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.

SARS-CoV-2 replication and drug discovery.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to wreak havoc across the globe. This sudden and deadly pandemic emphasizes the necessity for anti-viral drug development that can be rapidly administered to reduce morbidity, mortality, and virus propagation. Thus, lacking efficient anti-COVID-19 treatment, and especially given the lengthy drug development process as well as the critical death tool that has been associated with SARS-CoV-2 since its outbreak, drug repurposing (or repositioning) constitutes so far, the ideal and ready-to-go best approach in mitigating viral spread, containing the infection, and reducing the COVID-19-associated death rate. Indeed, based on the molecular similarity approach of SARS-CoV-2 with previous coronaviruses (CoVs), repurposed drugs have been reported to hamper SARS-CoV-2 replication. Therefore, understanding the inhibition mechanisms of viral replication by repurposed anti-viral drugs and chemicals known to block CoV and SARS-CoV-2 multiplication is crucial, and it opens the way for particular treatment options and COVID-19 therapeutics. In this review, we highlighted molecular basics underlying drug-repurposing strategies against SARS-CoV-2. Notably, we discussed inhibition mechanisms of viral replication, involving and including inhibition of SARS-CoV-2 proteases (3C-like protease, 3CLpro or Papain-like protease, PLpro) by protease inhibitors such as Carmofur, Ebselen, and GRL017, polymerases (RNA-dependent RNA-polymerase, RdRp) by drugs like Suramin, Remdesivir, or Favipiravir, and proteins/peptides inhibiting virus-cell fusion and host cell replication pathways, such as Disulfiram, GC376, and Molnupiravir. When applicable, comparisons with SARS-CoV inhibitors approved for clinical use were made to provide further insights to understand molecular basics in inhibiting SARS-CoV-2 replication and draw conclusions for future drug discovery research.

Efficacy and Safety of Ensitrelvir in Patients With Mild-to-Moderate Coronavirus Disease 2019: The Phase 2b Part of a Randomized, Placebo-Controlled, Phase 2/3 Study.

BACKGROUND: This phase 2b part of a randomized phase 2/3 study assessed the efficacy and safety of ensitrelvir for mild-to-moderate coronavirus disease 2019 (COVID-19) during the Omicron epidemic. METHODS: Patients were randomized (1:1:1) to orally receive ensitrelvir fumaric acid 125 mg (375 mg on day 1) or 250 mg (750 mg on day 1) or placebo once daily for 5 days. The co-primary endpoints were the change from baseline in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) titer on day 4 and time-weighted average change from baseline up to 120 hours in the total score of predefined 12 COVID-19 symptoms. Safety was assessed through adverse events. RESULTS: A total of 341 patients (ensitrelvir 125-mg group: 114; ensitrelvir 250-mg group: 116; and placebo group: 111; male: 53.5-64.9%; mean age: 35.3-37.3 years) were included in the efficacy analyses. The change from baseline in SARS-CoV-2 titer on day 4 was significantly greater with both ensitrelvir doses than with placebo (differences from placebo: -0.41 log10 50% tissue-culture infectious dose/mL; P < .0001 for both). The total score of the 12 COVID-19 symptoms did not show a significant difference between the ensitrelvir groups and placebo group. The time-weighted average change from baseline up to 120 hours was significantly greater with ensitrelvir versus placebo in several subtotal scores, including acute symptoms and respiratory symptoms. Most adverse events were mild in severity. CONCLUSIONS: Ensitrelvir treatment demonstrated a favorable antiviral efficacy and potential clinical benefit with an acceptable safety profile. CLINICAL TRIALS REGISTRATION: Japan Registry of Clinical Trials: jRCT2031210350 (https://jrct.niph.go.jp/en-latest-detail/jRCT2031210350).

Ensitrelvir is effective against SARS-CoV-2 3CL protease mutants circulating globally.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a public health concern worldwide. Ensitrelvir (S-217622) has been evaluated as an antiviral treatment for COVID-19, targeting SARS-CoV-2 3C-like protease (3CLpro). Ensitrelvir has been reported to have comparable antiviral activity against some of the SARS-CoV-2 variants: alpha, beta, gamma, delta, and omicron (BA.1.18). In this paper, we describe that ensitrelvir is effective against newly emerging SARS-CoV-2 variants and globally prevalent 3CLpro mutations. Ensitrelvir exhibited comparable antiviral activity against SARS-CoV-2 variants, including recently emerging ones: omicron (BA1.1, BA.2, BA.2.75, BA.4, BA.5, BQ.1.1, XBB.1, and XE), mu, lambda, and theta. Genetic surveillance of SARS-CoV-2 3CLpro, the target of ensitrelvir, was conducted using a public database and identified 11 major 3CLpro mutations circulating globally (G15S, T21I, T24I, K88R, L89F, K90R, P108S, P132H, A193V, H246Y, and A255V). The 3CLpro mutation from proline to histidine at amino acid position 132 was especially identified in the omicron variant, with prevalence of 99.69%. Enzyme kinetic assay revealed that these 3CLpro mutants have enzymatic activity comparable to that of the wild type (WT). Next, we assessed the inhibitory effect of ensitrelvir against mutated 3CLpro, with it showing inhibitory effects similar to that against the WT. These in vitro data suggest that ensitrelvir will be effective against currently circulating SARS-CoV-2 variants, including omicron variants and those carrying 3CLpro mutations, which emerging novel SARS-CoV-2 variants could carry.

Porcine Deltacoronavirus Infection Cleaves HDAC2 to Attenuate Its Antiviral Activity.

Protein acetylation plays an important role during virus infection. Thus, it is not surprising that viruses always evolve elaborate mechanisms to regulate the functions of histone deacetylases (HDACs), the essential transcriptional and epigenetic regulators for deacetylation. Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets and has the potential to infect humans. In this study, we found that PDCoV infection inhibited cellular HDAC activity. By screening the expressions of different HDAC subfamilies after PDCoV infection, we unexpectedly found that HDAC2 was cleaved. Ectopic expression of HDAC2 significantly inhibited PDCoV replication, while the reverse effects could be observed after treatment with an HDAC2 inhibitor (CAY10683) or the knockdown of HDAC2 expression by specific siRNA. Furthermore, we demonstrated that PDCoV-encoded nonstructural protein 5 (nsp5), a 3C-like protease, was responsible for HDAC2 cleavage through its protease activity. Detailed analyses showed that PDCoV nsp5 cleaved HDAC2 at glutamine 261 (Q261), and the cleaved fragments (amino acids 1 to 261 and 262 to 488) lost the ability to inhibit PDCoV replication. Interestingly, the Q261 cleavage site is highly conserved in HDAC2 homologs from other mammalian species, and the nsp5s encoded by seven tested mammalian coronaviruses also cleaved HDAC2, suggesting that cleaving HDAC2 may be a common strategy used by different mammalian coronaviruses to antagonize the antiviral role of HDAC2. IMPORTANCE As an emerging porcine enteropathogenic coronavirus that possesses the potential to infect humans, porcine deltacoronavirus (PDCoV) is receiving increasing attention. In this work, we found that PDCoV infection downregulated cellular histone deacetylase (HDAC) activity. Of particular interest, the viral 3C-like protease, encoded by the PDCoV nonstructural protein 5 (nsp5), cleaved HDAC2, and this cleavage could be observed in the context of PDCoV infection. Furthermore, the cleavage of HDAC2 appears to be a common strategy among mammalian coronaviruses, including the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to antagonize the antiviral role of HDAC2. To our knowledge, PDCoV nsp5 is the first identified viral protein that can cleave cellular HDAC2. Results from our study provide new targets to develop drugs combating coronavirus infection.

SARS-CoV-2 nsp5 Exhibits Stronger Catalytic Activity and Interferon Antagonism than Its SARS-CoV Ortholog.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an enormous threat to economic activity and public health worldwide. Previous studies have shown that the nonstructural protein 5 (nsp5, also called 3C-like protease) of alpha- and deltacoronaviruses cleaves Q231 of the NF-κB essential modulator (NEMO), a key kinase in the RIG-I-like receptor pathway, to inhibit type I interferon (IFN) production. In this study, we found that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleaved NEMO at multiple sites (E152, Q205, and Q231). Notably, SARS-CoV-2 nsp5 exhibited a stronger ability to cleave NEMO than SARS-CoV nsp5. Sequence and structural alignments suggested that an S/A polymorphism at position 46 of nsp5 in SARS-CoV versus SARS-CoV-2 may be responsible for this difference. Mutagenesis experiments showed that SARS-CoV-2 nsp5 (S46A) exhibited poorer cleavage of NEMO than SARS-CoV-2 nsp5 wild type (WT), while SARS-CoV nsp5 (A46S) showed enhanced NEMO cleavage compared with the WT protein. Purified recombinant SARS-CoV-2 nsp5 WT and SARS-CoV nsp5 (A46S) proteins exhibited higher hydrolysis efficiencies than SARS-CoV-2 nsp5 (S46A) and SARS-CoV nsp5 WT proteins in vitro. Furthermore, SARS-CoV-2 nsp5 exhibited stronger inhibition of Sendai virus (SEV)-induced interferon beta (IFN-ß) production than SARS-CoV-2 nsp5 (S46A), while introduction of the A46S substitution in SARS-CoV nsp5 enhanced suppression of SEV-induced IFN-ß production. Taken together, these data show that S46 is associated with the catalytic activity and IFN antagonism by SARS-CoV-2 nsp5. IMPORTANCE The nsp5-encoded 3C-like protease is the main coronavirus protease, playing a vital role in viral replication and immune evasion by cleaving viral polyproteins and host immune-related molecules. We showed that both SARS-CoV-2 nsp5 and SARS-CoV nsp5 cleave the NEMO at multiple sites (E152, Q205, and Q231). This specificity differs from NEMO cleavage by alpha- and deltacoronaviruses, demonstrating the distinct substrate recognition of SARS-CoV-2 and SARS-CoV nsp5. Compared with SARS-CoV nsp5, SARS-CoV-2 nsp5 encodes S instead of A at position 46. This substitution is associated with stronger catalytic activity, enhanced cleavage of NEMO, and increased interferon antagonism of SARS-CoV-2 nsp5. These data provide new insights into the pathogenesis and transmission of SARS-CoV-2.

Interaction of Laurusides 1 and 2 with the 3C-like Protease (Mpro) from Wild-Type and Omicron Variant of SARS-CoV-2: A Molecular Dynamics Study.

Laurus nobilis (bay laurel) is a natural source of biological compounds, and some of its extracts and phytocompounds are also endowed with antiviral activity toward the family of the severe acute respiratory syndrome (SARS)-associated ß-coronaviruses. Some glycosidic laurel compounds such as laurusides were proposed as inhibitors of important protein targets of SARS-CoV-2, which clearly recalls their potential as anti-COVID-19 drugs. Due to the frequent genomic variations of the ß-coronaviruses and the consequent importance of evaluating a new drug candidate with respect to the variants of the target ß-coronavirus, we decided to investigate at an atomistic level the molecular interactions of the potential laurel-derived drugs laurusides 1 and 2 (L01 and L02, respectively) toward a well-conserved and crucial target, the 3C-like protease (Mpro), using the enzymes of both the wild-type of SARS-CoV-2 and of the more recent Omicron variant. Thus, we performed molecular dynamic (MD) simulations of laurusides-SARS-CoV-2 protease complexes to deepen the knowledge on the stability of the interaction and compare the effects of the targeting among the two genomic variants. We found that the Omicron mutation does not significantly impact the lauruside binding and that L02 connects more stably with respect to L01 in the complexes from both variants, even though both compounds prevalently interact within the same binding pocket. Although purely in silico, the current study highlights the potential role of bay laurel phytocompounds in the antiviral and specifically anti-coronavirus research and shows their potential binding toward Mpro, corroborating the important commitment of bay laurel as functional food and disclosing novel scenarios of lauruside-based antiviral therapies.

A Randomized Phase 2/3 Study of Ensitrelvir, a Novel Oral SARS-CoV-2 3C-Like Protease Inhibitor, in Japanese Patients with Mild-to-Moderate COVID-19 or Asymptomatic SARS-CoV-2 Infection: Results of the Phase 2a Part.

This multicenter, double-blind, phase 2a part of a phase 2/3 study assessed the efficacy and safety of ensitrelvir, a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor, in Japanese patients with mild-to-moderate coronavirus disease 2019 (COVID-19) or asymptomatic SARS-CoV-2 infection. Sixty-nine patients were randomized (1:1:1) to orally receive 5-day ensitrelvir fumaric acid (375 mg on day 1 followed by 125 mg daily, or 750 mg on day 1 followed by 250 mg daily) or placebo and followed up until day 28. The primary outcome was the change from baseline in the SARS-CoV-2 viral titer. A total of 16, 14, and 17 patients in the ensitrelvir 125 mg, ensitrelvir 250 mg, and placebo groups, respectively, were included in the intention-to-treat population (mean age: 38.0 to 40.4 years). On day 4, the change from baseline in SARS-CoV-2 viral titer (log10 50% tissue culture infectious dose/mL) in patients with positive viral titer and viral RNA at baseline was greater with ensitrelvir 125 mg (mean [standard deviation], -2.42 [1.42]; P = 0.0712) and 250 mg (-2.81 [1.21]; P = 0.0083) versus placebo (-1.54 [0.74]); ensitrelvir treatment reduced SARS-CoV-2 RNA by -1.4 to -1.5 log10 copies/mL versus placebo. The viral titer and viral RNA were similar across groups on and after day 6. The median time to infectious viral clearance decreased by approximately 50 h with ensitrelvir treatment. All adverse events were mild to moderate. Ensitrelvir treatment demonstrated rapid SARS-CoV-2 clearance and was well tolerated (Japan Registry of Clinical Trials identifier: jRCT2031210350).

Crystal structure of SARS-CoV 3C-like protease with baicalein.

The 3C-like protease (Mpro, 3CLpro) plays a key role in the replication process in coronaviruses (CoVs). The Mpro is an essential enzyme mediates CoVs replication and is a promising target for development of antiviral drugs. Until now, baicalein has been shown the specific activity for SARS-CoV Mpro in vitro experiments. In this study, we resolved the SARS-CoV Mpro with baicalein by X-ray diffraction at 2.25 Å (PDB code 7XAX), which provided a structural basis for the research and development of baicalein as an anti-CoVs drug.

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.

In Vitro and in Silico Studies of Potential Coronavirus-Specific 3C-Like Protease Inhibitors.

We investigated similar compounds to ebselen and tideglusib, which exhibit strong activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using Molecular ACCess System (MACCS) keys. Four candidate compounds were identified. One of them, phenyl-benzothiazol-3-one, showed coronavirus-specific 3C-like (3CL) protease inhibitory activity. The results indicated that a similarity score above 0.81 is a good indicator of activity for ebselen-and-tideglusib-like compounds. Subsequently, we simulated the ring-cleavage Michael reaction of ebselen at the Se center, which is responsible for its 3CL protease inhibitory activity, and determined the activation free energy of the reaction. The results showed that reaction simulation is a useful tool for estimating the activity of inhibitory compounds that undergo Michael addition reactions with the relevant cysteine S atom of 3CL proteases.

Synthesis, spectroscopic, and computational studies on molecular charge-transfer complex of 2-((2-hydroxybenzylidene) amino)-2-(hydroxymethyl) propane-1, 3-diol with chloranilic acid: Potential antiviral activity simulation of CT-complex against SARS-CoV-2.

An innovative charge-transfer complex between the Schiff base 2-((2-hydroxybenzylidene) amino)-2-(hydroxymethyl) propane-1,3-diol [SAL-THAM] and the π-acceptor, chloranilic acid (CLA) within the mole ratio (1:1) was synthesized and characterized aiming to investigate its electronic transition spectra in acetonitrile (ACN), methanol (MeOH) and ethanol (EtOH) solutions. Applying Job`s method in the three solvents supported the 1:1 (CLA: SAL-THAM) mole ratio complex formation. The formation of stable CT- complex was shown by the highest values of charge-transfer complex formation constants, KCT, calculated using minimum-maximum absorbance method, with the sequence, acetonitrile > ethanol > methanol DFT study on the synthesized CT complex was applied based on the B3LYP method to evaluate the optimized structure and extract geometrical and reactivity parameters. Based on TD-DFT theory, the electronic properties, 1H and 13C NMR, IR, and UV-Vis spectra of the studied system in different solvents showing good agreement with the experimental studies. MEP map described the possibility of hydrogen bonding and charge transfer in the studied system. Finally, a computational approach for screening the antiviral activity of CT - complex towards SARS-CoV-2 coronavirus protease via molecular docking simulation was conducted and confirmed with molecular dynamic (MD) simulation.

From Repurposing to Redesign: Optimization of Boceprevir to Highly Potent Inhibitors of the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the betacoronavirus SARS-CoV-2 is an attractive target for the development of treatments for COVID-19. Structure-based design is a successful approach to discovering new inhibitors of the Mpro. Starting from crystal structures of the Mpro in complexes with the Hepatitis C virus NS3/4A protease inhibitors boceprevir and telaprevir, we optimized the potency of the alpha-ketoamide boceprevir against the Mpro by replacing its P1 cyclobutyl moiety by a γ-lactam as a glutamine surrogate. The resulting compound, MG-78, exhibited an IC50 of 13 nM versus the recombinant Mpro, and similar potency was observed for its P1' N-methyl derivative MG-131. Crystal structures confirmed the validity of our design concept. In addition to SARS-CoV-2 Mpro inhibition, we also explored the activity of MG-78 against the Mpro of the alphacoronavirus HCoV NL63 and against enterovirus 3C proteases. The activities were good (0.33 µM, HCoV-NL63 Mpro), moderate (1.45 µM, Coxsackievirus 3Cpro), and relatively poor (6.7 µM, enterovirus A71 3Cpro), respectively. The structural basis for the differences in activities was revealed by X-ray crystallo-graphy. We conclude that the modified boceprevir scaffold is suitable for obtaining high-potency inhibitors of the coronavirus Mpros but further optimization would be needed to target enterovirus 3Cpros efficiently.

What coronavirus 3C-like protease tells us: From structure, substrate selectivity, to inhibitor design.

The emergence of a variety of coronaviruses (CoVs) in the last decades has posed huge threats to human health. Especially, the ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 70 million infections and over 1.6 million of deaths worldwide in the past few months. None of the efficacious antiviral agents against human CoVs have been approved yet. 3C-like protease (3CLpro ) is an attractive target for antiviral intervention due to its essential role in processing polyproteins translated from viral RNA, and its conserved structural feature and substrate specificity among CoVs in spite of the sequence variation. This review focuses on all available crystal structures of 12 CoV 3CLpro s and their inhibitors, and intends to provide a comprehensive understanding of this protease from multiple aspects including its structural features, substrate specificity, inhibitor binding modes, and more importantly, to recapitulate the similarity and diversity among different CoV 3CLpro s and the structure-activity relationship of various types of inhibitors. Such an attempt could gain a deep insight into the inhibition mechanisms and drive future structure-based drug discovery targeting 3CLpro s.

Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease.

The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase's substrate specificities and the rational development of the antinidovirus drugs.IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The "noncanonical" substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

Identification of non-covalent 3C-like protease inhibitors against severe acute respiratory syndrome coronavirus-2 via virtual screening of a Korean compound library.

The outbreak of coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has turned into a pandemic. The enzyme 3C-like protease (3CLpro) is essential for the maturation of viral polyproteins in SARS-CoV-2 and is therefore regarded as a key drug target for treating the disease. To identify 3CLpro inhibitors that can suppress SARS-CoV-2 replication, we performed a virtual screening of 500,282 compounds in a Korean compound bank. We then subjected the top computational hits to inhibitory assays against 3CLpro in vitro, leading to the identification of a class of non-covalent inhibitors. Among these inhibitors, compound 7 showed an EC50 of 39.89 µM against SARS-CoV-2 and CC50 of 453.5 µM. This study provides candidates for the optimization of potent 3CLpro inhibitors showing antiviral effects against SARS-CoV-2.

Multistep rational molecular design and combined docking for discovery of novel classes of inhibitors of SARS-CoV-2 main protease 3CLpro.

The main protease (3CLpro) of SARS-CoV and SARS-CoV-2 is a promising target for discovery of novel antiviral agents. In this paper, new possible inhibitors of 3CLpro with high predicted binding affinity were detected through multistep computer-aided molecular design and bioisosteric replacements. For discovery of prospective 3CLpro binders several virtual ligand libraries were created and combined docking was performed. Moreover, the molecular dynamics simulation was applied for evaluation of protein-ligand complexes stability. Besides, important molecular properties and ADMET pharmacokinetic profiles of possible 3CLpro inhibitors were assessed by in silico prediction.

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro.

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CLpro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CLpro activity in vitro with IC50's of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC50's of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CLpro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CLpro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.

SARS-CoV-2 Main Protease Active Site Ligands in the Human Metabolome.

In late 2019, a global pandemic occurred. The causative agent was identified as a member of the Coronaviridae family, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we present an analysis on the substances identified in the human metabolome capable of binding the active site of the SARS-CoV-2 main protease (Mpro). The substances present in the human metabolome have both endogenous and exogenous origins. The aim of this research was to find molecules whose biochemical and toxicological profile was known that could be the starting point for the development of antiviral therapies. Our analysis revealed numerous metabolites-including xenobiotics-that bind this protease, which are essential to the lifecycle of the virus. Among these substances, silybin, a flavolignan compound and the main active component of silymarin, is particularly noteworthy. Silymarin is a standardized extract of milk thistle, Silybum marianum, and has been shown to exhibit antioxidant, hepatoprotective, antineoplastic, and antiviral activities. Our results-obtained in silico and in vitro-prove that silybin and silymarin, respectively, are able to inhibit Mpro, representing a possible food-derived natural compound that is useful as a therapeutic strategy against COVID-19.

Prioritisation of Compounds for 3CLpro Inhibitor Development on SARS-CoV-2 Variants.

COVID-19 represents a new potentially life-threatening illness caused by severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2 pathogen. In 2021, new variants of the virus with multiple key mutations have emerged, such as B.1.1.7, B.1.351, P.1 and B.1.617, and are threatening to render available vaccines or potential drugs ineffective. In this regard, we highlight 3CLpro, the main viral protease, as a valuable therapeutic target that possesses no mutations in the described pandemically relevant variants. 3CLpro could therefore provide trans-variant effectiveness that is supported by structural studies and possesses readily available biological evaluation experiments. With this in mind, we performed a high throughput virtual screening experiment using CmDock and the "In-Stock" chemical library to prepare prioritisation lists of compounds for further studies. We coupled the virtual screening experiment to a machine learning-supported classification and activity regression study to bring maximal enrichment and available structural data on known 3CLpro inhibitors to the prepared focused libraries. All virtual screening hits are classified according to 3CLpro inhibitor, viral cysteine protease or remaining chemical space based on the calculated set of 208 chemical descriptors. Last but not least, we analysed if the current set of 3CLpro inhibitors could be used in activity prediction and observed that the field of 3CLpro inhibitors is drastically under-represented compared to the chemical space of viral cysteine protease inhibitors. We postulate that this methodology of 3CLpro inhibitor library preparation and compound prioritisation far surpass the selection of compounds from available commercial "corona focused libraries".