Bioactive compounds of Jingfang Granules against SARS-CoV-2 virus proteases 3CLpro and PLpro.

OBJECTIVE: Jingfang Granules have been recommended for the prevention and treatment of corona virus disease 2019 (COVID-19). Through chemical analysis and bioactivity evaluation, this study aims to elucidate the potential effective components of Jingfang Granules. METHOD(S): The inhibitory acti-vities of Jingfang Granules extract against 3-chymotrypsin-like protease (3CLpro), papain like protease (PLpro), spike protein receptor-binding domain (S-RBD) and human cyclooxygenase-2 (COX-2) were evaluated using enzyme assay. The antitussive effects were evaluated using the classical ammonia-induced cough model. The chemical constituents of Jingfang Granules were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry (LC/MS). The 3CLpro and PLpro inhibitory activities of the major compounds were determined by enzyme assay, molecular docking, and site-directed mutagenesis. RESULT(S): Jingfang Granules exhibited 3CLpro and PLpro inhibitory activities, as well as COX-2 inhibitory and antitussive activities. By investigating the MS/MS behaviors of reference standards, a total of fifty-six compounds were characterized in Jingfang Granules. Sixteen of them were unambiguously identified by comparing with reference standards. The contents of the 16 major compounds were also determined, and their total contents were 2 498.8 mug/g. Naringin, nodakenin and neohesperidin were three dominating compounds in Jingfang Granules, and their contents were 688.8, 596.4 and 578.7 mug/g, respectively. In addition, neohesperidin and naringin exhibited PLpro inhibitory activities, and the inhibition rates at 8 mumol/L were 53.5% and 46.1%, respectively. Prim-O-glucosylcimifugin showed significant inhibitory activities against 3CLpro and PLpro, and the inhibitory rates at 8 mumol/L were 76.8% and 78.2%, respectively. Molecular docking indicated that hydrogen bonds could be formed between prim-O-glucosylcimifugin and amino acid residues H163, E166, Q192, T190 of 3CLpro (binding energy, -7.7 kcal/mol) and K157, D164, R166, E167, T301 of PLpro(-7.3 kcal/mol), respectively. Site-directed mutagenesis indicated amino acid residue K157 was a key active site for the interaction between prim-O-glucosylcimifugin and PLpro. CONCLUSION(S): Prim-O-glucosylcimifugin, neohesperidin, and naringin as the major compounds from Jingfang Granules could inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus proteases 3CLpro and PLpro. The results are valuable for rational clinical use of Jingfang Granules.

In silico screening of potential compounds from begonia genus as 3CL protease (3Cl pro) SARS-CoV-2 inhibitors

Background: The emergence of Coronavirus disease (COVID-19) has been declared a pandemic and made a medical emergency worldwide. Various attempts have been made, including optimizing effective treatments against the disease or developing a vaccine. Since the SARS-CoV-2 protease crystal structure has been discovered, searching for its inhibitors by in silico technique becomes possible. Objective(s): This study aims to virtually screen the potential of phytoconstituents from the Begonia genus as 3Cl pro-SARS-CoV- 2 inhibitors, based on its crucial role in viral replication, hence making these proteases "promising" for the anti-SARS-CoV-2 target. Method(s): In silico screening was carried out by molecular docking on the web-based program DockThor and validated by a retrospective method. Predictive binding affinity (Dock Score) was used for scoring the compounds. Further molecular dynamics on Desmond was performed to assess the complex stability. Result(s): Virtual screening protocol was valid with the area under curve value 0.913. Molecular docking revealed only beta-sitosterol-3-O-beta-D-glucopyranoside with a lower docking score of -9.712 kcal/mol than positive control of indinavir. The molecular dynamic study showed that the compound was stable for the first 30 ns simulations time with Root Mean Square Deviation <3 A, despite minor fluctuations observed at the end of simulation times. Root Mean Square Fluctuation of catalytic sites HIS41 and CYS145 was 0.756 A and 0.773 A, respectively. Conclusion(s): This result suggests that beta-sitosterol-3-O-beta-Dglucopyranoside might be a prospective metabolite compound that can be developed as anti-SARS-CoV-2.Copyright © 2023, Page Press Publications. All rights reserved.

Efficient synthesis of novel colchicine-magnolol hybrids and evaluation of their inhibitory activity on key proteases of 2019-nCoV replication and acute lung injury.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV), is a life-threatening infectious condition. Acute lung injury is a common complication in patients with COVID-19. 3-chymotrypsin-like protease (3CLpro) of 2019-nCoV and neutrophil elastase are critical targets of COVID-19 and acute lung injury, respectively. Colchicine and magnolol are reported to exert inhibitory effects on inflammatory response, the severe comorbidity in both COVID-19 and acute lung injury. We thus designed and synthesized a series of novel colchicine-magnolol hybrids based on a two-step synthetic sequence. It was found that these novel hybrids provided unexpected inhibition on 3CLpro and neutrophil elastase, a bioactivity that colchicine and magnolol did not possess. These findings not only provide perquisites for further in vitro and in vivo investigation to confirm the therapeutic potentiality of novel colchicine-magnolol hybrids, but also suggest that the concurrent inhibition of 3CLpro and neutrophil elastase may enable novel colchicine-magnolol hybrids as effective multi-target drug compounds.

Key Allosteric and Active Site Residues of SARS-CoV-2 3CLpro Are Promising Drug Targets.

The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3, and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.

Discovery of novel bicyclic[3.3.0]proline peptidyl α-ketoamides as potent 3CL-protease inhibitors for SARS-CoV-2.

The outbreak of SARS-CoV-2 has caused global crisis on health and economics. The multiple drug-drug interaction risk associated with ritonavir warrants specialized assessment before using Paxlovid. Here we report a multiple-round SAR study to provide a novel bicyclic[3.3.0]proline peptidyl α-ketoamide compound 4a, which is endowed with excellent antiviral activities and pharmacokinetic properties. Also, in vivo HCoV-OC43 neonatal mice model demonstrated compound 4a has good in vivo efficacy. Based on these properties, compound 4a worth further SAR optimization with the goal to develop compounds with better pharmacokinetic properties and finally to realize single agent efficacy in human.

Computational Screening of Inhibitory Compounds for SARS-Cov-2 3CL Protease with a Database Consisting of Approved and Investigational Chemicals.

Computational screening is one of the fundamental techniques in drug discovery. Each compound in a chemical database is bound to the target protein in virtual, and candidate compounds are selected from the binding scores. In this work, we carried out combinational computation of docking simulation to generate binding poses and molecular mechanics calculation to estimate binding scores. The coronavirus infectious disease has spread worldwide, and effective chemotherapy is strongly required. The viral 3-chymotrypsin-like (3CL) protease is a good target of low molecular-weight inhibitors. Hence, computational screening was performed to search for inhibitory compounds acting on the 3CL protease. As a preliminary assessment of the performance of this approach, we used 51 compounds for which inhibitory activity had already been confirmed. Docking simulations and molecular mechanics calculations were performed to evaluate binding scores. The preliminary evaluation suggested that our approach successfully selected the inhibitory compounds identified by the experiments. The same approach was applied to 8820 compounds in a database consisting of approved and investigational chemicals. Hence, docking simulations, molecular mechanics calculations, and re-evaluation of binding scores including solvation effects were performed, and the top 200 poses were selected as candidates for experimental assays. Consequently, 25 compounds were chosen for in vitro measurement of the enzymatic inhibitory activity. From the enzymatic assay, 5 compounds were identified to have inhibitory activities against the 3CL protease. The present work demonstrated the feasibility of a combination of docking simulation and molecular mechanics calculation for practical use in computational virtual screening.

Harnessing and bioprospecting botanical-based herbal medicines against potential drug targets for COVID-19: a review coupled molecular docking studies.

Since the end of February 2020, the world has come to a standstill due to the virus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Since then, the global scientific community has explored various remedies and treatments against this virus, including natural products that have always been a choice because of their many benefits. Various known phytochemicals are well documented for their antiviral properties. Research is being carried out to discover new natural plant products or existing ones as a treatment measure for this disease. The three important targets in this regard are-papain like protease (PLpro), spike protein, and 3 chymotrypsin like proteases (3CLpro). Various docking studies are also being elucidated to identify the phytochemicals that modulate crucial proteins of the virus. The paper is simultaneously a comprehensive review that covers recent advances in the domain of the effect of various botanically derived natural products as an alternative treatment approach against Coronavirus Disease 2019 (COVID-19). Furthermore, the docking analyses revealed that rutin (inhibitor of the major protease of SARS-CoV-2), gallocatechin (e.g., interacting with 03 hydrogen bonds with a spike-like protein), lycorine (showing the best binding affinity with amino acids GLN498, THR500 and GLY446 of the spike-like protein), and quercetrin (inhabiting at its residues ASP216, PHE219, and ILE259) are promising inhibitors of SARS­CoV­2.Communicated by Ramaswamy H. Sarma.

Structure-guided design of direct-acting antivirals that exploit the gem-dimethyl effect and potently inhibit 3CL proteases of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and middle east respiratory syndrome coronavirus (MERS-CoV).

The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 µM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1eand1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies.

Revealing Potential Binding Affinity of FDA Approved Therapeutics Targeting Main Protease (3CLpro) in Impairing Novel Coronavirus (SARS-CoV-2) Replication that Causes COVID-19

Background: Spread of COVID-19 attains a crucial transition in reveling its pandemic across the boundaries. In combating the infection caused by SARS-CoV-2, there is a spectrum of ideal strategies that have been adopted globally, of which repurposing of approved drugs considerably having high clinical relevance. 3-chymotrypsin-like protease (3CL pro) is considered to be the potential target for the researchers as it is highly essential for cleavage of polyprotein to get 16 nonstructural proteins (called nsp1-nsp16). These proteins are highly essential for viral replication and hence become a primary target for enzyme inhibitors. 3CL pro, having a structural projectile helical chain with biologically active site involved in processing viral polyproteins that are evolved from RNA genome translation. Objective(s): The major objective of the present investigation is to evaluate the enzyme inhibition potential of FDA approved therapeutic leads in targeting 3CLpro that medicates the viral replication. Method(s): Docking calculations were carried out for an array of FDA approved molecules which leads to a notable few molecules such as Emtricitabine, Oseltamivir, Ganciclovir, Chloroquine, Baricitinib, Favipiravir, Lopinavir, Ritonavir, Remdesivir, Ribavirin, Tenofovir, Umifenovir, Carbapenam, Ertap-enem and Imipenam which have both specificity and selectivity in terms of binding efficiency against 3CL proenzyme. Result(s): A combinatorial evaluation employing in-silico screening shows a major lead for remdesivir which possesses a substantial affinity to 3CL pro binding on core amino acid residues, such as Leu 27, His 41, Gly 143, Cys 145, His 164, Met 165, Glu 166, Pro 168 and His 172 which share the biological significance in mediating enzymatic action. Results of docking simulation by Autodock over a host of FDA approved molecules show high degree of selectivity and specificity in the increasing order of binding capacity;Remdesivir> Ertapenem> Imipenam> Tenofovir> Umifenovir> Chloroquine> Lopinavir> Ritonavir> Emtricitabine> Ganciclovir> Baricitinib> Ribavirin>Oseltamivir>Favipiravir> Carbapenam. Conclusion(s): Till date, there is no known cure attained for treating COVID-19 infection. In conclusion, lead molecules from already approved sources provoke promising potential which grabs the attention of the clinicians in availing potential therapeutic candidate as a drug of choice in the clinical management of COVID-19 time-dependently.Copyright © 2020 Bentham Science Publishers.

Synthesis of deuterated S-217622 (Ensitrelvir) with antiviral activity against coronaviruses including SARS-CoV-2.

S-217622 (Ensitrelvir) is a reversible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3-chymotrypsin-like protease (3CLpro) inhibitor which obtained emergency regulatory approval in Japan for the treatment of SARS-CoV-2 infection on Nov 22, 2022. Herein, analogs of S-271622 with deuterium-for-hydrogen replacement were synthesized for comparison of the antiviral activities and pharmacokinetic (PK) profiles. Compared to the parent compound, C11-d2-S-217622 compound YY-278 retained in vitro activity against 3CLpro and SARS-CoV-2. X-ray crystal structural studies showed similar interactions of SARS-CoV-2 3CLpro with YY-278 and S-271622. The PK profiling revealed the relatively favorable bioavailability and plasma exposure of YY-278. In addition, YY-278, as well as S-217622, displayed broadly anti-coronaviral activities against 6 other coronaviruses that infect humans and animals. These results laid the foundation for further research on the therapeutic potential of YY-278 against COVID-19 and other coronaviral diseases.

Effects of diarylbutane lignans from Schisandra chinensis fruit on SARS-CoV-2 3CLpro and PLpro and their in vitro anti-inflammatory properties.

Background: Schisandra chinensis fruit is a well-known traditional Chinese medicine (TCM), whose extract has a potent inhibitory effect on the severe acute respiratory syndrome-coronavirus-2 (SARS­CoV­2) 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). Purpose: This work aims to find the active components from the fruit of S. chinensis against SARS­CoV­2 3CLpro and PLpro. Materials and methods: The chemical constituents of the fruit of S. chinensis were retrieved based on the electronic databases, such as Web of Science, PubMed, Medline Plus, and CNKI. Molecular docking was used to screen the active components against SARS­CoV­2 3CLpro and PLpro. Potential hit compounds were further evaluated by enzymatic activity assay. Furthermore, the anti-inflammatory activities of the active compounds were further explored using the phorbol-12-myristate-13-acetate (PMA)-induced THP1 cells model. Results: In this work, we retrieved 75 components of S. chinensis fruit, including 62 dibenzocyclooctadiene-type lignans, 3 diarylbutane-type lignans, 2 tetrahydrofuran-type lignans, and 8 nortriterpenoids. Combining molecular docking study and in vitro experiments, we found that pregomisin (63), meso­dihydroguaiaretic acid (64), and nordihydroguaiaretic acid (65) could potently inhibit 3CLpro with IC50 values of 3.07 ± 0.38, 4.12 ± 0.38, and 6.06 ± 0.62 µM, respectively, and inhibit PLpro with IC50 values of 5.23 ± 0.33, 4.24 ± 0.46, and 16.28 ± 0.54 µM, respectively. Interestingly, compounds 63, 64, and 65 also have potent activities of regulating the inflammatory response in vitro. Conclusion: Our results suggest that compounds 63, 64, and 65 may be promising SARS-CoV-2 3CLpro and PLpro inhibitors and anti-inflammatory.

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.

Bacillus species; a potential source of anti-SARS-CoV-2 main protease inhibitors.

The COVID-19 being a preconized global pandemic by the World Health Organization needs persuasive immediate research for possible medications. The present study was carried out with a specific aim to computationally evaluate and identify compounds derived from Bacillus species as the plausible inhibitors against 3-chymotrypsin-like main protease (3CLpro) or main protease (MPro), which is a key enzyme in the life-cycle of coronavirus. The compounds were isolated from the crude extracts of Bacillus species. Among the isolated compounds, novel inhibitory leads were identified using in silico techniques. Molecular docking revealed that stigmasterol (-8.3 kcal/mol), chondrillasterol (-7.9 kcal/mol) and hexadecnoic acid (-6.9 kcal/mol)) among others bind in the substrate-binding pocket and also interacted with the catalytic dyad of the 3-CLpro. Further evaluation using 50 ns molecular dynamic simulation and MMPB-GBSA indicated that among the top three docking hits, hexadecanoic acid was found to be the most promising anti-COVID-19 lead against the main protease. Hexadecanoic acid might serve as a potent anti-SARS-CoV-2 compound to combat COVID-19, however, in vitro and in vivo validation and optimization is needed.Communicated by Ramaswamy H. Sarma.

Screening and identification of potential MERS-CoV papain-like protease (PLpro) inhibitors; Steady-state kinetic and Molecular dynamic studies.

MERS-CoV belongs to the coronavirus group. Recent years have seen a rash of coronavirus epidemics. In June 2012, MERS-CoV was discovered in the Kingdom of Saudi Arabia, with 2,591 MERSA cases confirmed by lab tests by the end of August 2022 and 894 deaths at a case-fatality ratio (CFR) of 34.5% documented worldwide. Saudi Arabia reported the majority of these cases, with 2,184 cases and 813 deaths (CFR: 37.2%), necessitating a thorough understanding of the molecular machinery of MERS-CoV. To develop antiviral medicines, illustrative investigation of the protein in coronavirus subunits are required to increase our understanding of the subject. In this study, recombinant expression and purification of MERS-CoV (PLpro), a primary goal for the development of 22 new inhibitors, were completed using a high throughput screening methodology that employed fragment-based libraries in conjunction with structure-based virtual screening. Compounds 2, 7, and 20, showed significant biological activity. Moreover, a docking analysis revealed that the three compounds had favorable binding mood and binding free energy. Molecular dynamic simulation demonstrated the stability of compound 2 (2-((Benzimidazol-2-yl) thio)-1-arylethan-1-ones) the strongest inhibitory activity against the PLpro enzyme. In addition, disubstitutions at the meta and para locations are the only substitutions that may boost the inhibitory action against PLpro. Compound 2 was chosen as a MERS-CoV PLpro inhibitor after passing absorption, distribution, metabolism, and excretion studies; however, further investigations are required.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Functional dynamics of SARS-CoV-2 3C-like protease as a member of clan PA.

SARS-CoV-2 3C-like protease (3CLpro), a potential therapeutic target for COVID-19, consists of a chymotrypsin fold and a C-terminal α-helical domain (domain III), the latter of which mediates dimerization required for catalytic activation. To gain further understanding of the functional dynamics of SARS-CoV-2 3CLpro, this review extends the scope to the comparative study of many crystal structures of proteases having the chymotrypsin fold (clan PA of the MEROPS database). First, the close correspondence between the zymogen-enzyme transformation in chymotrypsin and the allosteric dimerization activation in SARS-CoV-2 3CLpro is illustrated. Then, it is shown that the 3C-like proteases of family Coronaviridae (the protease family C30), which are closely related to SARS-CoV-2 3CLpro, have the same homodimeric structure and common activation mechanism via domain III mediated dimerization. The survey extended to order Nidovirales reveals that all 3C-like proteases belonging to Nidovirales have domain III, but with various chain lengths, and 3CLpro of family Mesoniviridae (family C107) has the same homodimeric structure as that of C30, even though they have no sequence similarity. As a reference, monomeric 3C proteases belonging to the more distant family Picornaviridae (family C3) lacking domain III are compared with C30, and it is shown that the 3C proteases are rigid enough to maintain their structures in the active state. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-022-01020-x.

Glycyrrhizin as a promising kryptonite against SARS-CoV-2: Clinical, experimental, and theoretical evidences.

COVID-19 is the most devastating disease in recent times affecting most people globally. The higher rate of transmissibility and mutations of SARS-CoV-2 along with the lack of potential therapeutics has made it a global crisis. Potential molecules from natural sources could be a fruitful remedy to combat COVID-19. This systematic review highlights the detailed therapeutic implication of naturally occurring glycyrrhizin and its related derivatives against COVID-19. Glycyrrhizin has already been established for blocking different biomolecular targets related to the SARS-CoV-2 replication cycle. In this article, several experimental and theoretical evidences of glycyrrhizin and related derivatives have been discussed in detail to evaluate their potential as a promising therapeutic strategy against COVID-19. Moreover, the implication of glycyrrhizin in traditional Chinese medicines for alleviating the symptoms of COVID-19 has been reviewed. The potential role of glycyrrhizin and related compounds in affecting various stages of the SARS-CoV-2 life cycle has also been discussed in detail. Derivatization of glycyrrhizin for designing potential lead compounds along with combination therapy with other anti-SARS-CoV-2 agents followed by extensive evaluation may assist in the formulation of novel anti-coronaviral therapy for better treatment to combat COVID-19.

[Bioactive compounds of Jingfang Granules against SARS-CoV-2 virus proteases 3CL(pro) and PL(pro)]

OBJECTIVE: Jingfang Granules have been recommended for the prevention and treatment of corona virus disease 2019 (COVID-19). Through chemical analysis and bioactivity evaluation, this study aims to elucidate the potential effective components of Jingfang Granules. METHODS: The inhibitory acti-vities of Jingfang Granules extract against 3-chymotrypsin-like protease (3CL(pro)), papain like protease (PL(pro)), spike protein receptor-binding domain (S-RBD) and human cyclooxygenase-2 (COX-2) were evaluated using enzyme assay. The antitussive effects were evaluated using the classical ammonia-induced cough model. The chemical constituents of Jingfang Granules were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry (LC/MS). The 3CL(pro) and PL(pro) inhibitory activities of the major compounds were determined by enzyme assay, molecular docking, and site-directed mutagenesis. RESULTS: Jingfang Granules exhibited 3CL(pro) and PL(pro) inhibitory activities, as well as COX-2 inhibitory and antitussive activities. By investigating the MS/MS behaviors of reference standards, a total of fifty-six compounds were characterized in Jingfang Granules. Sixteen of them were unambiguously identified by comparing with reference standards. The contents of the 16 major compounds were also determined, and their total contents were 2 498.8 μg/g. Naringin, nodakenin and neohesperidin were three dominating compounds in Jingfang Granules, and their contents were 688.8, 596.4 and 578.7 μg/g, respectively. In addition, neohesperidin and naringin exhibited PL(pro) inhibitory activities, and the inhibition rates at 8 μmol/L were 53.5% and 46.1%, respectively. Prim-O-glucosylcimifugin showed significant inhibitory activities against 3CL(pro) and PL(pro), and the inhibitory rates at 8 μmol/L were 76.8% and 78.2%, respectively. Molecular docking indicated that hydrogen bonds could be formed between prim-O-glucosylcimifugin and amino acid residues H163, E166, Q192, T190 of 3CL(pro) (binding energy, -7.7 kcal/mol) and K157, D164, R166, E167, T301 of PL(pro)(-7.3 kcal/mol), respectively. Site-directed mutagenesis indicated amino acid residue K157 was a key active site for the interaction between prim-O-glucosylcimifugin and PL(pro). CONCLUSION: Prim-O-glucosylcimifugin, neohesperidin, and naringin as the major compounds from Jingfang Granules could inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus proteases 3CL(pro) and PL(pro). The results are valuable for rational clinical use of Jingfang Granules.

Schaftoside inhibits 3CLpro and PLpro of SARS-CoV-2 virus and regulates immune response and inflammation of host cells for the treatment of COVID-19.

It is an urgent demand worldwide to control the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) are key targets to discover SARS-CoV-2 inhibitors. After screening 12 Chinese herbal medicines and 125 compounds from licorice, we found that a popular natural product schaftoside inhibited 3CLpro and PLpro with IC50 values of 1.73 ± 0.22 and 3.91 ± 0.19 µmol/L, respectively, and inhibited SARS-CoV-2 virus in Vero E6 cells with EC50 of 11.83 ± 3.23 µmol/L. Hydrogen-deuterium exchange mass spectrometry analysis, quantum mechanics/molecular mechanics calculations, together with site-directed mutagenesis indicated the antiviral activities of schaftoside were related with non-covalent interactions with H41, G143 and R188 of 3CLpro, and K157, E167 and A246 of PLpro. Moreover, proteomics analysis and cytokine assay revealed that schaftoside also regulated immune response and inflammation of the host cells. The anti-inflammatory activities of schaftoside were confirmed on lipopolysaccharide-induced acute lung injury mice. Schaftoside showed good safety and pharmacokinetic property, and could be a promising drug candidate for the prevention and treatment of COVID-19.

The SARS-CoV-2 main protease (Mpro): Structure, function, and emerging therapies for COVID-19.

The main proteases (Mpro), also termed 3-chymotrypsin-like proteases (3CLpro), are a class of highly conserved cysteine hydrolases in ß-coronaviruses. Increasing evidence has demonstrated that 3CLpros play an indispensable role in viral replication and have been recognized as key targets for preventing and treating coronavirus-caused infectious diseases, including COVID-19. This review is focused on the structural features and biological function of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease Mpro (also known as 3CLpro), as well as recent advances in discovering and developing SARS-CoV-2 3CLpro inhibitors. To better understand the characteristics of SARS-CoV-2 3CLpro inhibitors, the inhibition activities, inhibitory mechanisms, and key structural features of various 3CLpro inhibitors (including marketed drugs, peptidomimetic, and non-peptidomimetic synthetic compounds, as well as natural compounds and their derivatives) are summarized comprehensively. Meanwhile, the challenges in this field are highlighted, while future directions for designing and developing efficacious 3CLpro inhibitors as novel anti-coronavirus therapies are also proposed. Collectively, all information and knowledge presented here are very helpful for understanding the structural features and inhibitory mechanisms of SARS-CoV-2 3CLpro inhibitors, which offers new insights or inspiration to medicinal chemists for designing and developing more efficacious 3CLpro inhibitors as novel anti-coronavirus agents.