Structure-based virtual identification of natural inhibitors of SARS-CoV-2 and its Delta and Omicron variant proteins

Plain language summaryMutant strains of SARS-CoV-2 called 'variants of concern' (VOCs) are linked to a good ability to infect, re-infect and spread among people. They are also linked to poor ability to fight the disease and reduced effectiveness of vaccines. Delta and Omicron are important VOCs because they are difficult to control and treat. Specific resistance to some drugs used to treat COVID-19 poses a further challenge. Therefore, discovering natural or plant-derived drugs with no known resistance would be valuable to the treatment of COVID-19. In this study, we screen and identify seven plant-derived compounds that may be useful to treating COVID-19 - we identify Quercetin-3-acetyl-glucoside, Rutin, Kaempferol, Catechin, Orientin, Obetrioside and Neridienone A as potential candidates. Orientin, Obetrioside, Catechin and Neridienone A are identified as candidates against Delta and Omicron for the first time. Aim: Structure-based identification of natural compounds against SARS-CoV-2, Delta and Omicron target proteins. Materials & methods: Several known antiviral natural compounds were subjected to molecular docking and MD simulation against SARS-CoV-2 Mpro, Helicase and Spike, including Delta and Omicron Spikes. Results: Of the docked ligands, 20 selected for each complex exhibited overall good binding affinities (-7.79 to -5.06 kcal/mol) with acceptable physiochemistry following Lipinski's rule. Finally, two best ligands from each complex upon simulation showed structural stability and compactness. Conclusion: Quercetin-3-acetyl-glucoside, Rutin, Kaempferol, Catechin, Orientin, Obetrioside and Neridienone A were identified as potential inhibitors of SARS-CoV-2 Mpro, Helicase and Spike, while Orientin and Obetrioside also showed good binding-affinities with Omicron Spike. Catechin and Neridienone A formed stable complexes with Delta Spike. Tweetable We report structure-based identification of natural compounds viz., Quercetin-3-acetyl-glucoside, Rutin, Kaempferol, Catechin, Orientin, Obetrioside and Neridienone A against SARS-CoV-2 Mpro, Helicase and Spike as well as Delta and Omicron Spike proteins.

Coronaviruses SARS-CoV, MERS-CoV, and SARS-CoV-2 helicase inhibitors: a systematic review of invitro studies.

Introduction: The recent outbreak of SARS-CoV-2 has significantly increased the need to find inhibitors that target the essential enzymes for viral replication in host cells. This systematic review was conducted to identify potential inhibitors of SARS-CoV, MERS-CoV, and SARS-CoV-2 helicases that have been tested by in vitro methods. Their inhibitory mechanisms are discussed in this review, in addition to their cytotoxic and protective properties. Methods: The databases PUBMED/MEDLINE, EMBASE, SCOPUS, and Web of Science were searched using different combinations of the keywords "helicase", "nsp13", "inhibitors", "coronaviridae", "coronaviruses", "virus replication", "replication", and "antagonists and inhibitors". Results: A total of 6854 articles were identified. Thirty-one were included into this review. These studies reported on the inhibitory effects of 309 compounds on SARS-CoV, MERS-CoV, and SARS-CoV-2 helicase activities measured by invitro methods. Helicase inhibitors were categorized according to the type of coronavirus and tested enzymatic activity, nature, approval, inhibition level, cytotoxicity, and viral infection protective effects. These inhibitors are classified according to the site of their interaction with coronavirus helicases into four types: zinc-binding site inhibitors, nucleic acid-binding site inhibitors, nucleotide-binding site inhibitors, and inhibitors with no clear interaction site. Conclusion: Evidence from in vitro studies suggests that helicase inhibitors have a high potential as antiviral agents. Several show good antiviral activity while maintaining moderate cytotoxicity. These inhibitors should be clinically investigated to determine their efficacy in treating coronavirus infections, particularly SARS-CoV-2.

Unwinding mechanism of SARS-CoV helicase (nsp13) in the presence of Ca2+, elucidated by biochemical and single-molecular studies.

The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.

SARS-CoV-2 Nsp13 Catalytic Efficiency is Regulated by ATP: Mg2+ Stoichiometry and Functional Cooperativity Among Nsp13 Molecules

Coronavirus disease 19 (COVID-19) is a highly contagious and lethal disease caused by the SARS-CoV-2 positive-strand RNA virus. Nonstructural protein 13 (Nsp13) is the highly conserved ATPase/helicase required for replication of the SARS-CoV-2 genome which allows for the infection and transmission of COVID-19. We biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein expressed using a eukaryotic cell-based system and characterized its catalytic functions, focusing on optimization of its reaction conditions and assessment of functional cooperativity among Nsp13 molecules during unwinding of duplex RNA substrates. These studies allowed us to carefully determine the optimal reaction conditions for binding and unwinding various nucleic acid substrates. Previously, ATP concentration was suggested to be an important factor for optimal helicase activity by recombinant SARS-CoV-1 Nsp13. Apart from a single study conducted using fixed concentrations of ATP, the importance of the essential divalent cation for Nsp13 helicase activity had not been examined. Given the importance of the divalent metal ion cofactor for ATP hydrolysis and helicase activity, we assessed if the molar ratio of ATP to Mg2+ was important for optimal SARS-CoV-2 Nsp13 RNA helicase activity. We determined that Nsp13 RNA helicase activity was dependent on ATP and Mg2+ concentrations with an optimum of 1 mM Mg2+ and 2 mM ATP. Next, we examined Nsp13 helicase activity as a function of equimolar ATP:Mg2+ ratio and determined that helicase activity decreased as the equimolar concentration increased, especially above 5 mM. We determined that Nsp13 catalytic functions are sensitive to Mg2+ concentration suggesting a regulatory mechanism for ATP hydrolysis, duplex unwinding, and protein remodeling, processes that are implicated in SARS-CoV-2 replication and proofreading to ensure RNA synthesis fidelity. Evidence is presented that excess Mg2+ impairs Nsp13 helicase activity by dual mechanisms involving both allostery and ionic strength. In addition, using single-turnover reaction conditions, Nsp13 unwound partial duplex RNA substrates of increasing doublestranded regions (16-30 base pairs) with similar kinetic efficiency, suggesting the enzyme unwinds processively in this range under optimal reaction conditions. Furthermore, we determined that Nsp13 displayed sigmoidal behavior for helicase activity as a function of enzyme concentration, suggesting that functional cooperativity and oligomerization are important for optimal activity. The observed functional cooperativity of Nsp13 protomers suggests the essential coronavirus RNA helicase has roles in RNA processing events beyond its currently understood involvement in the SARS-CoV-2 replication-transcription complex (RTC), in which it was suggested that only one of the two Nsp13 subunits has a catalytic function, whereas the other has only a structural role in complex stability. Altogether, the intimate regulation of Nsp13 RNA helicase by divalent cation and protein oligomerization suggests drug targets for modulation of enzymatic activity that may prove useful for the development of novel anti-coronavirus therapeutic strategies. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

The SARS-CoV-2 Nsp13 Helicase Required for Coronavirus Replication Interacts with Structured Nucleic Acids in a Strand-Specific Manner and Catalyzes a Novel Protein-RNA Remodeling Activity Implicated in the Proofreading Mechanism of the Replication-Transc

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded, positive-sense RNA virus responsible for COVID-19, requires a set of virally encoded nonstructural proteins that compose a replication-transcription complex (RTC) to replicate its 30 kilobase genome. One such nonstructural protein within the RTC is Nsp13, a highly conserved molecular motor ATPase/helicase. Upon purification of the recombinant SARS-CoV-2 Nsp13 protein expressed using a eukaryotic cell-based system, we biochemically characterized the enzyme by examining its catalytic functions, nucleic acid substrate specificity, and putative protein-nucleic acid remodeling activity. We determined that Nsp13 preferentially interacts with single-stranded (ss) DNA compared to ssRNA during loading to unwind with greater efficiency a partial duplex helicase substrate. The binding affinity of Nsp13 to nucleic acid was confirmed through electrophoretic mobility shift assays (EMSA) by determining that Nsp13 binds to DNA substrates with significantly greater efficiency than RNA. These results demonstrate strand-specific interactions of SARS-CoV-2 Nsp13 that dictate its ability to load and unwind structured nucleic acid substrates. We next determined that Nsp13 catalyzed unwinding of double-stranded (ds) RNA forked duplexes on substrates containing a backbone disruption (neutrally charged polyglycol linker (PGL)) was strongly inhibited when the PGL was positioned in the 5' ssRNA overhang, suggesting an unwinding mechanism in which Nsp13 is strictly sensitive to perturbation of the translocating strand sugar-phosphate backbone integrity. Furthermore, we demonstrated for the first time the ability of the coronavirus Nsp13 helicase to disrupt a high-affinity nucleic acid-protein interaction, i.e., a streptavidin tetramer bound to biotinylated RNA or DNA substrate, in a uni-directional manner and with a preferential displacement of the streptavidin complex from biotinylated ssDNA versus ssRNA. In contrast to the poorly hydrolysable ATP-gamma-S or non-hydrolysable AMP-PNP, ATP supports Nsp13-catalyzed disruption of the nucleic acidprotein complex, suggesting that nucleotide binding by Nsp13 is not sufficient for protein-RNA disruption and the chemical energy of nucleoside triphosphate hydrolysis is required to fuel remodeling of protein bound to RNA or DNA. Our results build upon structural studies of the SARS-CoV-2 RTC in which it was suggested that Nsp13 pushes the RNA polymerase (Nsp12) backward on the template RNA strand. Experimental evidence from our studies demonstrate that Nsp13 helicase efficiently remodels a large high affinity protein-RNA complex in a manner dependent on its intrinsic ATP hydrolysis function. We proposed that this novel biochemical activity of Nsp13 is relevant to its role in SARS-CoV-2 RNA processing functions and replication. It was proposed that Nsp13 facilitates proofreading during coronavirus replication when a mismatched base is inadvertently incorporated into the SARS-CoV-2 genome during replication to reposition the RTC so that the proofreading nuclease complex (Nsp14-Nsp10) can gain access and remove the nascently synthesized nucleotide to ensure polymerase fidelity. Our findings implicate a direct catalytic role of Nsp13 in protein-RNA remodeling during coronavirus genome replication beyond its duplex strand separation or structural stabilization of the RTC, yielding new insight into the proofreading mechanism. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Sirna-A Potential Therapy for Covid-19 Infection

Background: The deadly virus COVID-19 has affected more than 1 crore people and claimed more than 5 lakh lives worldwide according to the World health organization. Though there are numerous treatment modalities available including anti-bacterials, antivirals, vaccines etc., none of them can be considered as effective cure for SARS CoV-2 virus as they are mostly non-specific in action. Aim(s): siRNA therapy can be considered as a significant treatment modality due to its specificity in action. The aim of this review is to explore siRNA as a potential treatment strategy for the treatment of COVID-19. Material(s) and Method(s): In this review we shall explore the targets of siRNA therapy which includes viral RNA-dependent RNA polymerase, helicase, protease and nucleoprotein N. siRNA related patents provide solutions for novel RNAi techniques, high expense of chemically synthetic siRNA, techniques for restraining SARS-CoV by disturbing RNA etc., siRNA-based drug delivery systems and limitations of nanocarrier delivery system were reviewed. siRNA is a gene silencer that targets highly conserved sequences which codes for protease 3CL (nsp5) and viral helicase (from 16-18 kbp). Conclusion(s): Thus, siRNA-based therapy is considered highly efficacious as it can hit the highly conserved regions of SARS-CoV-2 RNA.Copyright © 2023, Advanced Scientific Research. All rights reserved.

Dermatomyositis with Specific Autoantibodies Appearing after Sars-Cov-2 Infection or Mrna Vaccination: Hints at a Shared Pathogenetic Mechanism

Background. Environmental factors such as infections and vaccines are known to trigger dermatomyositis (DM), and during the recent SARS-CoV-2 pandemic this has become even clearer. SARS-CoV-2 infection may share features with anti-MDA5 DM, such as rapidly progressive lung involvement, cutaneous lesions and cytokine release syndrome. A few case reports of DM following SARSCoV-2 vaccination have been published, suggesting the onset of an aberrant immune response leading to DM with specific autoantibody signatures and severe organ impairment. Methods. Clinical and laboratory data of the 2 case reports were obtained from electronic clinical charts in Humanitas Research Hospital (Rozzano, Milan, Italy). Autoantibody analysis was performed by protein-immunoprecipitation for anti-MDA5 and immunoblot for anti-Ro52 and TIF1gamma antibodies as per protocol. Results. Case report 1 is a 71-year-old woman who developed fever, cough, and anosmia, which resolved spontaneously in two weeks, but did not undergo a nasopharyngeal swab, while her relatives were diagnosed with SARS-CoV-2 infection. When symptoms improved, she developed arthralgia and skin lesions on her face, chest, and hands for which she started topical treatment, with negative SARSCoV-2 nasopharyngeal swab and positive serum test for IgG against SARS-CoV-2 spike protein. For the persistence of the skin rash and arthralgia, she was admitted to our Department in March 2021. Blood tests showed mild elevation of C reactive protein (2.1 mg/L -normal value NV<5), aspartate (84 UI/L) and alanine aminotransferase (133 UI/L -NV<35), ferritin (595 ng/ml -NV<306), troponin I (19 ng/L -NV<14), and BNP (251 pg/ml -NV<100) with normal complete blood cell count, creatine kinase, C3 and C4. IgG antibodies for SARS-CoV-2 spike protein were confirmed to be elevated (96 AU/ml -NV<15). Autoantibodies associated with connective tissue diseases were tested and only anti-MDA5 antibodies were positive at immunoprecipitation. A punch biopsy of a Gottron-like lesion on the left hand showed leukocytoclastic vasculitis. We observed reduced capillary density with neoangiogenesis and ectasic capillaries at the nailfold capillaroscopy. EKG and ecocardiography were normal, while cardiac magnetic resonance detected abnormalities in the parametric sequences, consistent with signs of previous myocarditis. A lung CT scan revealed pulmonary emphysema while respiratory function tests demonstrated reduced volumes (FVC 82%, FEV1 64%, inadequate compliance CO diffusion test). Based on the biochemical and clinical findings, a diagnosis of anti-MDA5-associated DM with skin and heart involvement was made and treatment with low-dose methylprednisolone (0.25 mg/kg daily) and azathioprine 100 mg was started, then switched to mycophenolate because not effective on skin lesions. Case report 2 is an 84-year-old woman with history of colon cancer (surgical treatment) and oral lichen treated with low doses steroids in the last 2 years. After the 2nd dose of SARS-CoV-2 mRNA vaccination, in March 2021 she developed skin rash with V-sign, Gottron's papules, periungueal ulcers, muscle weakness and fatigue, thus she performed a rheumatologic evaluation. Blood tests showed mild elevation of creatine kinase (484 UI/L, NV <167), CK-MB (9.6ng/ml, NV <3.4), BNP (215 pg/ml -NV<100) with normal values of complete blood cell count, C3 and C4. Anti-Ro52kDa and TIF1gamma were positive at immunoblot, thus we confirmed a diagnosis of DM. The clinical evaluation also showed active scleroderma pattern at nailfold capillaroscopy, normal echocardiography, bronchiectasia but not interstitial lung disease at lung CT, and normal respiratory function tests (FVC 99%, FEV1 99%, DLCO 63%, DLCO/VA 81%). A PET-CT scan was performed to exclude paraneoplastic DM, and treatment with steroids and mycophenolate was started. Conclusions. SARS-CoV-2 may induce mechanisms for escaping the innate immunity surveillance and causing autoimmune diseases, but more clinical and functional studies are needed to demonstrate this possible association.

Impact of Sars-Cov-2 on the Clinical Presentation of Juvenile Idiopathic Inflammatory Myopathies

Background. Viruses are thought to play a role in triggering juvenile idiopathic inflammatory myopathies (JIIM), which include juvenile dermatomyositis (JDM), juvenile polymyositis (JPM), and overlap myositis. There is growing evidence that infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can trigger autoimmune diseases in genetically susceptible individuals, including idiopathic inflammatory myopathies (IIM). Studies have shown similarities between SARS-CoV-2 infection and anti-melanoma differentiation-associated gene 5 (MDA5) antibody-related dermatomyositis, suggesting possible shared underlying autoimmune and/or inflammatory mechanisms. To date, there are few studies describing individual cases of JIIM following SARS-CoV-2 infection, and, to our knowledge, none have explored the effects of SARS-CoV-2 on the clinical presentation of JIIM. In this study, we aim to investigate the impact of SARS-CoV-2 on JIIM by comparing the onset of new JIIM cases, as well as clinical and laboratory characteristics at disease onset, in patients diagnosed before and after onset of the Coronavirus Disease 2019 pandemic (COVID 19). Methods. Patients diagnosed with JIIM prior to age 19 at The Children's Hospital at Montefiore were eligible for study inclusion. Demographic, clinical, and laboratory data, as well as evidence of exposure to SARS-CoV-2, were collected retrospectively by manual chart review. Patients were grouped into pre-COVID 19 (defined as prior to January 1, 2020) and post-COVID 19 (defined as January 1, 2020, or later). Descriptive statistics were used to summarize each variable. Given the small sample size, non-parametric testing was performed using Fischer's exact test and Wilcoxon rank sum test. Results. Forty-four patients were included in the analysis (Table I). Thirty-four patients (77.3%) were diagnosed pre-COVID 19 and ten patients (22.7%) were diagnosed post-COVID 19. Of the ten patients diagnosed post-COVID 19, five (50%) had known exposure to or infection with SARS-CoV-2. Patients diagnosed with JIIM post-COVID 19 were more likely to be of non-Hispanic Black or Asian descent (p=0.041), develop disease at an older age (p=0.009), and present with non-classic cutaneous manifestations (as opposed to classic findings of Gottron's papules/sign or Heliotrope rash) (p=0.031), despite similar frequencies of JDM versus overlap myositis. While presence of muscle weakness did not differ between the groups, patients diagnosed post-COVID 19 tended to have more severe weakness, though results were not statistically significant. Interestingly, despite delays to diagnosis reported during the pandemic, there was no difference between time from symptom onset to diagnosis. Conclusion. This is the first study to explore the effects of SARS-CoV-2 on the clinical presentation of JIIM. In our center, we found that patients diagnosed with JIIM after COVID-19 were more likely to be racial minorities, older at onset, and present with non-classic cutaneous manifestations. While there were no significant differences in myositis specific or associated antibodies, patients diagnosed post-COVID 19 did not have complete autoantibody investigation performed at the time of this study. Clinicians should consider JIIM even in the absence of classic cutaneous manifestations, particularly in the post-COVID 19 era. Patients should be followed longitudinally to explore long-term impacts of SARS-CoV-2 on JIIM. Further investigation is warranted to identify the mechanisms by which SARS-CoV-2 impacts JIIM and how these differ from the effects of other viruses.

Effective Binding Affinity of Inhibitor - [3-(Carbamoylamino) Phenyl] Acetamide Against the SARS-CoV-2 NSP13 Helicase

The ongoing outbreak of COVID-19 has become a global health emergency. The SARS-CoV-2 NSP13 helicase plays an important role in SARS-Co V-2 replication and could serve as a target for antivirals to develop potential COVID-19 treatment. The objective of the study is to used Homology modeling and docking analysis of SARS-CoV-2 NSP13 helicase as drug target. The results of this study establish N-[3-( carbamoylamino) phenyl] acetamide as a valuable lead molecule with great potential for SARS-CoV-2 NSP13 helicase inhibitor. The structure and function of SARS-CoV- 2 NSP13 helicase predicted by in silica modeling studies. The SWISS-MODEL Structure Assessment tool was used for homology modeling and visual analysis of the crystal structure of the protein. The validation for structure models was performed using PROCHECK. Model quality estimates based on the QMEAN and ProSA. The MCULE-1-Click docking and InterEvDock-2.0 server were used for proteinlig and docking.The SARS-CoV-2 NSP13 helicase model corresponds to probability confirmation with 90.9% residue of the core section that specifies the accuracy of the predicted model. ProS A Z-score of -9.17;indicates the good quality of the model. Inhibitor N-[3-(carbamoylamino) phenyl] acetamide exhibited effective binding affinity against the NSP13 helicase. The docking results revealed the Lys-146, Leu-147, Ile-151, Tyr-185, Lys-195, Tyr-224, Val-226, Leu- 227, Ser-229 residues exhibit good binding interactions with inhibitor ligand.

Case report of an antimelanoma differentiationassociated protein 5 antibody-associated amyopathic dermatomyositis

Autoantibodies against melanoma differentiation-associated protein 5 (MDA5) associated with dermatomyositis have recently been described in Asians with rapidly progressive respiratory disease. Here we report the case of a middle-aged white woman with anti-MDA5 antibody-associated amyopathic dermatomyositis with interstitial lung disease (ILD), which is stable with minimal immune suppression. A 55-year-old woman was referred to a virtual dermatology clinic during the COVID-19 pandemic suspected of having widespread eczema involving the chest, face, arm and hands on the background of atopy. On direct questioning, she admitted to having constitutional symptoms, exertional dyspnoea, joint pain and symptoms of proximal muscle weakness. On clinical suspicion of possible connective tissue disorder, she was urgently reviewed in the hospital, where she was found to have a photodistributed rash involving cutaneous ulceration and violaceous plaques. Hand examination showed mechanic's hand mimicking hand eczema, ragged nail cuticles and acute tenosynovitis in the left index finger. Her upper and lower limb muscle power was normal and respiratory examination revealed bi-basal fine end-expiratory crepitation. Her repeated biochemical, haematological and muscle enzymes remained normal. Skin biopsy taken from photosensitive rash over the wrist showed hypergranulosis, Civatte body formation, colloid bodies and dyskeratotic keratinocytes, in keeping with severe lichenoid eruption. Superficial dermis showed patchy red-cell extravasation, perivascular chronic infiltration, dermal oedema and serum on the surface, in keeping with ulceration secondary to severe inflammatory processes. There were no eosinophils and eccrine coils were free of inflammation, raising the suspicion of a drug eruption. Her antinuclear antibody and double-stranded DNA were repeatedly negative. Myositisspecific antibody panel was performed owing to a high clinical suspicion of photosensitive dermatoses, both clinically and histologically. Histology revealed positive anti-MDA5 antibodies;repeated positive testing confirmed this. Although lung function was normal, computed tomography revealed evidence of ILD. We made a diagnosis of anti-MDA5 antibodyassociated amyopathic dermatomyositis with ILD. Her malignancy screening was negative. The patient was started on lowdose prednisolone and hydroxychloroquine 200 mg twice daily, with topical steroid applications, which resulted in remarkable clinical improvement. Anti-MDA5 associated dermatomyositis has characteristic cutaneous lesions consisting of skin ulceration and tender palmar papules, mechanic's hands, inflammatory arthritis and rapidly progressive ILD, which is frequently fatal. Although our patient had ILD, she was relatively stable on minimal immunosuppression. It is important for clinicians to have an increased awareness of this disease as it could have a highly variable clinical presentation in the white population.

Alveolar macrophages from people with severe eosinophilic asthma show differential antiviral responses to human rhinovirus 16 and SARS-CoV-2

Background: Rhinovirus is the most common trigger for exacerbations of asthma. Alveolar macrophages (AM) are a major site of RV infection and can also be infected by SARS-CoV-2. The pandemic caused by the SARS-CoV-2 raised concerns that patients with severe asthma (SA) would be at particularly high risk of developing severe disease. To date, evidence for poor outcomes in asthma remains limited suggesting a differential immune response to these two viruses. Method(s): Alveolar macrophages (AM) were isolated from bronchoalveolar lavage samples from patients with SA and infected with RV (n=13), SARS-CoV-2 alpha (B.1.1.7) (n=9) and delta (B.1.627.2)(n=8) variants. Antiviral mediators representing NF-KB-induced interferon-driven mRNAs (IL6 and IL8, RIGI and MDA5, respectively) were measured by qPCR, normalised to GAPDH and compared between infected AM and controls. Result(s): RV infected AM showed significant increases in mRNA expression of RIGI (4.39 fold change +/-4.68, p<0.001 vs control), MDA5 (2.96 fold change +/- 2.93, p=0.002 vs control) and IL6 (1.88 fold change +/- 0.98, p=0.006) compared to AM treated with control media alone, whilst IL8 did not significantly change. However, AM infected with SARS-CoV-2 alpha or delta variants showed no difference in levels of antiviral mediators compared to controls. Longitudinal analysis of AMs infected with SARS-CoV-2 alpha or delta variants showed no antiviral response. Conclusion(s): AM from subjects with severe asthma produce a pattern of anti-viral responses following RV infection that is absent when exposed to SARS-CoV-2 variants currently in circulation.

Novel Coronavirus Disease 2019 (COVID-19) Current Update: Perspective on Epidemiology, Diagnosis, Drug Targets and Vaccines

Background: A novel coronavirus disease, 2019-nCoV (COVID-19), was reported first in Wuhan, the capital of Hubei, China, in late December 2019 and subsequently reached pandemic level affecting around 213 countries. As of 24th May 2020, the total number of positive cases confirmed is 5,446,514 and 344,754 death reports worldwide. COVID-19 infection causes pneumonia-like severe respiratory infection and acute lung failure. Severe acute respiratory syndrome coron-avirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA beta coronavirus that is a confirmed causative agent of COVID-19. SARS-CoV-2 may use angiotensin-converting enzyme 2 (ACE2), unlike the receptor utilized by SARS-CoV (emerged in 2002) to infect humans. People with a history of hypertension, chronic obstructive pulmonary disease, diabetes, cardiovascular disease are more susceptible to SARS-CoV-2. Objective(s): The purpose of this review was to help the society to distinguish and deal with SARS-CoV-2, and make available a reference for forthcoming studies. Method(s): Recently, diagnostic primer sets on the SARS-CoV-2 genome have been identified. The receptor-binding domain of SARS-COV-2 highlighted the mode by which beta-CoV recognizes ACE2. Various diagnostic tools are available to differentiate and identify SARS-CoV-2 infection as RT-PCR, antigen detection assay, and antibody detection assay. Different strategies have been employed to control the SARS-CoV-2, considering various drug targets like the main protease (3-CLPro), papain-like protease (PLpro), helicase (NSP13), RNA dependent RNA polymerase (RdR-p), and viral envelope (E) protein. Conclusion(s): In the present review, we have updated details of transmission, pathogenesis, genome structure, diagnostic criteria, clinical characteristics, therapeutics, and vaccine development of the SARS-CoV-2 infection, which may be significant in the control and response to the COVID-19 out-break.Copyright © 2021 Bentham Science Publishers.

Virtual Screening of Potential Therapeutic Inhibitors Against Spike, Heli-case, and Polymerase of SARS-CoV-2 (COVID-19)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected around 13 million people and has caused more than 5.7 lakh deaths worldwide since December 2019. In the absence of FDA approved drugs for its treatment, only symptomatic management is done. Method(s): We attempted to uncover potential therapeutic targets of spike, helicase, and RNA dependent RNA polymerase (RdRp) proteins of the SARS-CoV-2 employing a computational approach. The PDB structure of spike and RdRp and predicted structure of helicase proteins were docked with 100 approved anti-viral drugs, natural compounds, and some other chemical compounds. Result(s): The anti-SARS ligands EK1 and CID 23631927, and NCGC00029283 are potential entry in-hibitors as they showed affinity with immunogenic Receptor Binding Domain (RBD) of the spike pro-tein. This RBD interacts with Angiotensin Converting Enzyme (ACE2) receptor, facilitating the entry of virion in the host cells. The FDA approved drugs, including Nelfinavir, Saquinavir, Tipranavir, Setrobu-vir, Indinavir, and Atazanavir, showed potential inhibitory activity against targeted domains and thus, may act as entry or replication inhibitor or both. Furthermore, several anti-HCoV natural compounds, including Amentoflavone, Rutin, and Tannin, are also potential entry and replication inhibitors as they showed affinity with RBD, P-loop containing nucleoside triphosphate hydrolase, and the catalytic domain of the respective protein. Dithymoquinone showed significant inhibitory potential against the fusion peptide of S2 domain. Importantly, Tannin, Dithymoquinone, and Rutin can be extracted from Nig-ella sativa seeds and thus, may prove to be one of the most potential anti-SARS-CoV-2 inhibitors. Conclusion(s): Several potential ligands were identified with already known anti-HCoVs activities. Fur-thermore, as this study showed that some of the ligands acted as both entry and replication inhibitors against SARS-CoV-2, it is envisaged that a combination of either inhibitor with a dual mode of action would prove to be a much desired therapeutic option against this viral infection.Copyright © 2021 Bentham Science Publishers.

Discovering potential inhibitors against SARS-CoV-2 by targeting Nsp13 Helicase.

The rise in the incidence of COVID-19 as a result of SARS-CoV-2 infection has threatened public health globally. Till now, there have been no proper prophylactics available to fight COVID-19, necessitating the advancement and evolution of effective curative against SARS-CoV-2. This study aimed at the nonstructural protein 13 (nsp13) helicase as a promising target for drug development against COVID-19. A unique collection of nucleoside analogs was screened against the SARS-CoV-2 helicase protein, for which a molecular docking experiment was executed to depict the selected ligand's binding affinity with the SARS-CoV-2 helicase proteins. Simultaneously, molecular dynamic simulations were performed to examine the protein's binding site's conformational stability, flexibility, and interaction with the ligands. Key nucleoside ligands were selected for pharmacokinetic analysis based on their docking scores. Selected ligands (cordycepin and pritelivir) showed excellent pharmacokinetics and were well stabilized at the proteins' binding site throughout the MD simulation. We have also performed binding free energy analysis or the binding characteristics of ligands with Nsp13 by using MM-PBSA and MM-GBSA. Free energy calculation by MM-PBSA and MM-GBSA analysis suggests that pritelivir may work as viable therapeutics for efficient drug advancement against SARS-CoV-2 Nsp13 helicase, potentially arresting the SARS-CoV-2 replication.Communicated by Ramaswamy H. Sarma.

In Silico Binding of 2-Aminocyclobutanones to SARS-CoV-2 Nsp13 Helicase and Demonstration of Antiviral Activity.

The landscape of viral strains and lineages of SARS-CoV-2 keeps changing and is currently dominated by Delta and Omicron variants. Members of the latest Omicron variants, including BA.1, are showing a high level of immune evasion, and Omicron has become a prominent variant circulating globally. In our search for versatile medicinal chemistry scaffolds, we prepared a library of substituted ɑ-aminocyclobutanones from an ɑ-aminocyclobutanone synthon (11). We performed an in silico screen of this actual chemical library as well as other virtual 2-aminocyclobutanone analogs against seven SARS-CoV-2 nonstructural proteins to identify potential drug leads against SARS-CoV-2, and more broadly against coronavirus antiviral targets. Several of these analogs were initially identified as in silico hits against SARS-CoV-2 nonstructural protein 13 (Nsp13) helicase through molecular docking and dynamics simulations. Antiviral activity of the original hits as well as ɑ-aminocyclobutanone analogs that were predicted to bind more tightly to SARS-CoV-2 Nsp13 helicase are reported. We now report cyclobutanone derivatives that exhibit anti-SARS-CoV-2 activity. Furthermore, the Nsp13 helicase enzyme has been the target of relatively few target-based drug discovery efforts, in part due to a very late release of a high-resolution structure accompanied by a limited understanding of its protein biochemistry. In general, antiviral agents initially efficacious against wild-type SARS-CoV-2 strains have lower activities against variants due to heavy viral loads and greater turnover rates, but the inhibitors we are reporting have higher activities against the later variants than the wild-type (10-20X). We speculate this could be due to Nsp13 helicase being a critical bottleneck in faster replication rates of the new variants, so targeting this enzyme affects these variants to an even greater extent. This work calls attention to cyclobutanones as a useful medicinal chemistry scaffold, and the need for additional focus on the discovery of Nsp13 helicase inhibitors to combat the aggressive and immune-evading variants of concern (VOCs).

Interaction of SARS-CoV-2 Nucleocapsid Protein and Human RNA Helicases DDX1 and DDX3X Modulates Their Activities on Double-Stranded RNA.

The nucleocapsid protein Np of SARS-CoV-2 is involved in the replication, transcription, and packaging of the viral genome, but it also plays a role in the modulation of the host cell innate immunity and inflammation response. Ectopic expression of Np alone was able to induce significant changes in the proteome of human cells. The cellular RNA helicase DDX1 was among the proteins whose levels were increased by Np expression. DDX1 and its related helicase DDX3X were found to physically interact with Np and to increase 2- to 4-fold its affinity for double-stranded RNA in a helicase-independent manner. Conversely, Np inhibited the RNA helicase activity of both proteins. These functional interactions among Np and DDX1 and DDX3X highlight novel possible roles played by these host RNA helicases in the viral life cycle.

Mechanism-based approaches of 1,3,4 thiadiazole scaffolds as potent enzyme inhibitors for cytotoxicity and antiviral activity

Significant progress in understanding cancer pathogenesis, it remains one of the leading causes of death after cardiovascular diseases. Similarly viral infections have emerged from wildlife or re-emerged, generating serious threats to the global health. As a result, there is an urgent need for the development of novel, more effective anticancer and antiviral therapeutics. Scientists, medicinal chemists and researchers are continuously finding novel targets, mechanisms and molecules against theses severe and dangerous infections. Therefore, ongoing extensively study and research emphasizes 1,3,4 thiadiazole pharmacophore have versatile pharmacological actions. Due to mesoionic behaviour of 1,3,4 thiadiazole pharmacophore allows to enter and easily cross biological membrane which allow to interact various biological proteins. In this review study an attempt has been made of various mechanisms involved in cancer and viral prevalence with updated studies done so far. This review study also findings the role of 1,3,4 thiadiazole motif in the management of various cancers and viral infection. This study also highlighting research statics on clinical trials and various patents containing 1,3,4 thiadiazole derivatives. © 2022 The Author(s)

Dermatomyositis: severe acute respiratory syndrome coronavirus 2, an initiator or a trigger?

Dermatomyositis is an immune-mediated inflammatory myopathy. In adults, the common triggering factors for its onset include viral infections, malignancy, and drugs. The clinical manifestation of these group of disorders may be cutaneous, neurological, pulmonary, or a combination of all. The cutaneous manifestations are helpful in the early clinical diagnosis. The detection of myositis-specific autoantibodies serves as specific biomarkers for the diagnosis and helps in predicting the prognosis. We are presenting two cases of dermatomyositis, temporally related to the severe acute respiratory syndrome coronavirus 2 infection and vaccination. Copyright © 2022 Journal of the Egyptian Women's Dermatologic Society.

Biochemical analysis of SARS-CoV-2 Nsp13 helicase implicated in COVID-19 and factors that regulate its catalytic functions.

Replication of the 30-kilobase genome of SARS-CoV-2, responsible for COVID-19, is a key step in the coronavirus life cycle that requires a set of virally encoded nonstructural proteins such as the highly conserved Nsp13 helicase. However, the features that contribute to catalytic properties of Nsp13 are not well established. Here, we biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein, focusing on its catalytic functions, nucleic acid substrate specificity, nucleotide/metal cofactor requirements, and displacement of proteins from RNA molecules proposed to be important for its proofreading role during coronavirus replication. We determined that Nsp13 preferentially interacts with single-stranded DNA compared with single-stranded RNA to unwind a partial duplex helicase substrate. We present evidence for functional cooperativity as a function of Nsp13 concentration, which suggests that oligomerization is important for optimal activity. In addition, under single-turnover conditions, Nsp13 unwound partial duplex RNA substrates of increasing double-stranded regions (16-30 base pairs) with similar efficiency, suggesting the enzyme unwinds processively in this range. We also show Nsp13-catalyzed RNA unwinding is abolished by a site-specific neutralizing linkage in the sugar-phosphate backbone, demonstrating continuity in the helicase-translocating strand is essential for unwinding the partial duplex substrate. Taken together, we demonstrate for the first time that coronavirus helicase Nsp13 disrupts a high-affinity RNA-protein interaction in a unidirectional and ATP-dependent manner. Furthermore, sensitivity of Nsp13 catalytic functions to Mg2+ concentration suggests a regulatory mechanism for ATP hydrolysis, duplex unwinding, and RNA protein remodeling, processes implicated in SARS-CoV-2 replication and proofreading.