 > 25 µM). In addition, both compounds showed acceptable results in metabolic stabilities, hERG binding affinities, CYP inhibitions, and preliminary PK studies.


Subject(s)
Antiviral Agents/chemical synthesis , Drug Design , Middle East Respiratory Syndrome Coronavirus/drug effects , Quinazolinones/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , Cell Line , Cell Survival/drug effects , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Half-Life , Humans , Inhibitory Concentration 50 , Mice , Microsomes/metabolism , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Quinazolinones/chemistry , Quinazolinones/metabolism , Quinazolinones/therapeutic use , Rats , SARS-CoV-2/isolation & purification , Structure-Activity Relationship
19.
J Microbiol Biotechnol ; 31(3): 358-367, 2021 03 28.
Article in English | MEDLINE | ID: covidwho-1006913

ABSTRACT

The World Health Organization (WHO) has declared the coronavirus disease 2019 (COVID-19) as an international health emergency. Current diagnostic tests are based on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method, which is the gold standard test that involves the amplification of viral RNA. However, the RT-qPCR assay has limitations in terms of sensitivity and quantification. In this study, we tested both qPCR and droplet digital PCR (ddPCR) to detect low amounts of viral RNA. The cycle threshold (CT) of the viral RNA by RT-PCR significantly varied according to the sequences of the primer and probe sets with in vitro transcript (IVT) RNA or viral RNA as templates, whereas the copy number of the viral RNA by ddPCR was effectively quantified with IVT RNA, cultured viral RNA, and RNA from clinical samples. Furthermore, the clinical samples were assayed via both methods, and the sensitivity of the ddPCR was determined to be equal to or more than that of the RT-qPCR. However, the ddPCR assay is more suitable for determining the copy number of reference materials. These findings suggest that the qPCR assay with the ddPCR defined reference materials could be used as a highly sensitive and compatible diagnostic method for viral RNA detection.


Subject(s)
COVID-19/diagnosis , Nucleic Acid Probes/genetics , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Animals , COVID-19/virology , Cell Line , Chlorocebus aethiops , Gene Dosage/genetics , Humans , RNA, Viral/genetics , Sensitivity and Specificity , Vero Cells
20.
Phytomedicine ; 86: 153440, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-978384

ABSTRACT

BACKGROUND: Highly effective novel treatments need to be developed to suppress emerging coronavirus (CoV) infections such as COVID-19. The RNA dependent RNA polymerase (RdRp) among the viral proteins is known as an effective antiviral target. Lycorine is a phenanthridine Amaryllidaceae alkaloid isolated from the bulbs of Lycoris radiata (L'Hér.) Herb. and has various pharmacological bioactivities including antiviral function. PURPOSE: We investigated the direct-inhibiting action of lycorine on CoV's RdRp, as potential treatment for emerging CoV infections. METHODS: We examined the inhibitory effect of lycorine on MERS-CoV, SARS-CoV, and SARS-CoV-2 infections, and then quantitatively measured the inhibitory effect of lycorine on MERS-CoV RdRp activity using a cell-based reporter assay. Finally, we performed the docking simulation with lycorine and SARS-CoV-2 RdRp. RESULTS: Lycorine efficiently inhibited these CoVs with IC50 values of 2.123 ± 0.053, 1.021 ± 0.025, and 0.878 ± 0.022 µM, respectively, comparable with anti-CoV effects of remdesivir. Lycorine directly inhibited MERS-CoV RdRp activity with an IC50 of 1.406 ± 0.260 µM, compared with remdesivir's IC50 value of 6.335 ± 0.731 µM. In addition, docking simulation showed that lycorine interacts with SARS-CoV-2 RdRp at the Asp623, Asn691, and Ser759 residues through hydrogen bonding, at which the binding affinities of lycorine (-6.2 kcal/mol) were higher than those of remdesivir (-4.7 kcal/mol). CONCLUSIONS: Lycorine is a potent non-nucleoside direct-acting antiviral against emerging coronavirus infections and acts by inhibiting viral RdRp activity; therefore, lycorine may be a candidate against the current COVID-19 pandemic.


Subject(s)
Amaryllidaceae Alkaloids/pharmacology , Antiviral Agents/pharmacology , Phenanthridines/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Chlorocebus aethiops , Hydrogen Bonding , Middle East Respiratory Syndrome Coronavirus/drug effects , Molecular Docking Simulation , SARS Virus/drug effects , Vero Cells , Viral Proteins
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