Molecular epidemiology and genetic diversity of canine coronavirus from domestic dogs in Chengdu, China from 2020 to 2021 using a multiplex RT-PCR.

Recent reports on identification of canine coronavirus (CCoV) in humans have emphasized the urgency to strengthen surveillance of animal CoVs. The fact that recombinations between CCoV with feline, porcine CoVs brought about new types of CoVs indicated that more attention should be paid to domestic animals like dogs, cats and pigs, and the CoVs they carried. However, there are about ten kinds of CoVs that infect above animals, and thus representative CoVs with zoonotic potentials were considered in this study. Multiplex RT-PCR against CCoV, Feline coronavirus (FCoV), porcine deltacoronavirus and porcine acute diarrhea syndrome coronavirus was developed to investigate the prevalence of CoVs from domestic dogs in Chengdu, Southwest China. Samples from a total of 117 dogs were collected from a veterinary hospital, and only CCoV (34.2%, 40/117) was detected. Therefore, this study focused on CCoV and its characteristics of S, E, M, N and ORF3abc genes. Compared with CoVs that are capable of infecting humans, CCoV strains showed highest nucleotide identity with the novel canine-feline recombinant detected from humans (CCoV-Hupn-2018). Phylogenetic analysis based on S gene, CCoV strains were not only clustered with CCoV-II strains, but also closely related to FCoV-II strains ZJU1617 and SMU-CD59/2018. As for assembled ORF3abc, E, M, N sequences, CCoV strains had the closest relationship with CCoV-II (B203_GZ_2019, B135_JS_2018 and JS2103). What's more, specific amino acid variations were found, especially in S and N proteins, and some mutations were consistent with FCoV, TGEV strains. Altogether, this study provided a novel insight into the identification, diversification and evolution of CoVs from domestic dogs. It is of top priority to recognize zoonotic potential of CoVs, and continued comprehensive surveillance will help better understand the emergence, spreading, and ecology of animal CoVs.

Are Non-Structural Proteins From SARS-CoV-2 the Target of Hydroxychloroquine? An in Silico Study

COVID-19 is caused by SARS-CoV-2 which has structural and non-structural proteins (NSP) essential for infection and viral replication. There is a possible binding of SARS-CoV-2 to the beta-1 chain of hemoglobin in red blood cells and thus, decreasing the oxygen transport capacity. Since hydroxychloroquine (HCQ) can accumulate in red cells, there is a chance of interaction of this drug with the virus. To analyze possible interactions between SARS-CoV-2 NSP and hemoglobin with the HCQ using molecular docking and implications for the infected host. This research consisted of a study using bioinformatics tools. The files of the protein structures and HCQ were prepared using the AutoDock Tools software. These files were used to perform molecular docking simulations by AutoDock Vina. The binding affinity report of the generated conformers was analyzed using PyMol software, as well as the chemical bonds formed. The results showed that HCQ is capable of interacting with both SARS-CoV-2 NSP and human hemoglobin. The HCQ/NSP3 conformer, HCQ/NSP5, HCQ/NSP7-NSP8-NSP12, HCQ/NSP9, HCQ/NSP10-NSP16 showed binding affinity. In addition, the interaction between HCQ and hemoglobin resulted in polar bonds. Interaction between SARS-CoV-2 NSP and HCQ indicates that this drug possibly acts by preventing the continuity of infection.Copyright © 2023 Tehran University of Medical Sciences.

TARGETING THE HOST-VIRUS INTERFACE TO BLOCK SARS-CoV-2 ASSEMBLY IN AIRWAY CELLS

Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.

Unusual Myositis of the Temporalis Muscle Occurring with Acute Covid-19

Background. SARS-CoV-2 infection can be accompanied by neuromuscular disorders. Rhabdomyolysis and Guillain-Barre syndrome have been described repeatedly. There are case reports of inflammatory myopathies manifesting during COVID-19, presenting as dermatomyositis, polymyositis or immune-mediated necrotizing myopathy, with dermatomyositis-like presentations most commonly reported. Larger cases series are from postmortem examinations of COVID-19 patients, where variable inflammatory pathology of the skeletal muscle has been found frequently but without local detection of the actual virus. Thus, autoimmune mechanisms or the systemic interferon response are discussed as causes. We report a case of focal inflammatory myopathy with perimysial pathology of the temporalis muscle occurring with acute, but mild COVID-19. Methods. Case report of clinical observations, cranial MRI, histopathological, and laboratory findings. 3T cranial MRI was performed with gadolinium contrast. Open temporalis muscle biopsy was performed. The sample underwent standard cryohistological studies as well as immunohistochemistry with antibodies against MHC-I and II, CD3, CD4, CD19, CD68, anti-MAC, p62 and MxA. Testing for auto-antibodies was based on immunoblots or ELISA. RT-PCR for SARS-CoV-2 was run with RNA extracted from cryopreserved muscle. Results. A Caucasian woman in her 60s with no history of autoimmune or muscle complaints developed swelling and pain of the right jaw musculature five days after testing positive for SARS-CoV-2 due to respiratory tract symptoms. In addition, she experienced trismus, but no further neuromuscular complaints. The course of respiratory tract symptoms stayed mild. She had been vaccinated previously with single shot SARS-CoV-2 vector vaccine. Due to persistent swelling and complaints, giant cells arteritis was excluded by unresponsiveness to five days oral steroids and sonography of the temporal artery. Cranial MRI was performed nearly four weeks after the SARS-CoV-2 infection and showed marked swelling and oedema of the temporalis muscle. Its biopsy showed numerous CD68 and acid phosphatase positive cells infiltrating from perimysial perivascular foci towards the endomysium with perimysial damage but little damage of adjacent, perifascicular muscle fibres. Muscle fibres did not react with anti-MHC-II, anti-MAC or -MxA. Capillaries did not react with anti-MAC or -MxA. SARS-CoV-2 RNA was not detected in muscle tissue. Serum creatine kinase was not elevated in the subacute phase. Slightly elevated ANA titre led to detection of autoantibodies against proliferating cell nuclear antigen (PCNA). No pathological results for other autoantibodies, including myositis-specific antibodies and anti-ds-DNA, were found in blood. Neither were antibodies against hepatitis C and B viruses. Retesting 15 weeks after infection, anti-PCNA immunoblot was still positive, but ELISA did not indicate a pathologic titre. The swelling, myalgia and trismus regressed spontaneously a month after onset, yet the latter still persists at the time of reporting. Conclusion. Our case diverges from the majority of COVID-19 associated my-ositis reports in the unusual location of the focal myositis and the histopathological pattern of predominantly perimysial damage and histiocytic infiltration. It concurs with the literature as no SARS-CoV2 RNA could be detected in the muscle. Anti-PCNA is associated very rarely with myositis. Other associated disorder (systemic lupus erythematosus, chronic viral hepatitis B or C) were not found. Increased levels of autoantibodies are reported in COVID-19 and mostly attributed to loss of self-tolerance during the acute disease phase. Interestingly, the structural protein M of SARS-CoV-2 appears to interact notably with PCNA in infected cells. Still, the causal connection between the myositis and COVID-19 in this case is based on the close temporal association in the absence of alternative, competing explanations from the medical history and findings.

Exploring the pharmacological aspects of natural phytochemicals against SARS-CoV-2 Nsp14 through an in silico approach.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), possesses an important bifunctional nonstructural protein (nsp14) with a C-terminal N7-methyltransferase (N7-MTase) domain and an N-terminal domain with exoribonuclease (ExoN) activity that is required for maintaining high-fidelity viral replication. Viruses use the error-prone replication mechanism, which results in high mutation rates, to adapt quickly to stressful situations. The efficiency with which nsp14 removes mismatched nucleotides due to the presence of ExoN activity protects viruses from mutagenesis. We investigated the pharmacological role of the phytochemicals (Baicalein, Bavachinin, Emodin, Kazinol F, Lycorine, Sinigrin, Procyanidin A2, Tanshinone IIA, Tanshinone IIB, Tomentin A, and Tomentin E) against the highly conserved nsp14 protein using docking-based computational analyses in search of new potential natural drug targets. The selected eleven phytochemicals failed to bind the active site of N7-Mtase in the global docking study, while the local docking study identified the top five phytochemicals with high binding energy scores ranging from - 9.0 to - 6.4 kcal/mol. Procyanidin A2 and Tomentin A showed the highest docking score of - 9.0 and - 8.1 kcal/mol, respectively. Local docking of isoform variants was also conducted, yielding the top five phytochemicals, with Procyanidin A1 having the highest binding energy value of - 9.1 kcal/mol. The phytochemicals were later tested for pharmacokinetics and pharmacodynamics analysis for Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) which resulted in choosing Tomentin A as a potential candidate. The molecular dynamics simulations studies of nsp14 revealed significant conformational changes upon complex formation with the identified compound, implying that these phytochemicals could be used as safe nutraceuticals which will impart long-term immunological competence in the human population against CoVs. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00143-7.

COVID-19 Pandemic: An Overview of its Origin, Current Status, and Ongoing Clinical Trials

The COVID-19 pandemic is raging across the globe, with the total active cases increas-ing each day. Globally over 63 million COVID-19cases and more than 1.4 million deaths have been reported to WHO. Throughout the world, academicians, clinicians and scientists are working tirelessly on developing a treatment to combat this pandemic. The origin of novel SARS-CoV-2 virus still remains foggy but is believed to have originated from a bat coronavirus RaTG13 with which it shares approximately 96% sequence similarity. In the present review, the authors have pro-vided an overview of the COVID-19 pandemic, epidemiology, transmission, developments related to diagnosis, drugs and vaccines, along with the genetic diversity and lifecycle of the SARS-CoV-2 based on the current studies and information available.Copyright © 2022 Bentham Science Publishers.

Bioinformatics analysis identifies a small orf in the genome of fish nidoviruses of genus oncotshavirus predicted to encode a novel integral protein

Genome sequence analysis of Atlantic salmon bafinivirus (ASBV) revealed a small open reading frame (ORF) predicted to encode a Type I membrane protein with an N-terminal cleaved signal sequence (110 aa), likely an envelope (E) protein. Bioinformatic analyses showed that the predicted protein is strikingly similar to the coronavirus E protein in structure. This is the first report to identify a putative E protein ORF in the genome of members of the Oncotshavirus genus (subfamily Piscavirinae, family Tobaniviridae, order Nidovirales) and, if expressed would be the third family (after Coronaviridae and Arteriviridae) within the order to have the E protein as a major structural protein.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

An overview of potential inhibitors targeting non-structural proteins 3 (PLpro and Mac1) and 5 (3CLpro/Mpro) of SARS-CoV-2.

There is an urgent need to develop effective treatments for coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid spread of SARS-CoV-2 has resulted in a global pandemic that has not only affected the daily lives of individuals but also had a significant impact on the global economy and public health. Although extensive research has been conducted to identify inhibitors targeting SARS-CoV-2, there are still no effective treatment strategies to combat COVID-19. SARS-CoV-2 comprises two important proteolytic enzymes, namely, the papain-like proteinase, located within non-structural protein 3 (nsp3), and nsp5, both of which cleave large replicase polypeptides into multiple fragments that are required for viral replication. Moreover, a domain within nsp3, known as the macrodomain (Mac1), also plays an important role in viral replication. Inhibition of their functions should be able to significantly interfere with the replication cycle of the virus, and therefore these key proteins may serve as potential therapeutic targets. The functions of the above viral targets and their corresponding inhibitors have been summarized in the current review. This review provides comprehensive updates of nsp3 and nsp5 inhibitor development and would help advance the discovery of novel anti-viral therapeutics against SARS-CoV-2.

Structural screening into the recognition of a potent inhibitor against non-structural protein 16: a molecular simulation to inhibit SARS-CoV-2 infection.

COVID-19 infection is caused by endemic crown infection (SARS-CoV-2) and is associated with lung damage and severe immune response. Non-Structural Proteins are the central components of coronaviral transcription and replication machinery in SARS-CoV-2 and also stimulate mRNA cap methylation to avoid the immune response. Non-Structural Protein 16 (NSP16) is one of the primary targets for the drug discovery of coronaviruses. Discovering an effective inhibitor against the NSP16 in comparison with Sinefungin was the main purpose of this investigation. Binding free-energy calculations, computational methods of molecular dynamics, docking, and virtual screening were utilized in this study. The ZINC and PubChem databases were applied to screen some chemical compounds regarding Sinefungin as a control inhibitor. Based on structural similarity to Sinefungin, 355 structures were obtained from the mentioned databases. Subsequently, this set of compounds were monitored by AutoDock Vina software, and ultimately the potent inhibitor (PUBCHEM512713) was chosen. At the next stage, molecular dynamics were carried out by GROMACS software to evaluate the potential elected compounds in a simulated environment and in a timescale of 100 nanoseconds. MM-PBSA investigation exhibited that the value of binding free energy for PUBCHEM512713 (-30.829 kJ.mol-1) is more potent than Sinefungin (-11.941 kJ.mol-1). Furthermore, the results of ADME analysis illustrated that the pharmacokinetics, drug-likeness, and lipophilicity parameters of PUBCHEM512713 are admissible for human utilization. Finally, our data suggested that PUBCHEM512713 is an effective drug candidate for inhibiting the NSP16 and is suitable for in vitro and in vivo studies.Communicated by Ramaswamy H. Sarma.

Suramin, penciclovir, and anidulafungin exhibit potential in the treatment of COVID-19 via binding to nsp12 of SARS-CoV-2.

COVID-19, for which no confirmed therapeutic agents are available, has claimed over 48,14,000 lives globally. A feasible and quicker method to resolve this problem may be 'drug repositioning'. We investigated selected FDA and WHO-EML approved drugs based on their previously promising potential as antivirals, antibacterials or antifungals. These drugs were docked onto the nsp12 protein, which reigns the RNA-dependent RNA polymerase activity of SARS-CoV-2, a key therapeutic target for coronaviruses. Docked complexes were reevaluated using MM-GBSA analysis and the top three inhibitor-protein complexes were subjected to 100 ns long molecular dynamics simulation followed by another round of MM-GBSA analysis. The RMSF plots, binding energies and the mode of physicochemical interaction of the active site of the protein with the drugs were evaluated. Suramin, Penciclovir, and Anidulafungin were found to bind to nsp12 with similar binding energies as that of Remdesivir, which has been used as a therapy for COVID-19. In addition, recent experimental evidences indicate that these drugs exhibit antiviral efficacy against SARS-CoV-2. Such evidence, along with the significant and varied physical interactions of these drugs with the key viral enzyme outlined in this investigation, indicates that they might have a prospective therapeutic potential in the treatment of COVID-19 as monotherapy or combination therapy with Remdesivir.

Homology Modeling of Coronavirus Structural Proteins and Molecular Docking of Potential Drug Candidates for the Treatment of COVID-19

Background: The discovery of a novel strain of coronavirus in 2019 (COVID-19) has triggered a series of tragic events in the world with thousands of deaths recorded daily. Despite the huge resources committed to the discovery of vaccines against this highly pathogenic virus, scientists are still unable to find suitable treatments for the disease. Understanding the structure of coronavirus proteins could provide a basis for the development of cheap, potent and, less toxic vaccines. Objective(s): This study was therefore designed to model coronavirus spike (S) glycoprotein and envelope (E) protein as well as to carry out molecular docking of potential drugs to the homologs and coronavirus main protease (Mpro). Method(s): Homology modeling of coronavirus spike (S) glycoprotein and envelope (E) protein was car-ried out using sequence deposited in the Uniprot database. The topological features of the model's catalytic site were evaluated using the CASTp server. Compounds reported as potential drugs against COVID-19 were docked to S glycoprotein, E protein, and coronavirus main protease (Mpro) to determine the best ligands and the mode of interaction. Result(s): Homology modeling of the proteins revealed structures with 91-98% sequence similarity with PDB entries. The catalytic site of the modeled proteins contained conserved residue involved in ligand binding. In addition, remdesivir, lopinavir, and ritonavir have a high binding affinity for the three proteins studied interacting with key residues in the protein's catalytic domain. Conclusion(s): Results from the study revealed that remdesivir, lopinavir, and ritonavir are inhibitors of key coronavirus proteins and therefore qualify for further studies as a potential treatment for coronavi-rus.Copyright © 2021 Bentham Science Publishers.

Study of the Effects of Several SARS-CoV-2 Structural Proteins on Antiviral Immunity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike (S) protein is a critical viral antigenic protein that enables the production of neutralizing antibodies, while other structural proteins, including the membrane (M), nucleocapsid (N) and envelope (E) proteins, have unclear roles in antiviral immunity. In this study, S1, S2, M, N and E proteins were expressed in 16HBE cells to explore the characteristics of the resultant innate immune response. Furthermore, peripheral blood mononuclear cells (PBMCs) from mice immunized with two doses of inactivated SARS-CoV-2 vaccine or two doses of mRNA vaccine were isolated and stimulated by these five proteins to evaluate the corresponding specific T-cell immune response. In addition, the levels of humoral immunity induced by two-dose inactivated vaccine priming followed by mRNA vaccine boosting, two homologous inactivated vaccine doses and two homologous mRNA vaccine doses in immunized mice were compared. Our results suggested that viral structural proteins can activate the innate immune response and elicit a specific T-cell response in mice immunized with the inactivated vaccine. However, the existence of the specific T-cell response against M, N and E is seemingly insufficient to improve the level of humoral immunity.

Crystal structure of the membrane (M) protein from a bat betacoronavirus.

The membrane (M) protein is the most abundant structural protein of coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2, and plays a central role in virus assembly through its interaction with various partner proteins. However, mechanistic details about how M protein interacts with others remain elusive due to lack of high-resolution structures. Here, we present the first crystal structure of a betacoronavirus M protein from Pipistrellus bat coronavirus HKU5 (batCOV5-M), which is closely related to MERS-CoV, SARS-CoV, and SARS-CoV-2 M proteins. Furthermore, an interaction analysis indicates that the carboxy-terminus of the batCOV5 nucleocapsid (N) protein mediates its interaction with batCOV5-M. Combined with a computational docking analysis an M-N interaction model is proposed, providing insight into the mechanism of M protein-mediated protein interactions.

SARS-CoV-2 Nsp2 Contributes to Inflammation by Activating NF-κB.

COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1ß and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNß expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNß promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor.

Repurposing of approved drug molecules for viral infectious diseases: a molecular modelling approach.

The identification of new viral drugs has become a task of paramount significance due to the frequent occurrence of viral infections and especially during the current pandemic. Despite the recent advancements, the development of antiviral drugs has not made parallel progress. Reduction of time frame and cost of the drug development process is the major advantage of drug repurposing. Therefore, in this study, a drug repurposing strategy using molecular modelling techniques, i.e. biological activity prediction, virtual screening, and molecular dynamics simulation was employed to find promising repurposing candidates for viral infectious diseases. The biological activities of non-redundant (4171) drug molecules were predicted using PASS analysis, and 1401 drug molecules were selected which showed antiviral activities in the analysis. These drug molecules were subjected to virtual screening against the selected non-structural viral proteins. A series of filters, i.e. top 10 drug molecules based on binding affinity, mean value of binding affinity, visual inspection of protein-drug complexes, and number of H-bond between protein and drug molecules were used to narrow down the drug molecules. Molecular dynamics simulation analysis was carried out to validate the intrinsic atomic interactions and binding conformations of protein-drug complexes. The binding free energies of drug molecules were assessed by employing MMPBSA analysis. Finally, nine drug molecules were prioritized, as promising repurposing candidates with the potential to inhibit the selected non-structural viral proteins.Communicated by Ramaswamy H. Sarma.

In silico evaluation of flavonoids as potential inhibitors of SARS-CoV-2 main nonstructural proteins (Nsps)-amentoflavone as a multitarget candidate.

Despite the development of vaccines against COVID-19 disease and the multiple efforts to find efficient drugs as treatment for this virus, there are too many social, political, economic, and health inconveniences to incorporate a fully accessible plan of prevention and therapy against SARS-CoV-2. In this sense, it is necessary to find nutraceutical/pharmaceutical drugs as possible COVID-19 preventives/treatments. Based on their beneficial effects, flavonoids are one of the most promising compounds. Therefore, using virtual screening, 478 flavonoids obtained from the KEGG database were evaluated against non-structural proteins Nsp1, Nsp3, Nsp5, Nsp12, and Nsp15, which are essential for the virus-host cell infection, searching for possible multitarget flavonoids. Amentoflavone, a biflavonoid found mainly in Ginkgo biloba, Lobelia chinensis, and Byrsonima intermedia, can interact and bind with the five proteins, suggesting its potential as a multitarget inhibitor. Molecular docking calculations and structural analysis (RMSD, number of H bonds, and clustering) performed from molecular dynamics simulations of the amentoflavone-protein complex support this potential. The results shown here are theoretical evidence of the probable multitarget inhibition of non-structural proteins of SARS-CoV-2 by amentoflavone, which has wide availability, low cost, no side effects, and long history of use. These results are solid evidence for future in vitro and in vivo experiments aiming to validate amentoflavone as an inhibitor of the Nsp1, 3, 5, 12, and 15 of SARS-CoV-2.

Discovery and Development Strategies for SARS-CoV-2 NSP3 Macrodomain Inhibitors.

The worldwide public health and socioeconomic consequences caused by the COVID-19 pandemic highlight the importance of increasing preparedness for viral disease outbreaks by providing rapid disease prevention and treatment strategies. The NSP3 macrodomain of coronaviruses including SARS-CoV-2 is among the viral protein repertoire that was identified as a potential target for the development of antiviral agents, due to its critical role in viral replication and consequent pathogenicity in the host. By combining virtual and biophysical screening efforts, we discovered several experimental small molecules and FDA-approved drugs as inhibitors of the NSP3 macrodomain. Analogue characterisation of the hit matter and crystallographic studies confirming binding modes, including that of the antibiotic compound aztreonam, to the active site of the macrodomain provide valuable structure-activity relationship information that support current approaches and open up new avenues for NSP3 macrodomain inhibitor development.

Molecular modelling and structure-activity relationship of a natural derivative of o-hydroxybenzoate as a potent inhibitor of dual NSP3 and NSP12 of SARS-CoV-2: in silico study.

The nsp3 macrodomain and nsp12 (RdRp) enzymes are strongly implicated in the virulent regulation of the host immune response and viral replication of SARS-CoV-2, making them plausible therapeutic targets for mitigating infectivity. Remdesivir remains the only FDA-approved small-molecule inhibitor of the nsp12 in clinical conditions while none has been approved yet for the nsp3 macrodomain. In this study, 69,067 natural compounds from the IBScreen database were screened for efficacious potentials with mechanistic multitarget-directed inhibitory pharmacology against the dual targets using in silico approaches. Standard and extra precision (SP and XP) Maestro glide docking analyses were employed to evaluate their inhibitory interactions against the enzymes. Four compounds, STOCK1N-45901, 03804, 83408, 08377 consistently showed high XP scores against the respective targets and interacted strongly with pharmacologically essential amino acid and RNA residues, in better terms than the standard, co-crystallized inhibitors, GS-441524 and remdesivir. Further assessments through the predictions of ADMET and mutagenicity distinguished STOCK1N-45901, a natural derivative of o-hydroxybenzoate as the most promising candidate. The ligand maintained a good conformational and thermodynamic stability in complex with the enzymes throughout the trajectories of 100 ns molecular dynamics, indicated by RMSD, RMSF and radius of gyration plots. Its binding free energy, MM-GBSA was recorded as -54.24 and -31.77 kcal/mol against the respective enzyme, while its structure-activity relationships confer high probabilities as active antiviral, anti-inflammatory, antiinfection, antitussive and peroxidase inhibitor. The IBScreen database natural product, STOCK1N-45901 (2,3,4,5,6-pentahydroxyhexyl o-hydroxybenzoate) is thus recommended as a potent inhibitor of dual nsp3 and nsp12 of SARS-CoV-2 for further study. Communicated by Ramaswamy H. Sarma.

Structural prediction of unknown SARS-CoV-2 protein through Homology Modelling

The COVID-19 pandemic has taken the world into a dark time. The number of infected people has crossed 12 crores and the death counts to more than 5 lakhs until July, 2020 worldwide[1]. Moreover, this lockdown is breaking the pillar of the economy of the countries and the condition is getting worse day by day. So, keeping these things in mind, a vaccine or medicine or some other method of cure is urgently needed. To make this possible, the study of the structure of the virus needs to be done very carefully. That is why we focused upon the structural proteins of the SARS-CoV-2 virus. We all know that in this short period of time it is nearly impossible to do XRD and to know the exact structures of all the proteins present in the discussed virus. So in this article, we have tried to predict the most accurate 3D structure of a yet-unmodelled protein of the SARS-CoV-2, so that in the future, our finding may appear helpful for researchers in case of performing XRD of this protein and further research. Copyright © 2020, Indian Pharmaceutical Association. All rights reserved.

Synergistic Immunity and Protection in Mice by Co-Immunization with DNA Vaccines Encoding the Spike Protein and Other Structural Proteins of SARS-CoV-2.

The emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated recurring worldwide infection outbreaks. These highly mutated variants reduce the effectiveness of current coronavirus disease 2019 (COVID-19) vaccines, which are designed to target only the spike (S) protein of the original virus. Except for the S of SARS-CoV-2, the immunoprotective potential of other structural proteins (nucleocapsid, N; envelope, E; membrane, M) as vaccine target antigens is still unclear and worthy of investigation. In this study, synthetic DNA vaccines encoding four SARS-CoV-2 structural proteins (pS, pN, pE, and pM) were developed, and mice were immunized with three doses via intramuscular injection and electroporation. Notably, co-immunization with two DNA vaccines that expressed the S and N proteins induced higher neutralizing antibodies and was more effective in reducing the SARS-CoV-2 viral load than the S protein alone in mice. In addition, pS co-immunization with either pN or pE + pM induced a higher S protein-specific cellular immunity after three immunizations and caused milder histopathological changes than pS alone post-challenge. The role of the conserved structural proteins of SARS-CoV-2, including the N/E/M proteins, should be investigated further for their applications in vaccine design, such as mRNA vaccines.