Coronavirus Main Protease Research Articles

Bioinformatics and 3D Structural Analysis of the Coronavirus Main Protease Active Site Diversity

Coronaviruses (Coronaviridae) such as SARS‐CoV‐2 (severe acute respiratory syndrome coronavirus) and MERS‐CoV (Middle East respiratory syndrome coronavirus) have been the source of recent outbreaks and global health concerns. While vaccines have been essential for controlling the SARS‐CoV‐2 (COVID‐19) pandemic, it is uncertain whether they will be effective against future coronavirus strains. Therefore, identification or design of a broad‐spectrum drug that targets highly conserved regions of the main protease of multiple coronavirus strains is essential in the long term. As part of a virtual summer research experience with the RCSB PDB, bioinformatics tools were employed to predict and construct 3D models of the coronavirus main protease (MPro) using SARS‐CoV‐2 as the template, with a focus on mutational trends and active sites. This study focused on the active sites of MPro, a cysteine protease essential for viral assembly and replication. Sequence alignments and structure modeling of MPro structures has identified conserved regions across multiple coronavirus strains. Inhibition of MPro halts coronavirus replication, making it an ideal drug target, and studies of MPro may foster and accelerate the discovery of high affinity broad‐spectrum drugs.

A VSV-based assay quantifies coronavirus Mpro/3CLpro/Nsp5 main protease activity and chemical inhibition

Protease inhibitors are among the most powerful antiviral drugs. However, for SARS-CoV-2 only a small number of protease inhibitors have been identified thus far and there is still a great need for assays that efficiently report protease activity and inhibition in living cells. Here, we engineer a safe VSV-based system to report both gain- and loss-of-function of coronavirus main protease (Mpro/3CLpro/Nsp5) activity in living cells. We use SARS-CoV-2 3CLpro in this system to confirm susceptibility to known inhibitors (boceprevir, GC376, PF-00835231, and PF-07321332/nirmatrelvir) and reevaluate other reported inhibitors (baicalein, ebselen, carmofur, ethacridine, ivermectin, masitinib, darunavir, and atazanavir). Moreover, we show that the system can be adapted to report both the function and the chemical inhibition of proteases from different coronavirus species as well as from distantly related viruses. Together with the fact that live cell assays also reflect compound permeability and toxicity, we anticipate that this system will be useful for both identification and optimization of additional coronavirus protease inhibitors.

SARS-CoV-2 3CLpro whole human proteome cleavage prediction and enrichment/depletion analysis

A novel coronavirus (SARS-CoV-2) has devastated the globe as a pandemic that has killed millions of people. Widespread vaccination is still uncertain, so many scientific efforts have been directed toward discovering antiviral treatments. Many drugs are being investigated to inhibit the coronavirus main protease, 3CLpro, from cleaving its viral polyprotein, but few publications have addressed this protease’s interactions with the host proteome or their probable contribution to virulence. Too few host protein cleavages have been experimentally verified to fully understand 3CLpro’s global effects on relevant cellular pathways and tissues. Here, I set out to determine this protease’s targets and corresponding potential drug targets. Using a neural network trained on cleavages from 392 coronavirus proteomes with a Matthews correlation coefficient of 0.985, I predict that a large proportion of the human proteome is vulnerable to 3CLpro, with 4898 out of approximately 20,000 human proteins containing at least one putative cleavage site. These cleavages are nonrandomly distributed and are enriched in the epithelium along the respiratory tract, brain, testis, plasma, and immune tissues and depleted in olfactory and gustatory receptors despite the prevalence of anosmia and ageusia in COVID-19 patients. Affected cellular pathways include cytoskeleton/motor/cell adhesion proteins, nuclear condensation and other epigenetics, host transcription and RNAi, ribosomal stoichiometry and nascent-chain detection and degradation, ubiquitination, pattern recognition receptors, coagulation, lipoproteins, redox, and apoptosis. This whole proteome cleavage prediction demonstrates the importance of 3CLpro in expected and nontrivial pathways affecting virulence, lead me to propose more than a dozen potential therapeutic targets against coronaviruses, and should therefore be applied to all viral proteases and subsequently experimentally verified.

An extended conformation of SARS-CoV-2 main protease reveals allosteric targets

SignificanceThe coronavirus main protease (Mpro) is required for viral replication. Here, we obtained the extended conformation of the native monomer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro by trapping it with nanobodies and found that the catalytic domain and the helix domain dissociate, revealing allosteric targets. Another monomeric state is termed compact conformation and is similar to one protomer of the dimeric form. We designed a Nanoluc Binary Techonology (NanoBiT)-based high-throughput allosteric inhibitor assay based on structural conformational change. Our results provide insight into the maturation, dimerization, and catalysis of the coronavirus Mpro and pave a way to develop an anticoronaviral drug through targeting the maturation process to inhibit the autocleavage of Mpro.

In Silico and In Vitro Identification of Pan-Coronaviral Main Protease Inhibitors from a Large Natural Product Library.

The main protease (Mpro or 3CLpro) in coronaviruses represents a promising specific drug target as it is essential for the cleavage of the virus polypeptide and has a unique cleavage site that does not exist in human host proteases. In this study, we explored potential natural pan-coronavirus drugs using in vitro and in silico approaches and three coronavirus main proteases as treatment targets. The PyRx program was used to screen 39,442 natural-product-like compounds from the ZINC database and 121 preselected phytochemicals from medicinal plants with known antiviral activity. After assessment with Lipinski’s rule of five, molecular docking was performed for the top 33 compounds of both libraries. Enzymatic assays were applied for the top candidates from both in silico approaches to test their ability to inhibit SARS-CoV-2 Mpro. The four compounds (hypericin, rosmarinic acid, isorhamnetin, and luteolin) that most efficiently inhibited SARS-CoV-2 Mpro in vitro were further tested for their efficacy in inhibiting Mpro of SARS-CoV-1 and MERS-CoV. Microscale thermophoresis was performed to determine dissociation constant (Kd) values to validate the binding of these active compounds to recombinant Mpro proteins of SARS-CoV-2, SARS-CoV-1, and MERS-CoV. The cytotoxicity of hypericin, rosmarinic acid, isorhamnetin, and luteolin was assessed in human diploid MRC-5 lung fibroblasts using the resazurin cell viability assay to determine their therapeutic indices. Sequence alignment of Mpro of SARS-CoV-2 demonstrated 96.08%, 50.83%, 49.17%, 48.51%, 44.04%, and 41.06% similarity to Mpro of other human-pathogenic coronaviruses (SARS-CoV-1, MERS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKU1, and HCoV-229E, respectively). Molecular docking showed that 12 out of 121 compounds were bound to SARS-CoV-2 Mpro at the same binding site as the control inhibitor, GC376. Enzyme inhibition assays revealed that hypericin, rosmarinic acid, isorhamnetin, and luteolin inhibited Mpro of SARS-CoV-2, while hypericin and isorhamnetin inhibited Mpro of SARS-CoV-1; hypericin showed inhibitory effects toward Mpro of MERS-CoV. Microscale thermophoresis confirmed the binding of these compounds to Mpro with high affinity. Resazurin assays showed that rosmarinic acid and luteolin were not cytotoxic toward MRC-5 cells, whereas hypericin and isorhamnetin were slightly cytotoxic. We demonstrated that hypericin represents a potential novel pan-anti-coronaviral agent by binding to and inhibiting Mpro of several human-pathogenic coronaviruses. Moreover, isorhamnetin showed inhibitory effects toward SARS-CoV-2 and SARS-CoV-1 Mpro, indicating that this compound may have some pan-coronaviral potential. Luteolin had inhibitory effects against SARS-CoV-2 Mpro.

Crystallization of Feline Coronavirus Mpro With GC376 Reveals Mechanism of Inhibition.

Coronaviruses infect a variety of hosts in the animal kingdom, and while each virus is taxonomically different, they all infect their host via the same mechanism. The coronavirus main protease (Mpro, also called 3CLpro), is an attractive target for drug development due to its essential role in mediating viral replication and transcription. An Mpro inhibitor, GC376, has been shown to treat feline infectious peritonitis (FIP), a fatal infection in cats caused by internal mutations in the feline enteric coronavirus (FECV). Recently, our lab demonstrated that the feline drug, GC373, and prodrug, GC376, are potent inhibitors of SARS-CoV-2 Mpro and solved the structures in complex with the drugs; however, no crystal structures of the FIP virus (FIPV) Mpro with the feline drugs have been published so far. Here, we present crystal structures of FIPV Mpro-GC373/GC376 complexes, revealing the inhibitors covalently bound to Cys144 in the active site, similar to SARS-CoV-2 Mpro. Additionally, GC376 has a higher affinity for FIPV Mpro with lower nanomolar Ki values compared to SARS-CoV and SARS-CoV-2 Mpro. We also show that improved derivatives of GC376 have higher potency for FIPV Mpro. Since GC373 and GC376 represent strong starting points for structure-guided drug design, determining the crystal structures of FIPV Mpro with these inhibitors are important steps in drug optimization and structure-based broad-spectrum antiviral drug discovery.

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (Mpro, also called 3CLpro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native Mpro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.

Antiviral activities of 4H-chromen-4-one scaffold-containing flavonoids against SARS–CoV–2 using computational and in vitro approaches

The widespread outbreak of the novel coronavirus called severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the main health challenge worldwide. This pandemic has attracted the attention of the research communities in various fields, prompting efforts to discover rapid drug molecules for the treatment of the life-threatening COVID-19 disease. This study is aimed at investigating 4H-chromen-4-one scaffold-containing flavonoids that combat the SARS-CoV-2 virus using computational and in vitro approaches. Virtual screening studies of the molecule’s library for 4H-chromen-4-one scaffold were performed with the recently reported coronavirus main protease (Mpro, also called 3CLpro) because it plays an essential role in the maturation and processing of the viral polyprotein. Based on the virtual screening, the top hit molecules such as isoginkgetin and afzelin molecules were selected for further estimating in vitro antiviral efficacies against SARS–CoV–2 in Vero cells. Additionally, these molecules were also docked with RNA-dependent RNA Polymerase (RdRp) to reveal the ligands-protein molecular interaction. In the in vitro study, isoginkgetin showed remarkable inhibition potency against the SARS-CoV-2 virus, with an IC50 value of 22.81 μM, compared to remdesivir, chloroquine, and lopinavir with IC50 values of 7.18, 11.63, and 11.49 μM, respectively. Furthermore, the complex stability of isoginkgetin with an active binding pocket of the SARS-CoV-2 Mpro and RdRp supports its inhibitory potency against the SARS-CoV-2. Thus, isoginkgetin is a potent leading drug candidate and needs to be used in in vivo trials for the treatment of SARS-CoV-2 infected patients.

A Simple and Efficient Approach to the Synthesis of 4-Aryl-2-dialkylphosphonomethyl-4-oxobutanenitrile

In this work, we describe a simple and easy synthetic approach to variously 4-aryl-2-alkylphosphonomethyl-4-oxobutanenitrile based on the reaction of aromatic aldehydes with phosphorylated Michael’s acceptors in good yields. A general mechanism for the reactions was also proposed. Characterization of the products was carried out by several spectroscopic tools, including Infrared and Nuclear Magnetic Resonance Spectroscopies (1H, 13C, and 31P-NMR). Molecular docking studies were conducted on the synthesized materials against (1UK4) the crystal structure of the SARS Coronavirus Main Proteinase (3CLpro) to study the antiviral activity of these compounds and against (1E3K) the Human Progesterone Receptor to study the anticancer activity of these compounds. We found that compound (5i) was the best one in both antiviral and anticancer activity (according to the binding energy values).

Curcumins and its derivatives as potential inhibitors of New Coronavirus (COVID-19) main protease: an in silico strategy

Coronavirus (COVID-19) disease outbreak caused a worldwide pandemic with a powerful lethal potential and still, there is no specific treatment to it. Natural bioactive molecules like curcumins were investigated in this work aiming to block the active site of COVID-19 Main protease (Mpro), since they present several biological activities, being more suitable in terms of fewer side effects, once this disease overloads the immune system of patients. Hereby, curcumin and several derivatives were screened for their ability to react with Mpro receptors (PDB: 6LU7). N3, Azithromycin (AZT), and Baracitinib (BRT) were evaluated as positive controls and in combined therapeutics possibilities with curcumins. N3, AZT, and BRT bound to different protein receptors, and also it was observed that N3 bound in the same site as hexahydrocurcumin and curcumin glucuronide bound at the AZT’s site and bisdemethoxycurcumin, curcumin, curcumin sulfate, cyclocurcumin, demethoxycurcumin, dihydrocurcumin and hexahydrocurcuminol bound at BRT’s site. All molecules analyzed have high force interaction fields. Once the viral activity is mainly intracellular, these compounds also were evaluated for their hydropathic abilities. All molecules were classified and considered capable of membrane cell invading. These results suggest that the therapeutic approach of the curcumin derivatives associated with AZT and the antiviral inhibitor N3 is promissory for future evaluation of their synergism in in vitro and in vivo tests to define their additional viability in the treatment of COVID-19.

In silico Molecular Docking, ADMET Property, Molecular Dynamic Simulation Evaluation of N,N′-bis(2-Hydroxybenzylidene)-1,2-diaminobenzene and its Metal Complexes against SARS-CoV-2

In search of potent inhibitors of SARS-CoV-2, well familiar N,N′-bis(2-hydroxybenzylidene)-1,2- diaminobenzene [HBDB]/salophen ligand and its corresponding metal complexes such as [VO(HBDB)], [Mo(O)2(HBDB)], [W(O)2(HBDB)], [Fe(H2O)2(HBDB)], [Pb(H2O)2(HBDB)], [Sn(Cl)2(HBDB)] have been accompanied with standard drug hydroxychloroquine employing CADD tools such as the molecular docking, ADMET toxicity assessment and molecular dynamics of the coronavirus main protease (PDB id: 6LZG) enzyme. The synthesized salophen metal complexes exhibited the greater binding energy than corresponding salophen Schiff base ligand and standard hydroxychloroquine drug. Consequently, salophen metal complexes could serve as potential lead molecules against COVID-19 for further optimization and drug development to combat the virulent SARS-CoV-2 syndrome. The molecular dynamic simulation studies for [Mo(O2)(HBDB)] complex shows an excellent binding free energy about -170.833 Kcal/mol. Subsequently, it is confirmed that salophen metal complexes could serve as potential drugs against COVID-19 for further optimization and drug development to combat the virulent SARS-CoV-2 syndrome.

Dietary polyphenols mitigate SARS-CoV-2 main protease (Mpro)–Molecular dynamics, molecular mechanics, and density functional theory investigations

The recent evolution of the SARS-like Coronavirus has ravaged the world. The deadly virus has claimed over millions of lives across the world and hence highlights the need to develop effective therapeutic drugs to contain the disease posed by this parasite. In this study, the inhibitory potential of fifty (50) dietary polyphenols against Coronavirus (SARS-CoV-2) main protease (Mpro) was conducted using the Autodock Vina Molecular docking tool. In the virtual screening process, the binding affinity of Remdesivir (-7.7 kcal/mol) currently used to treat COVID-19 patients was set as the cut-off value to screen out less probable inhibitors. Ellagic acid, Kievitone, and Punicalin were the only promising ligands with binding affinities (-8.9 kcal/mol, -8.0 kcal/mol and -7.9 kcal/mol respectively) lower than the set cut-off value. Furthermore, we validated Ellagic acid and Kievitone efficacy by subjecting them to molecular dynamics simulation and further stability was assessed at the molecular mechanics and quantum levels. The overall analysis indicates both compounds demonstrate higher stability and inhibitory potential to bind to the crucial His41 and Cys145 catalytic dyad of Mpro than the standard drug. However, further analysis of punicalin after evaluating its docking score was not conducted as the ligand pharmacokinetics properties suggests it could pose serious adverse effect to the health of participants in clinical trials. Hence, we employed a more safe approach by filtering out the compound during this study. Conclusively, while Ellagic acid and kievitone polyphenolic compounds have been demonstrated to be promising under this in silico research, further studies are needed to substantiate their clinical relevance.

A Patent Review on SARS Coronavirus Main Protease (3CLpro ) Inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic is an unprecedented global health emergency causing more than 4.2 million fatalities as of 30 July 2021. Only three antiviral therapies have been approved or granted emergency use authorization by the FDA. The SARS‐CoV‐2 3CL protease (3CLpro) is deemed an attractive drug target as it plays an essential role in viral polyprotein processing and pathogenesis, although no inhibitors have been approved. This patent review discusses SARS coronavirus 3CLpro inhibitors that have been filed up to 30 July 2021, giving an overview on the types of inhibitors that have generated commercial interest, especially amongst drug companies. Insights into the common structural motifs required for active site binding is also discussed.

High predictive QSAR models for predicting the SARS coronavirus main protease inhibition activity of ketone-based covalent inhibitors

In this research, a dataset including 29 ketone-based covalent inhibitors with SARS-CoV-1 3CLpro inhibition activity was used to develop high predictive QSAR models. Twenty-two molecules were put in train set and seven molecules in test set. By using stepwise MLR method for molecules in train set, four molecular descriptors including Mor26p, Hy, GATS7p and Mor04v were selected to build QSAR models. MLR and ANN methods were used to create QSAR models for predicting the activity of molecules in both train and test sets. Both QSAR models were validated by calculating several statistical parameters. R2 values for the test set of MLR and ANN models were 0.93 and 0.95, respectively, and RMSE values for their test sets were 0.24 and 0.17, respectively. Other calculated statistical parameters (especially Q_{F3}^{2} parameter) show that created ANN model has more predictive power with respect to developed MLR model (with four descriptor). Calculated leverages for all molecules show that predicted pIC50 (by both QSAR models) for all molecules is acceptable, and drawn residuals plots show that there is no systematic error in building both QSAR modes. Also, based on developed MLR model, used molecular descriptors were interpreted.

Food phytochemicals, epigallocatechin gallate and myricetin, covalently bind to the active site of the coronavirus main protease in vitro

SARS-CoV-2 main protease is a possible target for protection against viral infection. This study examined the inhibitory effect of food phytochemicals on the main protease of SARS-CoV-2 by determining a cleaved product after chromatographic separation. First, 37 phytochemicals, including glycosides and metabolites, were screened at 20 µM; epigallocatechin gallate, myricetin, theaflavin, herbacetin, piceatannol, myricitrin, and isothiocyanates inhibited the enzyme in varying degrees. The IC50 values were estimated from 0.4 to 33.3 µM against the 0.5-µM enzyme. The dose-dependent adduction of epigallocatechin gallate and myricetin was confirmed by quinone staining of protein blotted onto a membrane. The enzyme activity was decreased by increasing the concentration of the two phytochemicals, accompanied by increasing the respective adducted molecule estimated by intact mass spectrometry. Reduced glutathione canceled the formation of conjugate and the inhibitory effect of epigallocatechin gallate or myricetin on the enzyme, suggesting that the formation of the quinone moiety in the phytochemicals is critical for the inhibition. The covalent binding of epigallocatechin gallate or myricetin to the cysteine residue at the active site was confirmed by analyzing peptides from the chymotrypsin-digested main protease.

Repurposing drug molecule against SARS-Cov-2 (COVID-19) through molecular docking and dynamics: a quick approach to pick FDA-approved drugs.

A novel coronavirus known as severe acute respiratory syndrome is rapidly spreading worldwide. The international health authorities are putting all their efforts on quick diagnosis and placing the patients in quarantine. Although different vaccines have come for quick use as prophylactics, drug repurposing seems to be of paramount importance because of inefficient therapeutic options and clinical trial limitations. Here, we used structure-based drug designing approach to find and check the efficacy of the possible drug that can inhibit coronavirus main protease which is involved in polypeptide processing to functional protein. We performed virtual screening, molecular docking and molecular dynamics simulations of the FDA-approved drugs against the main protease of SARS-CoV-2. Using well-defined computational methods, we identified amprenavir, cefoperazone, riboflavin, diosmin, nadide and troxerutin approved for human therapeutic uses, as COVID-19 main protease inhibitors. These drugs bind to the SARS-CoV-2 main protease conserved residues of substrate-binding pocket and formed a remarkable number of non-covalent interactions. We have found diosmin as an inhibitor which binds covalently to the COVID-19 main protease. This study provides enough evidences for therapeutic use of these drugs in controlling COVID-19 after experimental validation and clinical demonstration.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00894-021-04923-w.

Inhibitory Effects and Surface Plasmon Resonance-Based Binding Affinities of Dietary Hydrolyzable Tannins and Their Gut Microbial Metabolites on SARS-CoV-2 Main Protease.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (Mpro) inhibitors are considered as potential treatments for coronavirus disease 2019, and dietary polyphenols show promise in SARS-CoV-2 Mpro inhibition based on in silico studies. In the present study, we utilize a combination of biochemical-, surface plasmon resonance-, and docking-based assays to evaluate the inhibition and binding affinities of a series of tannins and their gut microbial metabolites on SARS-CoV-2 Mpro. The tested compounds (2–50 μM) were hydrolyzable tannins, including ellagitannins (punicalagin and ellagic acid) and gallotannins (tannic acid, pentagalloyl glucose, ginnalin A, and gallic acid), and their gut microbial metabolites, urolithins and pyrogallol, respectively. They inhibited SARS-CoV-2 Mpro (by 6.6–100.0% at 50 μM) and bound directly to the Mpro protein (with dissociation constants from 1.1 × 10–6 to 5.3 × 10–5 M). This study sheds light on the inhibitory effects of tannins and their metabolites on SARS-CoV-2 Mpro.

Molecular Docking and ADMET Prediction of Natural Compounds towards SARS Spike Glycoprotein-Human Angiotensin-Converting Enzyme 2 and SARS-CoV-2 Main Protease.

More than a decade ago, a novel coronavirus that infects humans, bats, and certain other mammals termed severe acute respiratory syndrome coronavirus (SARS-CoV) caused an epidemic with ~ 10% case fatality, creating global panic and economic damage. Recently, another strain of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused an infectious disease (COVID-19) in humans which was detected for the first time in Wuhan, China. Presently, there is no specific therapy available for the treatment of COVID-19. However, social distancing, patient isolation, and supportive medical care make up the current management for this current infectious disease pandemic. The present in silico study evaluated the binding affinities of some natural products (resveratrol, xylopic acid, ellagic acid, kaempferol, and quercetin) to human angiotensin-converting enzyme 2 and coronavirus (SARS-CoV-2) main protease compared to chloroquine, an inhibitor known to prevent cellular entry and replication of the coronavirus. The respective binding energies of the selected natural compounds and chloroquine towards the proteins were computed using PyRx virtual screening tool. The pharmacodynamic and pharmacokinetic attributes of the selected compounds were predicted using admetSAR. Molecular docking analysis showed that the natural compounds had better scores towards the selected protein compared to chloroquine with polar amino acid residues present at the binding sites. The predicted ADMET properties revealed the lower acute oral toxicity of the natural products compared to chloroquine. The study provides evidence suggesting that the relatively less toxic compounds from the natural sources could be repositioned as anti-viral agents to prevent the entry and replication of SARS-CoV-2.

Effect of dihydromyricetin on SARS-CoV-2 viral replication and pulmonary inflammation and fibrosis

BackgroundCOVID-19 (Coronavirus Disease-2019) has spread widely around the world and impacted human health for millions. The lack of effective targeted drugs and vaccines forces scientific world to search for new effective antiviral therapeutic drugs. It has reported that flavonoids have potential inhibitory activity on SARS-CoV-2 Mpro and anti-inflammatory properties. Dihydromyricetin, as a flavonol, also has antiviral and anti-inflammatory potential. However, the inhibition of dihydromyricetin on SARS-CoV-2 Mpro and the protective effect of dihydromyricetin on pulmonary inflammation and fibrosis have not been proved and explained. PurposeThe coronavirus main protease (Mpro) is essential for SARS-CoV-2 replication and to be recognized as an attractive drug target, we expect to find the inhibitor of Mpro. Novel coronavirus infection can cause severe inflammation and even sequelae of pulmonary fibrosis in critically ill patients. We hope to find a drug that can not only inhibit virus replication but also alleviate inflammation and pulmonary fibrosis in patients. MethodsFRET-based enzymatic assay was used to evaluate the inhibit activity of dihydromyricetin on SARS-CoV-2 Mpro. Molecular docking was used to identify the binding pose of dihydromyricetin with SARS-CoV-2 Mpro. The protective effects of dihydromyricetin against BLM-induced pulmonary inflammation and fibrosis were investigated in C57BL6 mice. BALF and lung tissue were collected for inflammation cells count, ELISA, masson and HE staining, western blotting and immunohistochemistry to analyze the effects of dihydromyricetin on pulmonary inflammation and fibrosis. MTT, western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and wound healing were used to analyze the effects of dihydromyricetin on lung fibrosis mechanisms in Mlg cells. ResultsIn this study, we found that dihydromyricetin is a potent inhibitor targeting the SARS-CoV-2 Mpro with a half-maximum inhibitory concentration (IC50) of 1.716 ± 0.419 μM, using molecular docking and the FRET-based enzymatic assay. The binding pose of dihydromyricetin with SARS-CoV-2 Mpro was identified using molecular docking method. In the binding pocket of SARS-CoV-2 Mpro, the dihydrochromone ring of dihydromyricetin interact with the imidazole side chain of His163 through π-π stacking. The 1-oxygen of dihydromyricetin forms a hydrogen bond with the backbone nitrogen of Glu166. The 3-, 7-, 3’- and 4’-hydroxyl of dihydromyricetin interact with Gln189, Leu141, Arg188 and Thr190 through hydrogen bonds. Moreover, our results showed that dihydromyricetin can significantly alleviate BLM-induced pulmonary inflammation by inhibiting the infiltration of inflammation cells and the secretion of inflammation factors in the early process and also ameliorate pulmonary fibrosis by improving pulmonary function and down-regulate the expression of α-SMA and fibronectin in vivo. Our results also showed that dihydromyricetin inhibits the migration and activation of myofibroblasts and extracellular matrix production via transforming growth factor (TGF)-β1/Smad signaling pathways. ConclusionDihydromyricetin is an effective inhibitor for SARS-CoV-2 Mpro and it prevents BLM-induced pulmonary inflammation and fibrosis in mice. Dihydromyricetin will be a potential medicine for the treatment of COVID-19 and its sequelae.

Structure-Based Optimization of ML300-Derived, Noncovalent Inhibitors Targeting the Severe Acute Respiratory Syndrome Coronavirus 3CL Protease (SARS-CoV-2 3CLpro).

Starting from the MLPCN probe compound ML300, a structure-based optimization campaign was initiated against the recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CLpro). X-ray structures of SARS-CoV-1 and SARS-CoV-2 3CLpro enzymes in complex with multiple ML300-based inhibitors, including the original probe ML300, were obtained and proved instrumental in guiding chemistry toward probe compound 41 (CCF0058981). The disclosed inhibitors utilize a noncovalent mode of action and complex in a noncanonical binding mode not observed by peptidic 3CLpro inhibitors. In vitro DMPK profiling highlights key areas where further optimization in the series is required to obtain useful in vivo probes. Antiviral activity was established using a SARS-CoV-2-infected Vero E6 cell viability assay and a plaque formation assay. Compound 41 demonstrates nanomolar activity in these respective assays, comparable in potency to remdesivir. These findings have implications for antiviral development to combat current and future SARS-like zoonotic coronavirus outbreaks.