Syringa reticulata potently inhibits the activity of SARS-CoV-2 3CL protease.

The ongoing coronavirus infectious disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still urgently requires effective treatments. The 3C-like (3CL) protease of SARS-CoV-2 is a highly conserved cysteine protease that plays an important role in the viral life cycle and host inflammation, providing an ideal target for developing broad-spectrum antiviral drugs. Herein, we describe the discovery of a large number of herbs mainly produced in Heilongjiang Province, China, that exhibited different inhibitory activities against SARS-CoV-2 3CL protease. We confirmed that Syringa reticulata, which is used for clinical treatment of chronic bronchitis and asthma, is a specific and potent inhibitor of 3CL protease. A 70 % ethanol extract of S. reticulata dose-dependently inhibited the cleavage activity of 3CL protease in a fluorescence resonance energy transfer assay with an IC50 value of 0.0018 mg/mL, but had minimal effect in pseudovirus-based cell entry and luciferase-based RNA-dependent RNA polymerase assays. These results suggest that S. reticulata will be a potential leading candidate for COVID-19 treatment.

Discovery of Novel Chinese Medicine Compounds Targeting 3CL Protease by Virtual Screening and Molecular Dynamics Simulation.

The transmission and infectivity of COVID-19 have caused a pandemic that has lasted for several years. This is due to the constantly changing variants and subvariants that have evolved rapidly from SARS-CoV-2. To discover drugs with therapeutic potential for COVID-19, we focused on the 3CL protease (3CLpro) of SARS-CoV-2, which has been proven to be an important target for COVID-19 infection. Computational prediction techniques are quick and accurate enough to facilitate the discovery of drugs against the 3CLpro of SARS-CoV-2. In this paper, we used both ligand-based virtual screening and structure-based virtual screening to screen the traditional Chinese medicine small molecules that have the potential to target the 3CLpro of SARS-CoV-2. MD simulations were used to confirm these results for future in vitro testing. MCCS was then used to calculate the normalized free energy of each ligand and the residue energy contribution. As a result, we found ZINC15676170, ZINC09033700, and ZINC12530139 to be the most promising antiviral therapies against the 3CLpro of SARS-CoV-2.

The Ethanol Extract of Evodiae Fructus and Its Ingredient, Rutaecarpine, Inhibit Infection of SARS-CoV-2 and Inflammatory Responses.

COVID-19, derived from SARS-CoV-2, has resulted in millions of deaths and caused unprecedented socioeconomic damage since its outbreak in 2019. Although the vaccines developed against SARS-CoV-2 provide some protection, they have unexpected side effects in some people. Furthermore, new viral mutations reduce the effectiveness of the current vaccines. Thus, there is still an urgent need to develop potent non-vaccine therapeutics against this infectious disease. We recently established a series of detecting platforms to screen a large library of Chinese medicinal herbs and phytochemicals. Here, we reveal that the ethanolic extract of Evodiae Fructus and one of its components, rutaecarpine, showed promising potency in inhibiting the activity of 3C-like (3CL) protease, blocking the entry of the pseudo-typed SARS-CoV-2 (including wild-type and omicron) into cultured cells. In addition, inflammatory responses induced by pseudo-typed SARS-CoV-2 were markedly reduced by Evodiae Fructus extract and rutaecarpine. Together our data indicate that the herbal extract of Evodiae Fructus and rutaecarpine are potent anti-SARS-CoV-2 agents, which might be considered as a treatment against COVID-19 in clinical applications.

Polygoni multiflori radix extracts inhibit SARS-CoV-2 pseudovirus entry in HEK293T cells and zebrafish larvae.

BACKGROUND: Globally, COVID-19 has caused millions of deaths and led to unprecedented socioeconomic damage. There is therefore, in addition to vaccination, an urgent need to develop complementary effective treatments and/or protective and preventative therapies against this deadly disease. METHODS: Here, a multi-component testing platform was established to screen a library of herbal extracts from traditional Chinese medicine (TCM), to identify potent herbal extracts/phytochemicals as possible therapeutics for COVID-19. We utilized assays for spike protein (S-protein) binding to angiotensin-converting enzyme II (ACE2); the enzymatic inhibition of 3CL protease; and entry of the SARS-CoV-2 pseudovirus into cultured HEK293T cells and zebrafish larvae. RESULTS: Over a thousand herbal extracts were screened and approximately 20 positive hits were identified. Among these, we found that the water and ethanol extracts of Polygoni Multiflori Radix (PMR) significantly inhibited S-protein binding to ACE2, 3CL protease activity, and viral entry into the cell and fish models. The water extract was more effective than the ethanol extract, with IC50 values of 25 to 500 µg/ml. In addition, the polysaccharide-depleted fraction of the former, and epigallocatechin gallate (EGCG) which was found in both extracts, displayed significant antiviral activity. CONCLUSIONS: Our results indicate that the water and ethanol extracts of PMR have an inhibitory effect on SARS-CoV-2 pseudovirus host-cell entry. Furthermore, EGCG might be an active component of PMR, which blocks SARS-CoV-2 entry to cells. Taken together, our findings suggest that PMR might be considered as a potential treatment for COVID-19.

Efficient discovery of potential inhibitors for SARS-CoV-2 3C-like protease from herbal extracts using a native MS-based affinity-selection method.

The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the virus life cycle and is supposed to be a potential target for the treatment of coronaviral infection. Traditional Chinese medicines (TCMs) have played an impressive role in the treatment of COVID-19 in China. The effectiveness of TCM formulations prompts scientists to take continuous effort on searching for bioactive small molecules from the ancient resources. Herein, we developed a native mass spectrometry-based affinity-selection method for rapid screening of active small molecules from crude herbal extracts applied for COVID-19 therapy. Six common herbs named Lonicera japonica, Scutellaria baicalensis, Forsythia suspensa, Glycyrrhiza uralensis, Cirsium japonicum, and Andrographis paniculata were investigated. After preliminary separation of the crude extracts, the fractions were incubated with 3CLpro. A native MS-based affinity screening assay was then conducted to search for the protein-ligand complexes. A UHPLC-Q/TOF-MS with UNIFI data acquisition and data processing software was applied to identify the hit compounds. Standard compounds were used to verify the outcomes. Among the 16 hits, three flavonoids, baicalein, scutellarein and ganhuangenin, were identified as potential noncovalent inhibitors against 3CLpro with IC50 values of 0.94, 3.02, and 0.84 µM, respectively. Their binding affinities were further characterized by native MS, with Kd values being 1.43, 3.85, and 1.09 µM, respectively. Overall, we established an efficient native MS-based strategy for discovering 3CLpro ligands from crude mixtures, which supplies a potential strategy of small molecule lead discovery from TCMs.

The inhibitory effects of PGG and EGCG against the SARS-CoV-2 3C-like protease.

The coronavirus disease (COVID-19) pandemic, resulting from human-to-human transmission of a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has led to a global health crisis. Given that the 3 chymotrypsin-like protease (3CLpro) of SARS-CoV-2 plays an indispensable role in viral polyprotein processing, its successful inhibition halts viral replication and thus constrains virus spread. Therefore, developing an effective SARS-CoV-2 3CLpro inhibitor to treat COVID-19 is imperative. A fluorescence resonance energy transfer (FRET)-based method was used to assess the proteolytic activity of SARS-CoV-2 3CLpro using intramolecularly quenched fluorogenic peptide substrates corresponding to the cleavage sequence of SARS-CoV-2 3CLpro. Molecular modeling with GEMDOCK was used to simulate the molecular interactions between drugs and the binding pocket of SARS-CoV-2 3CLpro. This study revealed that the Vmax of SARS-CoV-2 3CLpro was about 2-fold higher than that of SARS-CoV 3CLpro. Interestingly, the proteolytic activity of SARS-CoV-2 3CLpro is slightly more efficient than that of SARS-CoV 3CLpro. Meanwhile, natural compounds PGG and EGCG showed remarkable inhibitory activity against SARS-CoV-2 3CLpro than against SARS-CoV 3CLpro. In molecular docking, PGG and EGCG strongly interacted with the substrate binding pocket of SARS-CoV-2 3CLpro, forming hydrogen bonds with multiple residues, including the catalytic residues C145 and H41. The activities of PGG and EGCG against SARS-CoV-2 3CLpro demonstrate their inhibition of viral protease activity and highlight their therapeutic potentials for treating SARS-CoV-2 infection.

Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. RESULTS: In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. CONCLUSIONS: This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

GC-MS, LC-MS/MS, Docking and Molecular Dynamics Approaches to Identify Potential SARS-CoV-2 3-Chymotrypsin-Like Protease Inhibitors from Zingiber officinale Roscoe.

This study aims to identify and isolate the secondary metabolites of Zingiber officinale using GC-MS, preparative TLC, and LC-MS/MS methods, to evaluate the inhibitory potency on SARS-CoV-2 3 chymotrypsin-like protease enzyme, as well as to study the molecular interaction and stability by using docking and molecular dynamics simulations. GC-MS analysis suggested for the isolation of terpenoids compounds as major compounds on methanol extract of pseudostems and rhizomes. Isolation and LC-MS/MS analysis identified 5-hydro-7, 8, 2'-trimethoxyflavanone (9), (E)-hexadecyl-ferulate (1), isocyperol (2), N-isobutyl-(2E,4E)-octadecadienamide (3), and nootkatone (4) from the rhizome extract, as well as from the leaves extract with the absence of 9. Three known steroid compounds, i.e., spinasterone (7), spinasterol (8), and 24-methylcholesta-7-en-3ß-on (6), were further identified from the pseudostem extract. Molecular docking showed that steroids compounds 7, 8, and 6 have lower predictive binding energies (MMGBSA) than other metabolites with binding energy of -87.91, -78.11, and -68.80 kcal/mole, respectively. Further characterization on the single isolated compound by NMR showed that 6 was identified and possessed 75% inhibitory activity on SARS-CoV-2 3CL protease enzyme that was slightly different with the positive control GC376 (77%). MD simulations showed the complex stability with compound 6 during 100 ns simulation time.

EGCG, a green tea polyphenol, inhibits human coronavirus replication in vitro.

COVID-19 pandemic results in record high deaths in many countries. Although a vaccine for SARS-CoV-2 is now available, effective antiviral drugs to treat coronavirus diseases are not available yet. Recently, EGCG, a green tea polyphenol, was reported to inhibit SARS-CoV-2 3CL-protease, however the effect of EGCG on coronavirus replication is unknown. In this report, human coronavirus HCoV-OC43 (beta coronavirus) and HCoV-229E (alpha coronavirus) were used to examine the effect of EGCG on coronavirus. EGCG treatment decreases 3CL-protease activity of HCoV-OC43 and HCoV-229E. Moreover, EGCG treatment decreased HCoV-OC43-induced cytotoxicity. Finally, we found that EGCG treatment decreased the levels of coronavirus RNA and protein in infected cell media. These results indicate that EGCG inhibits coronavirus replication.

Repurposing existing drugs: identification of SARS-CoV-2 3C-like protease inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19). Since its emergence, the COVID-19 pandemic has not only distressed medical services but also caused economic upheavals, marking urgent the need for effective therapeutics. The experience of combating SARS-CoV and MERS-CoV has shown that inhibiting the 3-chymotrypsin-like protease (3CLpro) blocks the replication of the virus. Given the well-studied properties of FDA-approved drugs, identification of SARS-CoV-2 3CLpro inhibitors in an FDA-approved drug library would be of great therapeutic value. Here, we screened a library consisting of 774 FDA-approved drugs for potent SARS-CoV-2 3CLpro inhibitors, using an intramolecularly quenched fluorescence (IQF) peptide substrate. Ethacrynic acid, naproxen, allopurinol, butenafine hydrochloride, raloxifene hydrochloride, tranylcypromine hydrochloride, and saquinavir mesylate have been found to block the proteolytic activity of SARS-CoV-2 3CLpro. The inhibitory activity of these repurposing drugs against SARS-CoV-2 3CLpro highlights their therapeutic potential for treating COVID-19 and other Betacoronavirus infections.

Optimization and evaluation of propolis liposomes as a promising therapeutic approach for COVID-19.

The present work aimed to develop an optimized liposomal formulation for enhancing the anti-viral activity of propolis against COVID-19. Docking studies were performed for certain components of Egyptian Propolis using Avigan, Hydroxychloroquine and Remdesivir as standard antivirals against both COVID-19 3CL-protease and S1 spike protein. Response surface methodology and modified injection method were implemented to maximize the entrapment efficiency and release of the liposomal formulation. The optimized formulation parameters were as follow: LMC of 60 mM, CH% of 20% and DL of 5 mg/ml. At those values the E.E% and released % were 70.112% and 81.801%, respectively with nanosized particles (117 ± 11 nm). Docking studies revealed that Rutin and Caffeic acid phenethyl ester showed the highest affinity to both targets. Results showed a significant inhibitory effect of the optimized liposomal formula of Propolis against COVID-3CL protease (IC50 = 1.183 ± 0.06) compared with the Egyptian propolis extract (IC50 = 2.452 ± 0.11), P < 0.001. Interestingly, the inhibition of viral replication of COVID-19 determined by RT_PCR has been significantly enhanced via encapsulation of propolis extract within the liposomal formulation (P < 0.0001) and was comparable to the viral inhibitory effect of the potent antiviral (remdesivir). These findings identified the potential of propolis liposomes as a promising treatment approach against COVID-19.

Uncovering Flexible Active Site Conformations of SARS-CoV-2 3CL Proteases through Protease Pharmacophore Clusters and COVID-19 Drug Repurposing.

The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease-ligand complex structures from ∼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 "PPC consensus anchors" (a distinct set observed in each PPC) were observed, of which three "PPC core anchors" EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived "PPC scoring S" to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 µM and an antiviral activity with EC50 of 49.89 µM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 µM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 µM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.

A traditional Chinese medicine formula NRICM101 to target COVID-19 through multiple pathways: A bedside-to-bench study.

COVID-19 is a global pandemic, with over 50 million confirmed cases and 1.2 million deaths as of November 11, 2020. No therapies or vaccines so far are recommended to treat or prevent the new coronavirus. A novel traditional Chinese medicine formula, Taiwan Chingguan Yihau (NRICM101), has been administered to patients with COVID-19 in Taiwan since April 2020. Its clinical outcomes and pharmacology have been evaluated. Among 33 patients with confirmed COVID-19 admitted in two medical centers, those (n = 12) who were older, sicker, with more co-existing conditions and showing no improvement after 21 days of hospitalization were given NRICM101. They achieved 3 consecutive negative results within a median of 9 days and reported no adverse events. Pharmacological assays demonstrated the effects of the formula in inhibiting the spike protein/ACE2 interaction, 3CL protease activity, viral plaque formation, and production of cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α. This bedside-to-bench study suggests that NRICM101 may disrupt disease progression through its antiviral and anti-inflammatory properties, offering promise as a multi-target agent for the prevention and treatment of COVID-19.

Tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3CL protease.

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. There is an urgent need to find new therapeutic methods to prevent and control PEDV. Not only is there a shortage of commercial anti-PEDV drugs, but available commercial vaccines fail to protect against highly virulent PEDV variants. We screened an FDA-approved library of 911 natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of PEDV replication in Vero and IPEC-J2 cells in vitro. Molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of PEDV 3CL protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). The inhibiting effect of tomatidine on 3CL protease was determined using cleavage visualization and FRET assay. Tomatidine-mediated blocking of 3CL protease activity in PEDV-infected cells was examined by western blot detection of the viral polyprotein in PEDV-infected cells. It indicates that tomatidine inhibits PEDV replication mainly by targeting 3CL protease. In addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), encephalo myocarditis virus (EMCV) and seneca virus A (SVA) in vitro. These results may be helpful in developing a new prophylactic and therapeutic strategy against PEDV and other swine disease infections.

Screening and evaluation of approved drugs as inhibitors of main protease of SARS-CoV-2.

The COVID-19 pandemic caused by SARS-CoV-2 has emerged as a global catastrophe. The virus requires main protease for processing the viral polyproteins PP1A and PP1AB translated from the viral RNA. In search of a quick, safe and successful therapeutic agent; we screened various clinically approved drugs for the in-vitro inhibitory effect on 3CLPro which may be able to halt virus replication. The methods used includes protease activity assay, fluorescence quenching, surface plasmon resonance (SPR), Thermofluor® Assay, Size exclusion chromatography and in-silico docking studies. We found that Teicoplanin as most effective drug with IC50 ~ 1.5 µM. Additionally, through fluorescence quenching Stern-Volmer quenching constant (KSV) for Teicoplanin was estimated as 2.5 × 105 L·mol-1, which suggests a relatively high affinity between Teicoplanin and 3CLPro protease. The SPR shows good interaction between Teicoplanin and 3CLPro with KD ~ 1.6 µM. Our results provide critical insights into the mechanism of action of Teicoplanin as a potential therapeutic against COVID-19. We found that Teicoplanin is about 10-20 fold more potent in inhibiting protease activity than other drugs in use, such as lopinavir, hydroxychloroquine, chloroquine, azithromycin, atazanavir etc. Therefore, Teicoplanin emerged as the best inhibitor among all drug molecules we screened against 3CLPro of SARS-CoV-2.

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

Flavonoids with inhibitory activity against SARS-CoV-2 3CLpro.

Coronavirus disease 2019 (COVID-19) has been a pandemic disease of which the termination is not yet predictable. Currently, researches to develop vaccines and treatments is going on globally to cope with this disastrous disease. Main protease (3CLpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the good targets to find antiviral agents before vaccines are available. Some flavonoids are known to inhibit 3CLpro from SARS-CoV which causes SARS. Since their sequence identity is 96%, a similar approach was performed with a flavonoid library. Baicalin, herbacetin, and pectolinarin have been discovered to block the proteolytic activity of SARS-CoV-2 3CLpro. An in silico docking study showed that the binding modes of herbacetin and pectolinarin are similar to those obtained from the catalytic domain of SARS-CoV 3CLpro. However, their binding affinities are different due to the usage of whole SARS-CoV-2 3CLpro in this study. Baicalin showed an effective inhibitory activity against SARS-CoV-2 3CLpro and its docking mode is different from those of herbacetin and pectolinarin. This study suggests important scaffolds to design 3CLpro inhibitors to develop antiviral agents or health-foods and dietary supplements to cope with SARS-CoV-2.

In silico and in vitro analysis of small molecules and natural compounds targeting the 3CL protease of feline infectious peritonitis virus.

The computational search of chemical libraries has been used as a powerful tool for the rapid discovery of candidate compounds. To find small molecules with anti-feline infectious peritonitis virus (FIPV) properties, we utilized a virtual screening technique to identify the active site on the viral protease for the binding of the available natural compounds. The protease 3CL (3CLpro) plays an important role in the replication cycle of FIPV and other viruses within the family Coronaviridae. The 15 best-ranked candidate consensus compounds, based on three docking tools, were evaluated for further assays. The protease inhibitor assay on recombinant FIPV 3CLpro was performed to screen the inhibitory effect of the candidate compounds with IC50 ranging from 6.36 ± 2.15 to 78.40 ± 2.60 µM. As determined by the cell-based assay, the compounds NSC345647, NSC87511, and NSC343256 showed better EC50 values than the broad-spectrum antiviral drug ribavirin and the protease inhibitor lopinavir, under all the test conditions including pre-viral entry, post-viral entry, and prophylactic activity. The NSC87511 particularly yielded the best selective index (>4; range of SI = 13.80-22.90). These results indicated that the natural small-molecular compounds specifically targeted the 3CLpro of FIPV and inhibited its replication. Structural modification of these compounds may generate a higher anti-viral potency for the further development of a novel therapy against FIP.

Potential SARS-CoV-2 3CL protease inhibitors selection from TCMSP platform by molecular docking approach / 中草药

Objective: To identify potential SARS-CoV-2 3CL protease inhibitors from the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) by molecular docking approach. Methods: To alternate extensive compounds experimental screening processes, a Computer-Aided Drug Design (CADD) based molecular docking technology was performed to explore existing drug repurposing possibilities. Molecular docking model with Schrodinger suit 2018 was used to evaluate the binding abilities between TCMSP 13 143 compounds and SARS-CoV-2 3CL protease receptor-binding domain (PBD ID 6LU7), which involving in mediating viral replication and transcription functions. According to the constructed docking system, potential compounds were screened according to docking score, oral bioavailability (OB), and drug-likeness (DL). At last, a compounds-herb-target organ-function network was constructed. Results: Compared with 6LU7 original ligand docking score (-7.734), a total of 498 compounds were identified with lower docking score against 6LU7 targets. These compounds were further reduced to 60 high-priority compounds, based on OB (more than 30) and DL (more than 0.18). Meanwhile, these 60 compounds were found to interact with the amino acid residues (GLU166, GLY143, ASP187, CYS145, GLN189, LEU141, etc.) which were critically involved in the 6LU7 domain mainly by hydrogen-bonded interaction. The network exploring results revealed that these potential compounds were mainly attributed to Glycyrrhizae Radix et Rhizoma, Mori Cortex, Rhododendron dauricum, Polygoni Cuspidati Rhizoma et Radix, and Plantaginis Herba, etc., which associates with acute lung syndromes induced by SARS-CoV-2, with the effect of clearing heat and removing toxin, relieving cough and dispelling phlegm and lung-draining and relieving asthma. Conclusion: Molecular docking method provides a useful tool for the screening of SARS-CoV-2 3CL protease inhibitors from TCMSP platform.

Traditional Chinese medicine herbal extracts of Cibotium barometz, Gentiana scabra, Dioscorea batatas, Cassia tora, and Taxillus chinensis inhibit SARS-CoV replication.

Development of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV) agents is pivotal to prevent the reemergence of the life-threatening disease, SARS. In this study, more than 200 extracts from Chinese medicinal herbs were evaluated for anti-SARS-CoV activities using a cell-based assay that measured SARS-CoV-induced cytopathogenic effect (CPE) in vitro on Vero E6 cells. Six herbal extracts, one each from Gentianae Radix ( lóng dǎn; the dried rhizome of Gentiana scabra), Dioscoreae Rhizoma ( shan yào; the tuber of Dioscorea batatas), Cassiae Semen ( jué míng zǐ; the dried seed of Cassia tora) and Loranthi Ramus ( sang jì sheng; the dried stem, with leaf of Taxillus chinensis) (designated as GSH, DBM, CTH and TCH, respectively), and two from Rhizoma Cibotii ( gǒu jǐ; the dried rhizome of Cibotium barometz) (designated as CBE and CBM), were found to be potent inhibitors of SARS-CoV at concentrations between 25 and 200 µg/ml. The concentrations of the six extracts needed to inhibit 50% of Vero E6 cell proliferation (CC50) and 50% of viral replication (EC50) were determined. The resulting selective index values (SI = CC50/EC50) of the most effective extracts CBE, GSH, DBM, CTH and TCH were > 59.4, > 57.5, > 62.1, > 59.4, and > 92.9, respectively. Among these extracts, CBM and DBM also showed significant inhibition of SARS-CoV 3CL protease activity with IC50 values of 39 µg/ml and 44 µg/ml, respectively. Our findings suggest that these six herbal extracts may have potential as candidates for future development of anti-SARS therapeutics.AbbreviationsSARS,severe acute respiratory syndromeCoV,coronavirusCPE,cytopathogenic effectTCM,traditional Chinese medicine.