Genetic conservation across SARS-CoV-2 non-structural proteins - Insights into possible targets for treatment of future viral outbreaks.

The majority of SARS-CoV-2 therapeutic development work has focussed on targeting the spike protein, viral polymerase and proteases. As the pandemic progressed, many studies reported that these proteins are prone to high levels of mutation and can become drug resistant. Thus, it is necessary to not only target other viral proteins such as the non-structural proteins (NSPs) but to also target the most conserved residues of these proteins. In order to understand the level of conservation among these viruses, in this review, we have focussed on the conservation across RNA viruses, conservation across the coronaviruses and then narrowed our focus to conservation of NSPs across coronaviruses. We have also discussed the various treatment options for SARS-CoV-2 infection. A synergistic melding of bioinformatics, computer-aided drug-design and in vitro/vivo studies can feed into better understanding of the virus and therefore help in the development of small molecule inhibitors against the viral proteins.

Targeting viral proteins for restraining SARS-CoV-2: focusing lens on viral proteins beyond spike for discovering new drug targets.

INTRODUCTION: Emergence of highly infectious SARS-CoV-2 variants are reducing protection provided by current vaccines, requiring constant updates in antiviral approaches. The virus encodes four structural and sixteen nonstructural proteins which play important roles in viral genome replication and transcription, virion assembly, release , entry into cells, and compromising host cellular defenses. As alien proteins to host cells, many viral proteins represent potential targets for combating the SARS-CoV-2. AREAS COVERED: Based on literature from PubMed and Web of Science databases, the authors summarize the typical characteristics of SARS-CoV-2 from the whole viral particle to the individual viral proteins and their corresponding functions in virus life cycle. The authors also discuss the potential and emerging targeted interventions to curb virus replication and spread in detail to provide unique insights into SARS-CoV-2 infection and countermeasures against it. EXPERT OPINION: Our comprehensive analysis highlights the rationale to focus on non-spike viral proteins that are less mutated but have important functions. Examples of this include: structural proteins (e.g. nucleocapsid protein, envelope protein) and extensively-concerned nonstructural proteins (e.g. NSP3, NSP5, NSP12) along with the ones with relatively less attention (e.g. NSP1, NSP10, NSP14 and NSP16), for developing novel drugs to overcome resistance of SARS-CoV-2 variants to preexisting vaccines and antibody-based treatments.

Expediting the drug discovery for ideal leads against SARS-CoV-2 via molecular docking of repurposed drugs.

SARS-CoV-2, the novel coronavirus spreading worldwide urges the need to repurpose drugs that can quickly enter clinical trials to combat the on-going global pandemic. A cluster of proteins are encoded for by the viral genome, each assuming a critical role in pathogen endurance inside the host. To handle the adverse circumstances, robust virtual strategies such as repurposing are coming to the fore due to being economical, efficient and rapid. Five FDA approved repurposed drugs proposed to act as inhibitors by targeting SARS-CoV-2 were used for initial evaluation via molecular docking. Moreover, a comparative analysis of the selected SARS-CoV-2 proteins against five ligands (Clemizole hydrochloride, Exemestane, Nafamostat, Pregnenolone and Umifenovir) was designed. In this regard, non-structural proteins (nsp3, nsp5, nsp10, nsp12 and nsp15), structural proteins (Spike, Nucleocapsid protein) and accessory proteins (ORF 3a, ORF 7a and ORF 9 b) were selected. Here, we aim to expedite the search for a potential drug from the five FDA approved repurposing drugs already in use for treatment of multiple diseases. Based on docking analysis, Umifenovir and Pregnenolone are suggested to show potential inhibitory effects against most of the SARS-CoV-2 proteins. These drugs are noteworthy since they exhibit high binding towards target proteins and should be used as lead compounds towards in vitro and in vivo studies. Communicated by Ramaswamy H. Sarma.

A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis.

The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health around the world. In-depth studies on the pathogenic mechanisms of SARS-CoV-2 is urgently necessary for pandemic prevention. However, most laboratory studies on SARS-CoV-2 have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting the progress of relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method to assemble a SARS-CoV-2 replication and transcription system in Vero E6 cells without virion envelope formation, thus avoiding the risk of coronavirus exposure. Furthermore, an improved real-time quantitative reverse transcription PCR (RT-qPCR) approach was used to distinguish the replication of full-length replicon RNAs and transcription of subgenomic RNAs (sgRNAs). Using the SARS-CoV-2 replicon, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 facilitates the transcription of sgRNAs in the discontinuous synthesis process. Moreover, two high-frequency mutants of N protein, R203K and S194L, can obviously enhance the transcription level of the replicon, hinting that these mutations likely allow SARS-CoV-2 to spread and reproduce more quickly. In addition, remdesivir and chloroquine, two well-known drugs demonstrated to be effective against coronavirus in previous studies, also inhibited the transcription of our replicon, indicating the potential applications of this system in antiviral drug discovery. Overall, we developed a bio-safe and valuable replicon system of SARS-CoV-2 that is useful to study the mechanisms of viral RNA synthesis and has potential in novel antiviral drug screening.

Variations of SARS-CoV-2 in the Iranian population and candidate putative drug-like compounds to inhibit the mutated proteins.

The first cases of the novel coronavirus, SARS-CoV-2, were detected in December 2019 in Wuhan, China. Nucleotide substitutions and mutations in the SARS-CoV-2 sequence can result in the evolution of the virus and its rapid spread across the world. Therefore, understanding genetic variants of SARS-CoV-2 and targeting the conserved elements responsible for viral replication have great benefits for detecting its infection sources and diagnosing and treating COVID-19. In this study, we used the SARS-CoV-2 sequence isolated from a 59-year-old man in Ardabil, Iran, in April 2020 and sequenced using Oxford Nanopore technology. A meta-analysis comparing the sequence under study with other sequences from Iran indicated long nucleotide insertions/deletions (indels) that code for NSP15, the NSP14-NSP10 complex, open reading frame ORF9b, and ORF1ab polyproteins. In addition, replicating the NSP8 protein in the study sequence is another topic that can affect viral replication. Then using the DNA structure of NSP8, NSP15, NSP14-NSP10 complex, and ORF1ab as a genetic target can help find drug-like compounds for COVID-19. Potential drug-like compounds reported in this study for their mechanism of action and interactions with SARS-CoV-2 genes using drug repurposing are resveratrol, erythromycin, chloramphenicol, indomethacin, ciclesonide, and PDE4 inhibitor. Ciclesonide appears to show the best results when docked with chosen viral proteins. Therefore, different proteins isolated from nucleotide mutations in the virus sequence can indicate distinct inducers for antibodies and are important in vaccine design.

l-Methionine may modulate the assembly of SARS-CoV-2 by interfering with the mechanism of RNA polymerase.

Coronaviruses have received worldwide attention following several severe acute respiratory syndrome (SARS) epidemics. In 2019, the first case of coronavirus disease (COVID-19) caused by a novel coronavirus (SARS-coronavirus 2 [CoV-2]) was reported. SARS-CoV-2 employs RNA-dependent RNA polymerase (RdRp) for genome replication and gene transcription. Recent studies have identified a sulfur (S) metal-binding site in the zinc center structures of the RdRp complex. This metal-binding site is essential for the proper functioning of the viral helicase. We hypothesize that the use of essential nutrients can permeabilize the cell membranes. The oxidation of the metal-binding site occurs via analogs of the essential S-containing amino acid, l-Methionine. l-Methionine can operate as a carrier, and its binding would cause the potential disassembly of RdRp via the S complex and drive methyl donors via a possible countercurrent exchange mechanism and electrical-chemical gradient leading to SARS-CoV-2 replication failure. Our previously published hypothesis on the control of cancer cell proliferation suggests that the presence of a novel disulfide/methyl- adenosine triphosphate pump as an energy source would allow this process. The S binding site in l-Methionine serves as a potential target cofactor for SARS-CoV RdRp, thus providing a possible avenue for the future development of vaccines and antiviral therapeutic strategies to combat COVID-19.

Health service utilisation and access for people who inject drugs during COVID-19.

INTRODUCTION: The wide-spread implementation of interventions to limit transmission and public health consequences of COVID-19 in the Australian state of Victoria had flow-on consequences for people who use and inject drugs. Consequences included the interruption of illicit drug supply and drug procurement, and the disruption to the delivery of health services. To inform strategies that can minimise the adverse outcomes of similar future disruptive events, this study explored how COVID-19 restrictions impacted access to harm reduction and drug treatment services for people who inject drugs in Melbourne, Victoria. METHODS: Qualitative semi-structured interviews were conducted via an online calling app, with 11 participants of a broader cohort study (the SuperMIX study) in April 2020. Interviews were focused on participants experiences of accessing and using harm reduction and drug treatment services. Data were thematically analysed using a process of blended coding. RESULTS: Findings revealed how disruptions in the delivery of harm reduction and drug treatment services-in response to COVID-19 restrictions-created barriers accessing sterile injecting equipment, increased risk of arrest by police and exacerbated social isolation. Participants reported difficulties adapting to changes in services access, with some increases in injecting risk behaviours. However, improvements in opioid agonist therapy prescriptions were noted as a beneficial outcome. DISCUSSION: By examining the impacts of COVID-19 and the resultant restrictions on people who inject drugs' access to health services in Melbourne, Victoria, findings provide guidance for future responses to the unanticipated large-scale effects of the COVID-19 pandemic, and similar disruptive events.

The current state of validated small molecules inhibiting SARS-CoV-2 nonstructural proteins.

The current COVID-19 outbreak has had a profound influence on public health and daily life. Despite all restrictions and vaccination programs, COVID-19 still can lead to fatality due to a lack of COVID-19-specific treatments. A number of studies have demonstrated the feasibility to develop therapeutics by targeting underlying components of the viral proteome. Here we reviewed recently developed and validated small molecule inhibitors of SARS-CoV-2's nonstructural proteins. We described the validation level of identified compounds specific for SARS-CoV-2 in the presence of in vitro and in vivo supporting data. The mechanisms of pharmacological activity, as well as approaches for developing improved SARS-CoV-2 NSP inhibitors have been emphasized.

In search of SARS CoV-2 replication inhibitors: Virtual screening, molecular dynamics simulations and ADMET analysis.

Severe acute respiratory syndrome has relapsed recently as novel coronavirus causing a life threat to the entire world in the absence of an effective therapy. To hamper the replication of the deadly SARS CoV-2 inside the host cells, systematic in silico virtual screening of total 267,324 ligands from Asinex EliteSynergy and BioDesign libraries has been performed using AutoDock Vina against RdRp. The molecular modeling studies revealed the identification of twenty-one macrocyclic hits (2-22) with better binding energy than remdesivir (1), marketed SARS CoV-2 inhibitor. Further, the analysis using rules for drug-likeness and their ADMET profile revealed the candidature of these hits due to superior oral bioavailability and druggability. Further, the MD simulation studies of top two hits (2 and 3) performed using GROMACS 2020.1 for 10 ns revealed their stability into the docked complexes. These results provide an important breakthrough in the design of macrocyclic hits as SARS CoV-2 RNA replicase inhibitor.

Mapping the Nonstructural Transmembrane Proteins of Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the disease coronavirus-19 disease (COVID-19) has wreaked havoc on the health and economy of humanity. In addition, the disease is observed in domestic and wild animals. The disease has impacted directly and indirectly every corner of the planet. Currently, there are no effective therapies for the treatment of COVID-19. Vaccination to protect against COVID-19 started in December 2020. SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of 29.8 kb. More than two-thirds of the genome comprise Orf1ab encoding 16 nonstructural proteins (nsps) followed by mRNAs encoding structural proteins, spike (S), envelop (E), membrane (M), and nucleocapsid (N). These genes are interspaced with several accessory genes (open reading frames [Orfs] 3a, 3b, 6, 7a, 7b, 8, 9b, 9c, and 10). The functions of these proteins are of particular interest for understanding the pathogenesis of SARS-CoV-2. Several of the nsps (nsp3, nsp4, and nsp6) and Orf3a are transmembrane proteins involved in regulating the host immunity, modifying host cell organelles for viral replication and escape and hence considered drug targets. In this paper, we report mapping the transmembrane structure of the nsps of SARS-CoV-2.

Screening of FDA-approved compound library identifies potential small-molecule inhibitors of SARS-CoV-2 non-structural proteins NSP1, NSP4, NSP6 and NSP13: molecular modeling and molecular dynamics studies.

COVID-19, the current global pandemic has caused immense damage to human lives and the global economy. It is instigated by the SARS-CoV-2 virus and there is an immediate need for the identification of effective drugs against this deadly virus. SARS-CoV-2 genome codes for four structural proteins, sixteen non-structural proteins (NSPs) and several accessory proteins for its survival inside the host cells. In the present study, through in silico approaches, we aim to identify compounds that are effective against the four NSPs namely, NSP1, NSP4, NSP6 and NSP13 of SARS-CoV-2. The selection criteria of these four NSP proteins are they are least explored and potential targets. First, we have modeled the 3D structures of these proteins using homology modeling methods. Further, through molecular docking studies, we have screened the FDA-approved compounds against these modeled proteins and reported their docking scores. To gain dynamic insights, molecular dynamics studies have also been carried out for the best scored ligand against the NSPs. This study can further pave way for exposing more number of compounds against these proteins and enhance COVID-19 treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42485-021-00067-w.

Current state of diagnostic, screening and surveillance testing methods for COVID-19 from an analytical chemistry point of view.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

Molecular basis for the repurposing of histamine H2-receptor antagonist to treat COVID-19.

With the world threatened by a second surge in the number of Coronavirus cases, there is an urgent need for the development of effective treatment for the novel coronavirus (COVID-19). Recently, global attention has turned to preliminary reports on the promising anti-COVID-19 effect of histamine H2-receptor antagonists (H2RAs), most especially Famotidine. Therefore, this study was designed to exploit a possible molecular basis for the efficacy of H2RAs against coronavirus. Molecular docking was performed between four H2RAs, Cimetidine, Famotidine, Nizatidine, Ranitidine, and three non-structural proteins viz. NSP3, NSP7/8 complex, and NSP9. Thereafter, a 100 ns molecular dynamics simulation was carried out with the most outstanding ligands to determine the stability. Thereafter, Famotidine and Cimetidine were subjected to gene target prediction analysis using HitPickV2 and eXpression2Kinases server to determine the possible network of genes associated with their anti-COVID activities. Results obtained from molecular docking showed the superiority of Famotidine and Cimetidine compared to other H2RAs with a higher binding affinity to all selected targets. Molecular dynamic simulation and MMPBSA results revealed that Famotidine as well as Cimetidine bind to non-structural proteins more efficiently with high stability over 100 ns. Results obtained suggest that Famotidine and Cimetidine could be a viable option to treat COVID-19 with a mechanism of action that involves the inhibition of viral replication through the inhibition of non-structural proteins. Therefore, Famotidineand Cimetidine qualify for further study as a potential treatment for COVID-19.

Insights into forsythia honeysuckle (Lianhuaqingwen) capsules: A Chinese herbal medicine repurposed for COVID-19 pandemic.

Background: In December 2019, a novel coronavirus, SARS-CoV-2 caused a series of acute atypical respiratory diseases worldwide. However, there is still a lack of drugs with clear curative effects, and the clinical trial research of vaccines has not been completely finished. Purpose: LH capsules are approved TCM patent medicine that are widely used for the treatment of respiratory tract infectious diseases caused by colds and flu. On April 12, 2020, LH capsules and granules were officially repurposed by the China Food and Drug Administration (CFDA) for patients with mild COVID-19 based on their safety and efficacy demonstrated through multicentre, randomized, controlled clinical trials. We hope to conduct a comprehensive review of it through modern pharmacy methods, and try to explain its possible mechanism. Methods: Using the full names of LH capsules Lianhuaqingwen, Lianhua Qingwen andSARS-COV-2, COVID-19 as the keywords of the search terms, systemically search for existing related papers in various databases such as Web of Science and PubMed. And completed the collection of clinical data in ClinicalTrials.gov and Chinese Clinical Trial Registry. Last but not least, we have sorted out the anti-inflammatory and antiviral mechanisms of LH capsules through literature and Selleck. Results: This review systematically sorted out the active ingredients in LH capsules. Furthermore, the related pharmacological and clinical trials of LH capsule on SARS-CoV-2, IAV and IBV were discussed in detail. Moreover, the present review provides the first summary of the potential molecular mechanism of specific substances in LH capsules involved in resistance to SARS-COV-2 infection and the inhibition of cytokine storm syndrome (CSS) caused by IL-6. Conclusion: This review summarizes the available reports and evidence that support the use of LH capsules as potential drug candidates for the prevention and treatment of COVID-19. However, TCM exerts its effects through multiple targets and multiple pathways, and LH capsules are not an exception. Therefore, the relevant mechanisms need to be further improved and experimentally verified.

Genetic characterization of structural and open reading Fram-8 proteins of SARS-CoV-2 isolates from different countries.

Since December 2019, a severe pandemic of pneumonia, COVID-19 associated with a novel coronavirus (SARS-CoV-2), have emerged in Wuhan, China and spreading throughout the world. As RNA viruses have a high mutation rate therefore we wanted to identify whether this virus is also prone to mutations. For this reason we selected four major structural (Spike protein (S), Envelope protein (E), Membrane glycoprotein (M), Nucleocapsid phosphoprotein (N)) and ORF8 protein of 100 different SARS-CoV-2 isolates of fifteen countries from NCBI database and compared these to the reference sequence, Wuhan NC_045512.2, which was the first isolate of SARS-CoV-2 that was sequenced. By multiple sequence alignment of amino acids, we observed substitutions and deletion in S protein at 13 different sites in the isolates of five countries (China, USA, Finland, India and Australia) as compared to the reference sequence. Similarly, alignment of N protein revealed substitutions at three different sites in isolates of China, Spain and Japan. M protein exhibits substitution only in one isolates from USA, however, no mutation was observed in E protein of any isolate. Interestingly, in ORF8 substitution of Leucine, a nonpolar to Serine a polar amino acid at same position (aa84 L to S) in 23 isolates of five countries i.e. China, USA, Spain, Taiwan and India were observed, which may affect the conformation of peptides. Thus, we observed several mutations in the isolates thereafter the first sequencing of SARS-CoV-2 isolate, NC_045512.2, which suggested that this virus might be a threat to the whole world and therefore further studies are needed to characterize how these mutations in different proteins affect the functionality and pathogenesis of SARS-CoV-2.

In silico structure modelling of SARS-CoV-2 Nsp13 helicase and Nsp14 and repurposing of FDA approved antiviral drugs as dual inhibitors.

The high mortality rate from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in humans and the lack of effective therapeutic regime for its treatment necessitates the identification of new antivirals. SARS-CoV-2 relies on non-structural proteins such as Nsp13 helicase and nsp14 which are the key components of the replication-transcription complex (RTC) to complete its infectious life cycle. Therefore, targeting these essential viral proteins with small molecules will most likely to halt the disease pathogenesis. The lack of experimental structures of these proteins deters the process of structure-based identification of their specific inhibitors. In the present study, the in silico models of SARS-CoV-2 nsp13 helicase and nsp14 protein were elucidated using a comparative homology modelling approach. These in silico model structures were validated using various parameters such as Ramachandran plot, Verify 3D score, ERRAT score, knowledge-based energy and Z-score. The in silico models were further used for virtual screening of the Food and Drug Administration (FDA) approved antiviral drugs. Simeprevir (SMV), Paritaprevir (PTV) and Grazoprevir (GZR) were the common leads identified which show higher binding affinity to both nsp13 helicase and nsp14 as compared to the control inhibitors and therefore, they might be potential dual-target inhibitors. The leads also establish a network of hydrogen bonds and hydrophobic interactions with the key residues lining the active site pockets. The present findings suggest that these FDA approved antiviral drugs can be subjected to repurposing against SARS-CoV-2 infection after verifying the in silico results through in vitro and in vivo studies.

Current approaches used in treating COVID-19 from a molecular mechanisms and immune response perspective.

Coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared by the World Health Organization (WHO) as a global pandemic on March 11, 2020. SARS-CoV-2 targets the respiratory system, resulting in symptoms such as fever, headache, dry cough, dyspnea, and dizziness. These symptoms vary from person to person, ranging from mild to hypoxia with acute respiratory distress syndrome (ARDS) and sometimes death. Although not confirmed, phylogenetic analysis suggests that SARS-CoV-2 may have originated from bats; the intermediary facilitating its transfer from bats to humans is unknown. Owing to the rapid spread of infection and high number of deaths caused by SARS-CoV-2, most countries have enacted strict curfews and the practice of social distancing while awaiting the availability of effective U.S. Food and Drug Administration (FDA)-approved medications and/or vaccines. This review offers an overview of the various types of coronaviruses (CoVs), their targeted hosts and cellular receptors, a timeline of their emergence, and the roles of key elements of the immune system in fighting pathogen attacks, while focusing on SARS-CoV-2 and its genomic structure and pathogenesis. Furthermore, we review drugs targeting COVID-19 that are under investigation and in clinical trials, in addition to progress using mesenchymal stem cells to treat COVID-19. We conclude by reviewing the latest updates on COVID-19 vaccine development. Understanding the molecular mechanisms of how SARS-CoV-2 interacts with host cells and stimulates the immune response is extremely important, especially as scientists look for new strategies to guide their development of specific COVID-19 therapies and vaccines.

Corona virus versus existence of human on the earth: A computational and biophysical approach.

SARS-CoV-2 has a positive sense RNA genome of 29.9 kb in size, showing high sequence similarity to the BAT-CoV, SARS-CoV, MERS-CoV. SARS-CoV-2 is composed of 14 open reading frames (ORFs), which encodes for a total of 27 proteins divided into structural and non-structural proteins (NSPs). The fundamental structural protein-encoding genes are a spike protein (S) gene, envelope protein (E) gene, a membrane protein (M) gene, and a nucleocapsid protein (N) gene. They make about 33% of the entire genome and are vital for the viral life cycle. Rest 67% is distributed among different NSPs (such as Mpro, helicase, and RNA-dependent RNA polymerase) encoding genes across the ORFs, which are involved in virus-cell receptor interactions during viral entry. Researchers are trying to formulate vaccines, therapeutic antibodies or protein-targeted antiviral drugs to control the spread. This review proceeds stepwise through the COVID-19 outbreak, structural and genomic organization, entry mechanism, pathogenesis, and finally highlighting the essential proteins involved at each step that might be potential targets for drug discovery. Currently, approved treatment modalities consist of only supportive care and oxygen supplementation. This review is established on the current knowledge that has expanded on structural motifs and topology of proteins and their functions.