Phytoconstituents of Artemisia Annua as potential inhibitors of SARS CoV2 main protease: an in silico study.

BACKGROUND: In November 2019, the world faced a pandemic called SARS-CoV-2, which became a major threat to humans and continues to be. To overcome this, many plants were explored to find a cure. METHODS: Therefore, this research was planned to screen out the active constituents from Artemisia annua that can work against the viral main protease Mpro as this non-structural protein is responsible for the cleavage of replicating enzymes of the virus. Twenty-five biocompounds belonging to different classes namely alpha-pinene, beta-pinene, carvone, myrtenol, quinic acid, caffeic acid, quercetin, rutin, apigenin, chrysoplenetin, arteannunin b, artemisinin, scopoletin, scoparone, artemisinic acid, deoxyartemisnin, artemetin, casticin, sitogluside, beta-sitosterol, dihydroartemisinin, scopolin, artemether, artemotil, artesunate were selected. Virtual screening of these ligands was carried out against drug target Mpro by CB dock. RESULTS: Quercetin, rutin, casticin, chrysoplenetin, apigenin, artemetin, artesunate, sopolin and sito-gluside were found as hit compounds. Further, ADMET screening was conducted which represented Chrysoplenetin as a lead compound. Azithromycin was used as a standard drug. The interactions were studied by PyMol and visualized in LigPlot. Furthermore, the RMSD graph shows fluctuations at various points at the start of simulation in Top1 (Azithromycin) complex system due to structural changes in the helix-coil-helix and beta-turn-beta changes at specific points resulting in increased RMSD with a time frame of 50 ns. But this change remains stable after the extension of simulation time intervals till 100 ns. On other side, the Top2 complex system remains highly stable throughout the time scale. No such structural dynamics were observed bu the ligand attached to the active site residues binds strongly. CONCLUSION: This study facilitates researchers to develop and discover more effective and specific therapeutic agents against SARS-CoV-2 and other viral infections. Finally, chrysoplenetin was identified as a more potent drug candidate to act against the viral main protease, which in the future can be helpful.

Drug repurposing: identification of SARS-CoV-2 potential inhibitors by virtual screening and pharmacokinetics strategies.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic caused global health, economic, and population loss. Variants of the coronavirus contributed to the severity of the disease and persistent rise in infections. This study aimed to identify potential drug candidates from fifteen approved antiviral drugs against SARS-CoV-2 (6LU7), SARS-CoV (5B6O), and SARS-CoV-2 spike protein (6M0J) using virtual screening and pharmacokinetics to gain insights into COVID-19 therapeutics. METHODOLOGY: We employed drug repurposing approach to analyze binding performance of fifteen clinically approved antiviral drugs against the main protease of SARS-CoV-2 (6LU7), SARS-CoV (5B6O), and SARS-CoV-2 spike proteins bound to ACE-2 receptor (6M0J), to provide an insight into the therapeutics of COVID-19. AutoDock Vina was used for docking studies. The binding affinities were calculated, and 2-3D structures of protein-ligand interactions were drawn. RESULTS: Rutin, hesperidin, and nelfinavir are clinically approved antiviral drugs with high binding affinity to proteins 6LU7, 5B6O, and 6M0J. These ligands have excellent pharmacokinetics, ensuring efficient absorption, metabolism, excretion, and digestibility. Hesperidin showed the most potent interaction with spike protein 6M0J, forming four H-bonds. Nelfinavir had a high human intestinal absorption (HIA) score of 0.93, indicating maximum absorption in the body and promising interactions with 6LU7. CONCLUSIONS: Our results indicated that rutin, hesperidin, and nelfinavir had the highest binding results against the proposed drug targets. The computational approach effectively identified SARS-CoV-2 inhibitors. COVID-19 is still a recurrent threat globally and predictive analysis using natural compounds might serve as a starting point for new drug development against SARS-CoV-2 and related viruses.

SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds.

The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Isolation, Virtual Screening, and Evaluation of Hazelnut-Derived Immunoactive Peptides for the Inhibition of SARS-CoV-2 Main Protease.

The aim of this study is to validate the activity of hazelnut (Corylus avellana L.)-derived immunoactive peptides inhibiting the main protease (Mpro) of SARS-CoV-2 and further unveil their interaction mechanism using in vitro assays, molecular dynamics (MD) simulations, and binding free energy calculations. In general, the enzymatic hydrolysis components, especially molecular weight < 3 kDa, possess good immune activity as measured by the proliferation ability of mouse splenic lymphocytes and phagocytic activity of mouse peritoneal macrophages. Over 866 unique peptide sequences were isolated, purified, and then identified by nanohigh-performance liquid chromatography/tandem mass spectrometry (NANO-HPLC-MS/MS) from hazelnut protein hydrolysates, but Trp-Trp-Asn-Leu-Asn (WWNLN) and Trp-Ala-Val-Leu-Lys (WAVLK) in particular are found to increase the cell viability and phagocytic capacity of RAW264.7 macrophages as well as promote the secretion of the cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß). Fluorescence resonance energy transfer assay elucidated that WWNLN and WAVLK exhibit excellent inhibitory potency against Mpro, with IC50 values of 6.695 and 16.750 µM, respectively. Classical all-atom MD simulations show that hydrogen bonds play a pivotal role in stabilizing the complex conformation and protein-peptide interaction. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculation indicates that WWNLN has a lower binding free energy with Mpro than WAVLK. Furthermore, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions illustrate favorable drug-likeness and pharmacokinetic properties of WWNLN compared to WAVLK. This study provides a new understanding of the immunomodulatory activity of hazelnut hydrolysates and sheds light on peptide inhibitors targeting Mpro.

Ciclesonide Inhibits SARS-CoV-2 Papain-Like Protease in Vitro.

The emergence of coronavirus disease 2019 (COVID-19), a novel identified pneumonia resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has significantly impacted and posed significant challenges to human society. The papain-like protease (PLpro) found in the nonstructural protein 3 of SARS-CoV-2 plays a vital role in viral replication. Moreover, PLpro disrupts the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 from host proteins. Consequently, PLpro has emerged as a promising drug target against SARS-CoV-2 infection. Computational studies have reported that ciclesonide can bind to SARS-CoV-2 PLpro. However, the inhibitory effects of ciclenoside on the PLpro have not been experimentally evaluated. Here, we evaluated the inhibitory effects of synthetic glucocorticoids (sGCs), including ciclesonide, on SARS-CoV-2 PLpro in vitro assay. Ciclesonide significantly inhibited the enzymatic activity of PLpro, compared with other sGCs and its IC50 was 18.4 ± 1.89 µM. These findings provide insights into the development of PLpro inhibitors.

Computational Estimation of Residues Involving Resistance to the SARS-CoV-2 Main Protease Inhibitor Ensitrelvir Based on Virtual Alanine Scan of the Active Site.

Ensitrelvir is a noncovalent inhibitor of the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2. Acquisition of drug resistance in virus-derived proteins is a serious therapeutic concern, and drug resistance occurs due to amino acid mutations. In this study, we computationally constructed 24 mutants, in which one residue around the active site was replaced with alanine and performed molecular dynamics simulations to the complex of Mpro and ensitrelvir to predict the residues involved in drug resistance. We evaluated the changes in the entire protein structure and ligand configuration in each of these mutants and estimated which residues were involved in ensitrelvir recognition. This method is called a virtual alanine scan. In nine mutants (S1A, T26A, H41A, M49A, L141A, H163A, E166A, V186A, and R188A), although the entire protein structure and catalytic dyad (cysteine (Cys)145 and histidine (His)41) were not significantly moved, the ensitrelvir configuration changed. Thus, it is considered that these mutants did not recognize ensitrelvir while maintaining Mpro enzymatic activities, and Ser1, Thr26, His41, Met49, Leu141, His163, Glu166, Val186, and Arg188 may be related to ensitrelvir resistance. The ligand shift noted in M49A was similar to that observed in M49I, which has been shown to be experimentally ensitrelvir resistant. These findings suggest that our research approach can predict mutations that incite drug resistance.

Accelerating reliable multiscale quantum refinement of protein-drug systems enabled by machine learning.

Biomacromolecule structures are essential for drug development and biocatalysis. Quantum refinement (QR) methods, which employ reliable quantum mechanics (QM) methods in crystallographic refinement, showed promise in improving the structural quality or even correcting the structure of biomacromolecules. However, vast computational costs and complex quantum mechanics/molecular mechanics (QM/MM) setups limit QR applications. Here we incorporate robust machine learning potentials (MLPs) in multiscale ONIOM(QM:MM) schemes to describe the core parts (e.g., drugs/inhibitors), replacing the expensive QM method. Additionally, two levels of MLPs are combined for the first time to overcome MLP limitations. Our unique MLPs+ONIOM-based QR methods achieve QM-level accuracy with significantly higher efficiency. Furthermore, our refinements provide computational evidence for the existence of bonded and nonbonded forms of the Food and Drug Administration (FDA)-approved drug nirmatrelvir in one SARS-CoV-2 main protease structure. This study highlights that powerful MLPs accelerate QRs for reliable protein-drug complexes, promote broader QR applications and provide more atomistic insights into drug development.

Predicting drug-Protein interaction with deep learning framework for molecular graphs and sequences: Potential candidates against SAR-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the COVID-19 disease, which represents a new life-threatening disaster. Regarding viral infection, many therapeutics have been investigated to alleviate the epidemiology such as vaccines and receptor decoys. However, the continuous mutating coronavirus, especially the variants of Delta and Omicron, are tended to invalidate the therapeutic biological product. Thus, it is necessary to develop molecular entities as broad-spectrum antiviral drugs. Coronavirus replication is controlled by the viral 3-chymotrypsin-like cysteine protease (3CLpro) enzyme, which is required for the virus's life cycle. In the cases of severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV), 3CLpro has been shown to be a promising therapeutic development target. Here we proposed an attention-based deep learning framework for molecular graphs and sequences, training from the BindingDB 3CLpro dataset (114,555 compounds). After construction of such model, we conducted large-scale screening the in vivo/vitro dataset (276,003 compounds) from Zinc Database and visualize the candidate compounds with attention score. geometric-based affinity prediction was employed for validation. Finally, we established a 3CLpro-specific deep learning framework, namely GraphDPI-3CL (AUROC: 0.958) achieved superior performance beyond the existing state of the art model and discovered 10 molecules with a high binding affinity of 3CLpro and superior binding mode.

Synthesis and in silico inhibitory action studies of azo-anchored imidazo[4,5-b]indole scaffolds against the COVID-19 main protease (Mpro).

The present work elicits a novel approach to combating COVID-19 by synthesizing a series of azo-anchored 3,4-dihydroimidazo[4,5-b]indole derivatives. The envisaged methodology involves the L-proline-catalyzed condensation of para-amino-functionalized azo benzene, indoline-2,3-dione, and ammonium acetate precursors with pertinent aryl aldehyde derivatives under ultrasonic conditions. The structures of synthesized compounds were corroborated through FT-IR, 1H NMR, 13C NMR, and mass analysis data. Molecular docking studies assessed the inhibitory potential of these compounds against the main protease (Mpro) of SARS-CoV-2. Remarkably, in silico investigations revealed significant inhibitory action surpassing standard drugs such as Remdesivir, Paxlovid, Molnupiravir, Chloroquine, Hydroxychloroquine (HCQ), and (N3), an irreversible Michael acceptor inhibitor. Furthermore, the highly active compound was also screened for cytotoxicity activity against HEK-293 cells and exhibited minimal toxicity across a range of concentrations, affirming its favorable safety profile and potential suitability. The pharmacokinetic properties (ADME) of the synthesized compounds have also been deliberated. This study paves the way for in vitro and in vivo testing of these scaffolds in the ongoing battle against SARS-CoV-2.

Detection of Active SARS-CoV-2 3CL Protease in Infected Cells Using Activity-Based Probes with a 2,6-Dichlorobenzoyloxymethyl Ketone Reactive Warhead.

The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe 2) or 2,6-dichlorobenzoyloxymethyl ketone (probe 3) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe 3 displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe 3 for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe 3, suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe 3 will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.

SARS-CoV-2 Mpro oligomerization as a potential target for therapy.

The main protease (Mpro) of SARS-CoV-2 is critical in the virus's replication cycle, facilitating the maturation of polyproteins into functional units. Due to its conservation across taxa, Mpro is a promising target for broad-spectrum antiviral drugs. Targeting Mpro with small molecule inhibitors, such as nirmatrelvir combined with ritonavir (Paxlovid™), which the FDA has approved for post-exposure treatment and prophylaxis, can effectively interrupt the replication process of the virus. A key aspect of Mpro's function is its ability to form a functional dimer. However, the mechanics of dimerization and its influence on proteolytic activity remain less understood. In this study, we utilized biochemical, structural, and molecular modelling approaches to explore Mpro dimerization. We evaluated critical residues, specifically Arg4 and Arg298, that are essential for dimerization. Our results show that changes in the oligomerization state of Mpro directly affect its enzymatic activity and dimerization propensity. We discovered a synergistic relationship influencing dimer formation, involving both intra- and intermolecular interactions. These findings highlight the potential for developing allosteric inhibitors targeting Mpro, offering promising new directions for therapeutic strategies.

New meroterpenoids and polyketides from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1 and their anti-inflammatory and SARS-CoV-2 Mpro inhibitory activities.

Seven new meroterpenoids, paraphaeones A-G (1-7), and two new polyketides, paraphaeones H-I (8-9), along with eight known compounds (10-17), were isolated from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1. The structures of 1-9 were identified through the analysis of 1H, 13C, and 2D NMR spectra, assisted by HR-ESI-MS data. Compounds 1 and 7 exhibited a dose-dependent decrease in lactate dehydrogenase levels, with IC50 values of 1.78 µM and 1.54 µM, respectively. Moreover, they inhibited the secretion of IL-1ß and CASP-1, resulting in a reduction in the activity levels of NLRP3 inflammasomes. Fluorescence microscopy results indicated that compound 7 concentration-dependently attenuated cell pyroptosis. Additionally, compounds 4 and 7 showed potential inhibitory effects on the severe acute respiratory syndrome coronavirus-2 main protease (SARS-CoV-2 Mpro), with IC50 values of 10.8 ± 0.9 µM and 12.9 ± 0.7 µM, respectively.

Non-peptidic inhibitors targeting SARS-CoV-2 main protease: A review.

The COVID-19 pandemic continues to pose a threat to global health, and sounds the alarm for research & development of effective anti-coronavirus drugs, which are crucial for the patients and urgently needed for the current epidemic and future crisis. The main protease (Mpro) stands as an essential enzyme in the maturation process of SARS-CoV-2, playing an irreplaceable role in regulating viral RNA replication and transcription. It has emerged as an ideal target for developing antiviral agents against SARS-CoV-2 due to its high conservation and the absence of homologous proteases in the human body. Among the SARS-CoV-2 Mpro inhibitors, non-peptidic compounds hold promising prospects owing to their excellent antiviral activity and improved metabolic stability. In this review, we offer an overview of research progress concerning non-peptidic SARS-CoV-2 Mpro inhibitors since 2020. The efforts delved into molecular structures, structure-activity relationships (SARs), biological activity, and binding modes of these inhibitors with Mpro. This review aims to provide valuable clues and insights for the development of anti-SARS-CoV-2 agents as well as broad-spectrum coronavirus Mpro inhibitors.

In vitro biological evaluation and in silico insights into the antiviral activity of standardized olive leaves extract against SARS-CoV-2.

There is still a great global need for efficient treatments for the management of SARS-CoV-2 illness notwithstanding the availability and efficacy of COVID-19 vaccinations. Olive leaf is an herbal remedy with a potential antiviral activity that could improve the recovery of COVID-19 patients. In this work, the olive leaves major metabolites were screened in silico for their activity against SARS-CoV-2 by molecular docking on several viral targets such as methyl transferase, helicase, Plpro, Mpro, and RdRp. The results of in silico docking study showed that olive leaves phytoconstituents exhibited strong potential antiviral activity against SARS-CoV-2 selected targets. Verbacoside demonstrated a strong inhibition against methyl transferase, helicase, Plpro, Mpro, and RdRp (docking scores = -17.2, -20, -18.2, -19.8, and -21.7 kcal/mol.) respectively. Oleuropein inhibited 5rmm, Mpro, and RdRp (docking scores = -15, -16.6 and -18.6 kcal/mol., respectively) respectively. Apigenin-7-O-glucoside exhibited activity against methyl transferase and RdRp (docking score = -16.1 and -19.4 kcal/mol., respectively) while Luteolin-7-O-glucoside inhibited Plpro and RdRp (docking score = -15.2 and -20 kcal/mol., respectively). The in vitro antiviral assay was carried out on standardized olive leaf extract (SOLE) containing 20% oleuropein and IC50 was calculated. The results revealed that 20% SOLE demonstrated a moderate antiviral activity against SARS-CoV-2 with IC50 of 118.3 µg /mL. Accordingly, olive leaf could be a potential herbal therapy against SARS-CoV-2 but more in vivo and clinical investigations are recommended.

Anti-Coronavirus Potential of Polyether Ionophores: The New Application of Veterinary Antibiotics in Livestock.

Coronaviruses have consistently posed a major global concern in the field of livestock industry and public health. However, there is currently a lack of efficient drugs with broad-spectrum antiviral activity to address the challenges presented by emerging mutated strains or drug resistance. Additionally, the method for identifying multitarget drugs is also insufficient. Aminopeptidase N (APN) and 3C-like proteinase (3CLpro) represent promising targets for host-directed and virus-directed strategies, respectively, in the development of effective drugs against various coronaviruses. In this study, maduramycin ammonium demonstrated a broad-spectrum antiviral effect by targeting both of the proteins. The binding domains 4 Å from the ligand of both target proteins shared a structural similarity, suggesting that screening and designing drugs based on these domains might exhibit broad-spectrum and highly effective antiviral activity. Furthermore, it was identified that the polyether ionophores' ability to carry zinc ion might be one of the reasons why they were able to target APN and exhibit antiviral effect. The findings of this experiment provide novel perspectives for future drug screening and design, while also offering valuable references for the utilization of polyether ionophores in the management of livestock health.

De novo design of potential peptide analogs against the main protease of Omicron variant using in silico studies.

SARS-CoV-2 and its variants are crossing the immunity barrier induced through vaccination. Recent Omicron sub-variants are highly transmissible and have a low mortality rate. Despite the low severity of Omicron variants, these new variants are known to cause acute post-infectious syndromes. Nowadays, novel strategies to develop new potential inhibitors for SARS-CoV-2 and other Omicron variants have gained prominence. For viral replication and survival the main protease of SARS-CoV-2 plays a vital role. Peptide-like inhibitors that mimic the substrate peptide have already proved to be effective in inhibiting the Mpro of SARS-CoV-2 variants. Our systematic canonical amino acid point mutation analysis on the native peptide has revealed various ways to improve the native peptide of the main protease. Multi mutation analysis has led us to identify and design potent peptide-analog inhibitors that act against the Mpro of the Omicron sub-variants. Our in-depth analysis of all-atom molecular dynamics studies has paved the way to characterize the atomistic behavior of Mpro in Omicron variants. Our goal is to develop potent peptide-analogs that could be therapeutically effective against Omicron and its sub-variants.

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease.

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro), a highly conserved protease among various CoVs, is essential for viral replication and pathogenesis, making it a prime target for antiviral drug development. Here, we leverage proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Among them, MPD2 was demonstrated to effectively reduce MPro protein levels in 293T cells, relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MPro protein levels in SARS-CoV-2-infected A549-ACE2 cells. MPD2 also displayed potent antiviral activity against various SARS-CoV-2 strains and exhibited enhanced potency against nirmatrelvir-resistant viruses. Overall, this proof-of-concept study highlights the potential of targeted protein degradation of MPro as an innovative approach for developing antivirals that could fight against drug-resistant viral variants.

Deep profiling of potential substrate atlas of porcine epidemic diarrhea virus 3C-like protease.

Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.

Inhibitory effects of senkyuchachosan on SARS-CoV-2 papain-like protease activity in vitro.

Papain-like protease (PLpro) enzyme plays a vital role in viral replication as it breaks down polyproteins and disrupts the host's immune response. There are few reports on Kampo formulas that focus on PLpro activity. In this study, we evaluated the inhibitory effects of senkyuchachosan, a traditional Japanese medicine, on PLpro of SARS-CoV-2, the virus responsible for causing COVID-19. We purified the PLpro enzyme and conducted in vitro enzymatic assays using specific substrates. Among the nine crude drugs present in senkyuchachosan, four (Cyperi Rhizoma, Schizonepetae Spica, Menthae Herba, and Camelliae sinensis Folium [CsF]) strongly inhibited PLpro activity. CsF, derived from Camellia sinensis (green tea), contains polyphenols, including catechins and tannins. To confirm that the PLpro inhibitory effects of senkyuchachosan predominantly stem from tannins, the tannins were removed from the decoction using polyvinylpolypyrrolidone (PVPP). The inhibitory effect of senkyuchachosan on PLpro activity was reduced by the removal of PVPP. In addition, the tannin fraction obtained from the CsF extracts showed significant PLpro inhibitory effects. These findings lay the groundwork for the potential development of therapeutic agents that target SARS-CoV-2 infection by intervening in proteolytic cleavage of the virus.

Exploring the Efficacy of Noncovalent SARS-CoV-2 Main Protease Inhibitors: A Computational Simulation Analysis Study.

The SARS-CoV-2 main protease, as a key target for antiviral therapeutics, is instrumental in maintaining virus stability, facilitating translation, and enabling the virus to evade innate immunity. Our research focused on designing non-covalent inhibitors to counteract the action of this protease. Utilizing a 3D-QSAR model and contour map, we successfully engineered eight novel non-covalent inhibitors. Further evaluation and comparison of these novel compounds through methodologies including molecular docking, ADMET analysis, frontier molecular orbital studies, molecular dynamics simulations, and binding free energy revealed that the inhibitors N02 and N03 demonstrated superior research performance (N02 ΔGbind=-206.648 kJ/mol, N03 ΔGbind=-185.602 kJ/mol). These findings offer insightful guidance for the further refinement of molecular structures and the development of more efficacious inhibitors. Consequently, future investigations can draw upon these findings to unearth more potent inhibitors, thereby amplifying their impact in the treatment and prevention of associated diseases.