Recombinant SARS-CoV-2 Spike Protein Stimulates Secretion of Chymase, Tryptase, and IL-1ß from Human Mast Cells, Augmented by IL-33.

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin-converting enzyme 2 (ACE2) and results in the production of multiple proinflammatory cytokines, especially in the lungs, leading to what is known as COVID-19. However, the cell source and the mechanism of secretion of such cytokines have not been adequately characterized. In this study, we used human cultured mast cells that are plentiful in the lungs and showed that recombinant SARS-CoV-2 full-length S protein (1-10 ng/mL), but not its receptor-binding domain (RBD), stimulates the secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) as well as the proteolytic enzymes chymase and tryptase. The secretion of IL-1ß, chymase, and tryptase is augmented by the co-administration of interleukin-33 (IL-33) (30 ng/mL). This effect is mediated via toll-like receptor 4 (TLR4) for IL-1ß and via ACE2 for chymase and tryptase. These results provide evidence that the SARS-CoV-2 S protein contributes to inflammation by stimulating mast cells through different receptors and could lead to new targeted treatment approaches.

Sulforaphane is a reversible covalent inhibitor of 3-chymotrypsin-like protease of SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a major public health threat worldwide and emphasizes an urgent need for effective therapeutics. Recently, Ordonez et al. identified sulforaphane (SFN) as a novel coronavirus inhibitor both in vitro and in mice, but the mechanism of action remains elusive. In this study, we independently discovered SFN for its inhibitory effect against SARS-CoV-2 using a target-based screening approach, identifying the viral 3-chymotrypsin-like protease (3CLpro ) as a target of SFN. Mechanistically, SFN inhibits 3CLpro in a reversible, mixed-type manner. Moreover, enzymatic kinetics studies reveal that SFN is a slow-binding inhibitor, following a two-step interaction. Initially, an encounter complex forms by specific binding of SFN to the active pocket of 3CLpro ; subsequently, the isothiocyanate group of SFN as "warhead" reacts covalently to the catalytic cysteine in a slower velocity, stabilizing the SFN-3CLpro complex. Our study has identified a new lead of the covalent 3CLpro inhibitors which has potential to be developed as a therapeutic agent to treat SARS-CoV-2 infection.

Role of mast cells in the pathogenesis of severe lung damage in COVID-19 patients.

BACKGROUND: There is still insufficient knowledge with regard to the potential involvement of mast cells (MCs) and their mediators in the pathology of coronavirus disease-2019 (COVID-19). Therefore, our study aimed to investigate the role of MCs, their activation and protease profiles in the pathogenesis of early and late lung damage in COVID-19 patients. METHODS: Formalin-fixed and paraffin embedded lung specimens from 30 patients who died from COVID-19 and 9 controls were used for histological detection of MCs and their proteases (tryptase, chymase) followed by morphometric quantification. RESULTS: Our results demonstrated increased numbers of MCs at early stage and further augmentation of MCs number during the late stage of alveolar damage in COVID-19 patients, as compared to the control group. Importantly, the percentage of degranulated (activated) MCs was higher during both stages of alveolar lesions in comparison to the controls. While there was no prominent alteration in the profile of tryptase-positive MCs, our data revealed a significant elevation in the number of chymase-positive MCs in the lungs of COVID-19 patients, compared to the controls. CONCLUSIONS: MCs are characterized by dysregulated accumulation and increased activation in the lungs of patients suffering from COVID-19. However, future profound studies are needed for precise analysis of the role of these immune cells in the context of novel coronavirus disease.

Severity of SARS-CoV-2 infection is associated with high numbers of alveolar mast cells and their degranulation.

Background: The systemic inflammatory response post-SARS-CoV-2 infection increases pro-inflammatory cytokine production, multi-organ damage, and mortality rates. Mast cells (MC) modulate thrombo-inflammatory disease progression (e.g., deep vein thrombosis) and the inflammatory response post-infection. Objective: To enhance our understanding of the contribution of MC and their proteases in SARS-CoV-2 infection and the pathogenesis of the disease, which might help to identify novel therapeutic targets. Methods: MC proteases chymase (CMA1), carboxypeptidase A3 (CPA3), and tryptase beta 2 (TPSB2), as well as cytokine levels, were measured in the serum of 60 patients with SARS-CoV-2 infection (30 moderate and 30 severe; severity of the disease assessed by chest CT) and 17 healthy controls by ELISA. MC number and degranulation were quantified by immunofluorescent staining for tryptase in lung autopsies of patients deceased from either SARS-CoV-2 infection or unrelated reasons (control). Immortalized human FcεR1+c-Kit+ LUVA MC were infected with SARS-CoV-2, or treated with its viral proteins, to assess direct MC activation by flow cytometry. Results: The levels of all three proteases were increased in the serum of patients with COVID-19, and strongly correlated with clinical severity. The density of degranulated MC in COVID-19 lung autopsies was increased compared to control lungs. The total number of released granules and the number of granules per each MC were elevated and positively correlated with von Willebrand factor levels in the lung. SARS-CoV-2 or its viral proteins spike and nucleocapsid did not induce activation or degranulation of LUVA MC in vitro. Conclusion: In this study, we demonstrate that SARS-CoV-2 is strongly associated with activation of MC, which likely occurs indirectly, driven by the inflammatory response. The results suggest that plasma MC protease levels could predict the disease course, and that severe COVID-19 patients might benefit from including MC-stabilizing drugs in the treatment scheme.

Mast cells promote viral entry of SARS-CoV-2 via formation of chymase/spike protein complex.

The pulmonary pathological findings associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) result from the release of multiple proinflammatory cytokines, which causes the subsequential damage of the lungs. The current study was undertaken to investigate the responses of mast cells to viral inoculation and their contribution to host defenses from the point of view of viral entry. Pseudovirions, in which the spike glycoprotein of SARS-CoV-2 was incorporated, triggered activation of mast cells, and a mast cell-derived chymase, MCP2, formed a complex with spike protein, which promoted protease-dependent viral entry. According to the quantification results of viral entry, 10 µM quercetin, a mast cell stabilizer, potentially potently inhibited 41.3% of viral entry, while 100 µM chymostatin, which served as a chymase inhibitor, suppressed 52.1% of viral entry, compared to non-treated cells. Study using mast cell-deficient mice showed that the absence of mast cells may influence early viral loading in the upper respiratory tract, which consequently increases the risk of viral invasion into the lower respiratory system. Furthermore, mast cell-deficient mice exhibited ongoing infection in the late phase post-viral inoculation, while clearance of virus-positive cells was observed in wild-type mice. In conclusion, mast cells act as a multifaceted immune modulator that is equipped with both protective effects and pathogenic influences on viral entry of SARS-CoV-2. The utility of mast cell stabilizers and chymase inhibitors in the treatment of SARS-CoV-2-induced acute respiratory syndrome should be optimized regarding the infection stage and the risk of cytokine storm.

Ginkgo biloba in the management of the COVID-19 severity.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is linked with inflammatory disorders and the development of oxidative stress in extreme cases. Therefore, anti-inflammatory and antioxidant drugs may alleviate these complications. Ginkgo biloba L. folium extract (EGb) is a herbal medicine containing various active constituents. This review aims to provide a critical discussion on the potential role of EGb in the management of coronavirus disease 2019 (COVID-19). The antiviral effect of EGb is mediated by different mechanisms, including blocking SARS-CoV-2 3-chymotrypsin-like protease that provides trans-variant effectiveness. Moreover, EGb impedes the development of pulmonary inflammatory disorders through the diminution of neutrophil elastase activity, the release of proinflammatory cytokines, platelet aggregation, and thrombosis. Thus, EGb can attenuate the acute lung injury and acute respiratory distress syndrome in COVID-19. In conclusion, EGb offers the potential of being used as adjuvant antiviral and symptomatic therapy. Nanosystems enabling targeted delivery, personalization, and booster of effects provide the opportunity for the use of EGb in modern phytotherapy.

Effect of tezepelumab on airway inflammatory cells, remodelling, and hyperresponsiveness in patients with moderate-to-severe uncontrolled asthma (CASCADE): a double-blind, randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Tezepelumab is a human monoclonal antibody that blocks the activity of thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine. In phase 2b and 3 studies, tezepelumab significantly reduced exacerbations versus placebo in patients with severe uncontrolled asthma, irrespective of baseline levels of type 2 inflammatory biomarkers. We investigated the mechanism of action of tezepelumab by assessing its effects on airway inflammatory cells, airway remodelling, and airway hyperresponsiveness. METHODS: CASCADE was an exploratory, double-blind, randomised, placebo-controlled, parallel-group, phase 2 study done in 27 medical centres in Canada, Denmark, Germany, the UK, and the USA. Adults aged 18-75 years with uncontrolled, moderate-to-severe asthma were randomly assigned (1:1) to receive tezepelumab 210 mg or placebo administered subcutaneously every 4 weeks for a planned 28 weeks, extended to up to 52 weeks if COVID-19-related disruption delayed participants' end-of-treatment assessments. Randomisation was balanced and stratified by blood eosinophil count. The primary endpoint was the change from baseline to the end of treatment in the number of airway submucosal inflammatory cells in bronchoscopic biopsy samples. Eosinophils, neutrophils, CD3+ T cells, CD4+ T cells, tryptase+ mast cells, and chymase+ mast cells were evaluated separately. This endpoint was also assessed in subgroups according to baseline type 2 inflammatory biomarker levels, including blood eosinophil count. Airway remodelling was assessed via the secondary endpoints of change from baseline in reticular basement membrane thickness and epithelial integrity (proportions of denuded, damaged, and intact epithelium). Exploratory outcomes included airway hyperresponsiveness to mannitol. All participants who completed at least 20 weeks of study treatment, had an end-of-treatment visit up to 8 weeks after the last dose of study drug, and had evaluable baseline and end-of-treatment bronchoscopies were included in the primary efficacy analysis. All participants who received at least one dose of study drug were included in the safety analyses. This study is registered with ClinicalTrials.gov, NCT03688074. FINDINGS: Between Nov 2, 2018, and Nov 16, 2020, 250 patients were enrolled, 116 of whom were randomly assigned (59 to tezepelumab, 57 to placebo). 48 in the tezepelumab group and 51 in the placebo group completed the study and were assessed for the primary endpoint. Treatment with tezepelumab resulted in a nominally significantly greater reduction from baseline to the end of treatment in airway submucosal eosinophils versus placebo (ratio of geometric least-squares means 0·15 [95% CI 0·05-0·41]; nominal p<0·0010), with the difference seen across all baseline biomarker subgroups. There were no significant differences between treatment groups in the other cell types evaluated (ratio of geometric least-squares means: neutrophils 1·36 [95% CI 0·94-1·97]; CD3+ T cells 1·12 [0·86-1·46]; CD4+ T cells 1·18 [0·90-1·55]; tryptase+ mast cells 0·83 [0·61-1·15]; chymase+ mast cells 1·19 [0·67-2·10]; all p>0·10). In assessment of secondary endpoints, there were no significant differences between treatment groups in reticular basement membrane thickness and epithelial integrity. In an exploratory analysis, the reduction in airway hyperresponsiveness to mannitol was significantly greater with tezepelumab versus placebo (least-squares mean change from baseline in interpolated or extrapolated provoking dose of mannitol required to induce ≥15% reduction in FEV1 from baseline: tezepelumab 197·4 mg [95% CI 107·9 to 286·9]; placebo 58·6 mg [-30·1 to 147·33]; difference 138·8 [14·2 to 263·3], nominal p=0·030). Adverse events were reported in 53 (90%) patients in the tezepelumab group and 51 (90%) patients in the placebo group, and there were no safety findings of concern. INTERPRETATION: The improvements in asthma clinical outcomes observed in previous studies with tezepelumab are probably driven, at least in part, by reductions in eosinophilic airway inflammation, as shown here by reduced airway eosinophil counts regardless of baseline blood eosinophil count. Tezepelumab also reduced airway hyperresponsiveness to mannitol, indicating that TSLP blockade might have additional benefits in asthma beyond reducing type 2 airway inflammation. FUNDING: AstraZeneca and Amgen.

Biochemical and biophysical characterization of the main protease, 3-chymotrypsin-like protease (3CLpro) from the novel coronavirus SARS-CoV 2.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is responsible for the novel coronavirus disease 2019 (COVID-19). An appealing antiviral drug target is the coronavirus 3C-like protease (3CLpro) that is responsible for the processing of the viral polyproteins and liberation of functional proteins essential for the maturation and infectivity of the virus. In this study, multiple thermal analytical techniques have been implemented to acquire the thermodynamic parameters of 3CLpro at different buffer conditions. 3CLpro exhibited relatively high thermodynamic stabilities over a wide pH range; however, the protease was found to be less stable in the presence of salts. Divalent metal cations reduced the thermodynamic stability of 3CLpro more than monovalent cations; however, altering the ionic strength of the buffer solution did not alter the stability of 3CLpro. Furthermore, the most stable thermal kinetic stability of 3CLpro was recorded at pH 7.5, with the highest enthalpy of activation calculated from the slope of Eyring plot. The biochemical and biophysical properties of 3CLpro explored here may improve the solubility and stability of 3CLpro for optimum conditions for the setup of an enzymatic assay for the screening of inhibitors to be used as lead candidates in the discovery of drugs and design of antiviral therapeutics against COVID-19.

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir.

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC50 value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

Structure-based virtual screening suggests inhibitors of 3-Chymotrypsin-Like Protease of SARS-CoV-2 from Vernonia amygdalina and Occinum gratissimum.

Antiviral culinary plants are potential bioresources for preventive nutraceuticals and/or antiviral drugs in COVID-19. Structure-based virtual screening was undertaken to screen 173 compounds previously reported from Vernonia amygdalina and Occinum gratissimum for direct interaction with the active site of the 3-Chymotrypsin-Like Protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on docking scores and comparison with reference inhibitors, a hit-list of 10 top phytocompounds was defined, which also had strong interactions with the catalytic centre of 3CLpro from three related strains of coronavirus (SARS-CoV, MERS-CoV, HKU4). Among these, six compounds (neoandrographolide, vernolide, isorhamnetin, chicoric acid, luteolin, and myricetin) exhibited the highest binding tendencies to the equilibrated conformers of SARS-CoV-2 3CLpro in an in-depth docking analysis to 5 different representative conformations from the cluster analysis of the molecular dynamics simulation (MDS) trajectories of the protein. In silico drug-likeness analyses revealed two drug-like terpenoids viz: neoandrographolide and vernolide as promising inhibitors of SARS-CoV-2 3CLpro. These structures were accommodated within the substrate-binding pocket; and interacted with the catalytic dyad (Cys145 and His41), the oxyanion loop (residues 138-145), and the S1/S2 sub-sites of the enzyme active site through the formation of an array of hydrogen bonds and hydrophobic interactions. Molecular dynamics simulation and binding free energy calculation revealed that the terpenoid-enzyme complexes exhibit strong interactions and structural stability. Therefore, these compounds may stabilize the conformation of the flexible oxyanion loop; and thereby interfere with the tetrahedral oxyanion intermediate formation during the proteolytic activity of the enzyme.

In silico analysis and identification of antiviral coumarin derivatives against 3-chymotrypsin-like main protease of the novel coronavirus SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is a pandemic viral disease caused by SARS-CoV-2 that generated serious damages for both the human population and the global economy. Therefore, it is currently considered as one of the most important global health problems of human societies and there is an urgent need for potent drugs or vaccines which can effectively combat this virus. The chymotrypsin-like protease (3CLpro) of SARS-CoV-2 plays a key role in the viral replication inside the host and thus is a promising drug target to design and develop effective antiviral drugs against SARS and other coronaviruses. This study evaluated some antiviral coumarin phytochemicals as potential inhibitors of coronaviruses 3CLpro by in silico approaches such as molecular docking, ADMET prediction, molecular dynamics simulation, and MM-PBSA binding energy calculation. Natural coumarin derivatives were docked to the 3CLpro of SARS-CoV-2 and for further investigation, docked to the 3CLpro of SARS-CoV and MERS-CoV. The docking scores of these natural compounds were compared with 3CLpro referenced inhibitors (ritonavir and lopinavir) and co-crystal inhibitor N3. Molecular docking studies suggested more than half of the coumarin phytochemicals had favorable interaction at the binding pocket of the coronaviruses 3CLpro and exhibited better binding affinities toward 3CLpro than ritonavir and lopinavir. Most antiviral phytochemicals interact strongly with one or both the catalytic dyad residues (His41 and Cys145) and the other key residues of SARS-CoV-2 main protease. Further, MD simulation and binding free energy calculations using MM-PBSA were carried out for three 3CLpro-coumarin complexes and 3CLpro-N3/lopinavir. The results confirmed that the 3CLpro-glycycoumarin, 3CLpro-oxypeucedanin hydrate, and 3CLpro-inophyllum P complexes were highly stable, experience fewer conformation fluctuations and share a similar degree of compactness. Also, the pharmacokinetics and drug-likeness studies showed good results for the selected coumarin phytochemicals.Therefore, the coumarin phytochemicals could be used as antiviral agents in the treatment of COVID-19 after further studies.

[Studies on Functional Molecules Based on Peptide Chemistry].

Studies on functional molecules starting from syntheses of cysteine-containing peptides and protein are described. Starting from evaluation of a cysteine specific side-reaction, a specific reaction for disulfide-bond formation was developed. The reaction made it possible to independently construct a disulfide bridge without effecting the existing disulfide bonds, which resulted in a unique approach for the synthesis of human insulin by site-specific disulfide bond formation. In a series of studies on sulfur-containing amino acids, another cysteine related un-natural amino acid, α-methyl cysteine, was used for the total syntheses of natural products containing a unique thiazorine/thiazole ring system. Chloroimidazolidium coupling reagent developed by us was effective for the successive couplings of the α-methyl cysteine residues. Based on these synthetic studies, design and evaluation of protease inhibitors were then studied, since a stereo-specific synthesis of the key structure is crucial to make the inhibitor an effective functional molecule in the interactions with its target protease. As the target proteases, ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) and chymotrypsin-like protease of severe acute respiratory syndrome (SARS 3CL protease) were selected: the former is a crucial enzyme for amyloid ß production and the latter is an essential enzyme for the re-construction of SARS corona virus in host cells. Structure optimization procedure of the respective inhibitors are described based on X-ray crystal structure analyses of the inhibitor-protease complex.

Existing antiviral options against SARS-CoV-2 replication in COVID-19 patients.

COVID-19 caused by SARS-CoV-2, is an international concern. This infection requires urgent efforts to develop new antiviral compounds. To date, no specific drug in controlling this disease has been identified. Developing the new treatment is usually time consuming, therefore using the repurposing broad-spectrum antiviral drugs could be an effective strategy to respond immediately. In this review, a number of broad-spectrum antivirals with potential efficacy to inhibit the virus replication via targeting the virus spike protein (S protein), RNA-dependent RNA polymerase (RdRp), 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) that are critical in the pathogenesis and life cycle of coronavirus, have been evaluated as possible treatment options against SARS-CoV-2 in COVID-19 patients.

Kinins and chymase: the forgotten components of the renin-angiotensin system and their implications in COVID-19 disease.

The unique clinical features of COVID-19 disease present a formidable challenge in the understanding of its pathogenesis. Within a very short time, our knowledge regarding basic physiological pathways that participate in SARS-CoV-2 invasion and subsequent organ damage have been dramatically expanded. In particular, we now better understand the complexity of the renin-angiotensin-aldosterone system (RAAS) and the important role of angiotensin converting enzyme (ACE)-2 in viral binding. Furthermore, the critical role of its major product, angiotensin (Ang)-(1-7), in maintaining microcirculatory balance and in the control of activated proinflammatory and procoagulant pathways, generated in this disease, have been largely clarified. The kallikrein-bradykinin (BK) system and chymase are intensively interwoven with RAAS through many pathways with complex reciprocal interactions. Yet, so far, very little attention has been paid to a possible role of these physiological pathways in the pathogenesis of COVID-19 disease, even though BK and chymase exert many physiological changes characteristic to this disorder. Herein, we outline the current knowledge regarding the reciprocal interactions of RAAS, BK, and chymase that are probably turned-on in COVID-19 disease and participate in its clinical features. Interventions affecting these systems, such as the inhibition of chymase or blocking BKB1R/BKB2R, might be explored as potential novel therapeutic strategies in this devastating disorder.

Caffeine and caffeine-containing pharmaceuticals as promising inhibitors for 3-chymotrypsin-like protease of SARS-CoV-2.

In December 2019, a new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the outbreak of a pulmonary disease called COVID-19, which killed thousands of people worldwide. Therefore, the necessity to find out the potential therapeutic pharmaceuticals is imperious. This study investigates the inhibitory effect of SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) using caffeine and caffeine-containing pharmaceuticals (3CPs) based on molecular dynamics simulations and free energy calculations by means of molecular mechanics-Poisson-Boltzmann surface area (MMPBSA) and molecular mechanics-generalized-Born surface area (MMGBSA). Of these 3CPs, seven drugs approved by the US-Food and Drug Administration have shown a good binding affinity to the catalytic residues of 3CLpro of His41 and Cys145: caffeine, theophylline, dyphylline, pentoxifylline, linagliptin, bromotheophylline and istradefylline. Their binding affinity score ranged from -4.9 to -8.6 kcal/mol. The molecular dynamic simulation in an aqueous solution of docked complexes demonstrated that the 3CPs conformations bound to the active sites of 3CLpro during 200 ns molecular dynamics simulations. The free energy of binding also confirms the stability of the 3CPs-3CLpro complexes. To our knowledge, this in silico study shows for the first time very inexpensive drugs available in large quantities that can be potential inhibitors against 3CLpro. In particular, the repurposing of linagliptin, and caffeine are recommended for COVID-19 treatment after in vitro, in vivo and clinical trial validation.Communicated by Ramaswamy H. Sarma.

Polyphenols vs. Coronaviruses: How Far Has Research Moved Forward?

The epidemic, caused by SARS-CoV-2 at the beginning of 2020, led us to a serious change in our lifestyle that for about three months has confined us to our homes, far from our laboratory routine. In this period, the belief that the work of a researcher should never stop has been the driving force in writing the present paper. It aims at reviewing the recent scientific knowledge about in vitro experimental data that focused on the antiviral role of phenols and polyphenols against different species of coronaviruses (CoVs), pointing up the viral targets potentially involved. In the current literature scenario, the papain-like and the 3-chymotrypsin-like proteases seem to be the most deeply investigated and a number of isolated natural (poly)phenols has been screened for their efficacy.

Potential anti-SARS-CoV-2 drug candidates identified through virtual screening of the ChEMBL database for compounds that target the main coronavirus protease.

A novel coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or 2019 novel coronavirus] has been identified as the pathogen of coronavirus disease 2019. The main protease (Mpro , also called 3-chymotrypsin-like protease) of SARS-CoV-2 is a potential target for treatment of COVID-19. A Mpro homodimer structure suitable for docking simulations was prepared using a crystal structure (PDB ID: 6Y2G; resolution 2.20 Å). Structural refinement was performed in the presence of peptidomimetic α-ketoamide inhibitors, which were previously disconnected from each Cys145 of the Mpro homodimer, and energy calculations were performed. Structure-based virtual screenings were performed using the ChEMBL database. Through a total of 1 485 144 screenings, 64 potential drugs (11 approved, 14 clinical, and 39 preclinical drugs) were predicted to show high binding affinity with Mpro . Additional docking simulations for predicted compounds with high binding affinity with Mpro suggested that 28 bioactive compounds may have potential as effective anti-SARS-CoV-2 drug candidates. The procedure used in this study is a possible strategy for discovering anti-SARS-CoV-2 drugs from drug libraries that may significantly shorten the clinical development period with regard to drug repositioning.

Potential inhibitors of coronavirus 3-chymotrypsin-like protease (3CLpro): an in silico screening of alkaloids and terpenoids from African medicinal plants.

The novel coronavirus disease 2019 (COVID-19) caused by SARS-COV-2 has raised myriad of global concerns. There is currently no FDA approved antiviral strategy to alleviate the disease burden. The conserved 3-chymotrypsin-like protease (3CLpro), which controls coronavirus replication is a promising drug target for combating the coronavirus infection. This study screens some African plants derived alkaloids and terpenoids as potential inhibitors of coronavirus 3CLpro using in silico approach. Bioactive alkaloids (62) and terpenoids (100) of plants native to Africa were docked to the 3CLpro of the novel SARS-CoV-2. The top twenty alkaloids and terpenoids with high binding affinities to the SARS-CoV-2 3CLpro were further docked to the 3CLpro of SARS-CoV and MERS-CoV. The docking scores were compared with 3CLpro-referenced inhibitors (Lopinavir and Ritonavir). The top docked compounds were further subjected to ADEM/Tox and Lipinski filtering analyses for drug-likeness prediction analysis. This ligand-protein interaction study revealed that more than half of the top twenty alkaloids and terpenoids interacted favourably with the coronaviruses 3CLpro, and had binding affinities that surpassed that of lopinavir and ritonavir. Also, a highly defined hit-list of seven compounds (10-Hydroxyusambarensine, Cryptoquindoline, 6-Oxoisoiguesterin, 22-Hydroxyhopan-3-one, Cryptospirolepine, Isoiguesterin and 20-Epibryonolic acid) were identified. Furthermore, four non-toxic, druggable plant derived alkaloids (10-Hydroxyusambarensine, and Cryptoquindoline) and terpenoids (6-Oxoisoiguesterin and 22-Hydroxyhopan-3-one), that bind to the receptor-binding site and catalytic dyad of SARS-CoV-2 3CLpro were identified from the predictive ADME/tox and Lipinski filter analysis. However, further experimental analyses are required for developing these possible leads into natural anti-COVID-19 therapeutic agents for combating the pandemic.Communicated by Ramaswamy H. Sarma.

Identification of chymotrypsin-like protease inhibitors of SARS-CoV-2 via integrated computational approach.

Recently, the world has witnessed outbreak of a novel Coronavirus (SARS-CoV-2), the virus which initially emerged in Wuhan, China has now made its way to a large part of the world, resulting in a public emergency of international concern. The functional importance of Chymotrypsin-like protease (3CLpro) in viral replication and maturation turns it into an attractive target for the development of effective antiviral drugs against SARS and other coronaviruses. At present, there is no standard drug regime nor any vaccine available against the infection. The rapid development and identification of efficient interventions against SARS-CoV-2 remains a major challenge. Based on the available knowledge of closely related coronavirus and their safety profiles, repurposing of existing antiviral drugs and screening of available databases is considered a near term strategic and economic way to contain the SARS-CoV-2 pandemic. Herein, we applied computational drug design methods to identify Chymotrypsin-like protease inhibitors from FDA approved antiviral drugs and our in-house database of natural and drug-like compounds of synthetic origin. As a result three FDA approved drugs (Remdesivir, Saquinavir and Darunavir) and two natural compounds (. flavone and coumarine derivatives) were identified as promising hits. Further, MD simulation and binding free energy calculations were performed to evaluate the dynamic behavior, stability of protein-ligand contact, and binding affinity of the hit compounds. Our results indicate that the identified compounds can inhibit the function of Chymotrypsin-like protease (3CLpro) of Coronavirus. Considering the severity of the spread of coronavirus, the current study is in-line with the concept of finding the new inhibitors against the vital pathway of the corona virus to expedite the process of drug discovery.Communicated by Ramaswamy H. Sarma.