Elucidation of novel compounds and epitope-based peptide vaccine design against C30 endopeptidase regions of SARS-CoV-2 using immunoinformatics approaches.

There has been progressive improvement in immunoinformatics approaches for epitope-based peptide design. Computational-based immune-informatics approaches were applied to identify the epitopes of SARS-CoV-2 to develop vaccines. The accessibility of the SARS-CoV-2 protein surface was analyzed, and hexa-peptide sequences (KTPKYK) were observed having a maximum score of 8.254, located between amino acids 97 and 102, whereas the FSVLAC at amino acids 112 to 117 showed the lowest score of 0.114. The surface flexibility of the target protein ranged from 0.864 to 1.099 having amino acid ranges of 159 to 165 and 118 to 124, respectively, harboring the FCYMHHM and YNGSPSG hepta-peptide sequences. The surface flexibility was predicted, and a 0.864 score was observed from amino acids 159 to 165 with the hepta-peptide (FCYMHHM) sequence. Moreover, the highest score of 1.099 was observed between amino acids 118 and 124 against YNGSPSG. B-cell epitopes and cytotoxic T-lymphocyte (CTL) epitopes were also identified against SARS-CoV-2. In molecular docking analyses, -0.54 to -26.21 kcal/mol global energy was observed against the selected CTL epitopes, exhibiting binding solid energies of -3.33 to -26.36 kcal/mol. Based on optimization, eight epitopes (SEDMLNPNY, GSVGFNIDY, LLEDEFTPF, DYDCVSFCY, GTDLEGNFY, QTFSVLACY, TVNVLAWLY, and TANPKTPKY) showed reliable findings. The study calculated the associated HLA alleles with MHC-I and MHC-II and found that MHC-I epitopes had higher population coverage (0.9019% and 0.5639%) than MHC-II epitopes, which ranged from 58.49% to 34.71% in Italy and China, respectively. The CTL epitopes were docked with antigenic sites and analyzed with MHC-I HLA protein. In addition, virtual screening was conducted using the ZINC database library, which contained 3,447 compounds. The 10 top-ranked scrutinized molecules (ZINC222731806, ZINC077293241, ZINC014880001, ZINC003830427, ZINC030731133, ZINC003932831, ZINC003816514, ZINC004245650, ZINC000057255, and ZINC011592639) exhibited the least binding energy (-8.8 to -7.5 kcal/mol). The molecular dynamics (MD) and immune simulation data suggest that these epitopes could be used to design an effective SARS-CoV-2 vaccine in the form of a peptide-based vaccine. Our identified CTL epitopes have the potential to inhibit SARS-CoV-2 replication.

Design, synthesis and biological evaluation of peptidomimetic benzothiazolyl ketones as 3CLpro inhibitors against SARS-CoV-2.

A series of peptidomimetic compounds containing benzothiazolyl ketone and [2.2.1] azabicyclic ring was designed, synthesized and evaluated in the hope of obtaining potent oral 3CLpro inhibitors with improved pharmacokinetic properties. Among the target compounds, 11b had the best enzymatic potency (IC50 = 0.110 µM) and 11e had the best microsomal stability (t1/2 > 120 min) and good enzyme activity (IC50 = 0.868 µM). Therefore, compounds 11b and 11e were chosen for further evaluation of pharmacokinetics in ICR mice. The results exhibited that the AUC(0-t) of 11e was 5143 h*ng/mL following single-dose oral administration of 20 mg/kg, and the F was 67.98%. Further structural modification was made to obtain compounds 11g-11j based on 11e. Among them, 11j exhibited the best enzyme inhibition activity against SARS-CoV-2 3CLpro (IC50 = 1.646 µM), the AUC(0-t) was 32473 h*ng/mL (20 mg/kg, po), and the F was 48.1%. In addition, 11j displayed significant anti-SARS-CoV-2 activity (EC50 = 0.18 µM) and low cytotoxicity (CC50 > 50 µM) in Vero E6 cells. All of the above results suggested that compound 11j was a promising lead compound in the development of oral 3CLpro inhibitors and deserved further research.

Investigation of Neolamarckia cadamba Phytoconstituents Against SARS-CoV-2 3CL Pro: An In-Silico Approach

In present study, the inhibitory potential of Neolamarckia cadamba phytoconstituents was investigated against SARS-CoV-2 3CL protease (3CL pro) (PDB ID: 6M2N). Molecular docking was analyzed using AutoDock Vina software by setting the grid parameter as X=-33.163, Y=-65.074 and Z= 41.434 with dimensions of the grid box 25 × 25 × 25 Å. Remdesivir was taken as the standard for comparative analysis along with inhibitor 5, 6, 7-trihydroxy-2-phenyl-4H-chromen-4-one. Furthermore, the exploration of 2 D Hydrogen-bond interactions was performed by Biovia Discovery Studio 4.5 program to identify the interactions between an amino acid of target and ligand followed by assessment of physicochemical properties using Lipinski's rule and Swiss ADME database. The decent bonding scores of secondary metabolites owing to hydrogen bonding with catalytic residues suggest the effectiveness of these phytochemicals towards 3CLpro. The results are further consolidated positively by Lipinski's rule and Swiss ADME prediction. Thus reasonably, observations with docking studies suggest possibility of phytochemicals from Neolamarckia cadamba to inhibit the 3CLpro and consequently would be explored further as agents for preventing COVID-19. © 2023, Universidade Federal de Mato Grosso do Sul, Departamento de Quimica. All rights reserved.

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.

High throughput screening for drugs that inhibit 3C-like protease in SARS-CoV-2.

The SARS coronavirus 2 (SARS-CoV-2) pandemic remains a major problem in many parts of the world and infection rates remain at extremely high levels. This high prevalence drives the continued emergence of new variants, and possibly ones that are more vaccine-resistant and that can drive infections even in highly vaccinated populations. The high rate of variant evolution makes clear the need for new therapeutics that can be clinically applied to minimize or eliminate the effects of COVID-19. With a hurdle of 10 years, on average, for first in class small molecule therapeutics to achieve FDA approval, the fastest way to identify therapeutics is by drug repurposing. To this end, we developed a high throughput cell-based screen that incorporates the essential viral 3C-like protease and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or FDA-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Medicinal chemistry efforts to optimize the hits identified Tranilast as a potential lead. Here, we report the rapid screening and identification of potentially relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral 3C-like protease.

Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2.

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC50 values ranging from 0.118 to 0.582 µM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC50 value of 7.249 µM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.

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 epidemic diarrhea virus 3CLpro causes apoptosis and collapse of mitochondrial membrane potential requiring its protease activity and signaling through MAVS.

Porcine epidemic diarrhea (PED) is a highly contagious and virulent intestinal infectious disease characterized by diarrhea, vomiting and dehydration. Although PEDV-induced apoptosis has been characterized in vitro and vivo, the functional proteins related to this event and the mechanism still need further research. Here, we firstly demonstrated that PEDV epidemic strain JS2013 could trigger apoptosis in a dose- and time-dependent manner. Then, PEDV 3CLpro was further identified as a crucial inducer of PEDV-triggered apoptosis. In addition, using site-directed mutagenesis to disrupt the protease activity of 3CLpro by His41 and Cys144 mutations, we found that 3CLpro-induced apoptosis and mitochondrial damage significantly reduced, suggesting that the protease activity of 3CLpro was essential for apoptosis and mitochondrial damage. Furthermore, PEDV 3CLpro could synergistically promote MAVS-mediated apoptosis and MAVS was involved in the signaling pathway of 3CLpro-induced apoptosis, but no direct interaction between PEDV 3CLpro and MAVS was detected by immunoprecipitation assays. Our findings provide important insights into the role of 3CLpro in the pathogenicity of PEDV.

Potential common mechanism of four Chinese patent medicines recommended by diagnosis and treatment protocol for COVID-19 in medical observation period.

The global epidemic has been controlled to some extent, while sporadic outbreaks still occur in some places. It is essential to summarize the successful experience and promote the development of new drugs. This study aimed to explore the common mechanism of action of the four Chinese patent medicine (CPMs) recommended in the Medical Observation Period COVID-19 Diagnostic and Treatment Protocol and to accelerate the new drug development process. Firstly, the active ingredients and targets of the four CPMs were obtained by the Chinese medicine composition database (TCMSP, TCMID) and related literature, and the common action targets of the four TCMs were sorted out. Secondly, the targets of COVID-19 were obtained through the gene-disease database (GeneCards, NCBI). Then the Venn diagram was used to intersect the common drug targets with the disease targets. And GO and KEGG pathway functional enrichment analysis was performed on the intersected targets with the help of the R package. Finally, the results were further validated by molecular docking and molecular dynamics analysis. As a result, a total of 101 common active ingredients and 21 key active ingredients of four CPMs were obtained, including quercetin, luteolin, acacetin, kaempferol, baicalein, naringenin, artemisinin, aloe-emodin, which might be medicinal substances for the treatment of COVID-19. TNF, IL6, IL1B, CXCL8, CCL2, IL2, IL4, ICAM1, IFNG, and IL10 has been predicted as key targets. 397 GO biological functions and 166 KEGG signaling pathways were obtained. The former was mainly enriched in regulating apoptosis, inflammatory response, and T cell activation. The latter, with 92 entries related to COVID-19, was mainly enriched to signaling pathways such as Coronavirus disease-COVID-19, Cytokine-cytokine receptor interaction, IL-17 signaling pathway, and Toll-like receptor signaling pathway. Molecular docking results showed that 19/21 of key active ingredients exhibited strong binding activity to recognized COVID-19-related targets (3CL of SARS-CoV-2, ACE2, and S protein), even better than one of these four antiviral drugs. Among them, shinflavanone had better affinity to 3CL, ACE2, and S protein of SARS-CoV-2 than these four antiviral drugs. In summary, the four CPMs may play a role in the treatment of COVID-19 by binding flavonoids such as quercetin, luteolin, and acacetin to target proteins such as ACE2, 3CLpro, and S protein and acting on TNF, IL6, IL1B, CXCL8, and other targets to participate in broad-spectrum antiviral, immunomodulatory and inflammatory responses.

Discovery and mechanism of action of Thonzonium bromide from an FDA-approved drug library with potent and broad-spectrum inhibitory activity against main proteases of human coronaviruses.

Although the effective drugs or vaccines have been developed to prevent the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), their efficacy may be limited for the viral evolution and immune escape. Thus, it is urgently needed to develop the novel broad-spectrum antiviral agents to control the coronavirus disease 2019 (COVID-19) global pandemic. The 3C-like protease (3CLpro) is a highly conserved cysteine proteinase that plays a pivotal role in processing the viral polyprotein to create non-structural proteins (nsps) for replication and transcription of SARS-CoV-2, making it an attractive antiviral target for developing broad-spectrum antiviral agents against SARS-CoV-2. In this study, we identified Thonzonium bromide as an inhibitor of SARS-CoV-2 3CLpro with an IC50 value of 2.04 ± 0.25 µM by fluorescence resonance energy transfer (FRET)-based enzymatic inhibition assay from the FDA-approved drug library. Next, we determined the inhibitory activity of Thonzonium bromide analogues against SARS-CoV-2 3CLpro and analyzed their structure-activity relationship (SAR). Interestingly, Thonzonium bromide showed better inhibitory activity than other analogues. Further fluorescence quenching assay, enzyme kinetics analysis, circular dichroism (CD) analysis and molecular docking studies showed that Thonzonium bromide inhibited SARS-CoV-2 3CLpro activity by firmly occupying the catalytic site and inducing conformational changes of the protease. In addition, Thonzonium bromide didn't exhibit inhibitory activity on human chymotrypsin C (CTRC) and Dipeptidyl peptidase IV (DPP-IV), indicating that it had a certain selectivity. Finally, we measured the inhibitory activities of Thonzonium bromide against 3CLpro of SARS-CoV, MERS-CoV and HCoV-229E and found that it had the broad-spectrum inhibitory activity against the proteases of human coronaviruses. These results provide the possible mechanism of action of Thonzonium bromide, highlighting its potential efficacy against multiple human coronaviruses.

Garcinia cambogia Phenolics as Potent Anti-COVID-19 Agents: Phytochemical Profiling, Biological Activities, and Molecular Docking.

COVID-19 is a disease caused by the coronavirus SARS-CoV-2 and became a pandemic in a critically short time. Phenolic secondary metabolites attracted much attention from the pharmaceutical industries for their easily accessible natural sources and proven antiviral activity. In our mission, a metabolomics study of the Garcinia cambogia Roxb. fruit rind was performed using LC-HRESIMS to investigate its chemical profile, especially the polar aspects, followed by a detailed phytochemical analysis, which led to the isolation of eight known compounds. Using spectrometric techniques, the isolated compounds were identified as quercetin, amentoflavone, vitexin, rutin, naringin, catechin, p-coumaric, and gallic acids. The antiviral activities of the isolated compounds were investigated using two assays; the 3CL-Mpro enzyme showed that naringin had a potent effect with IC50 16.62 µg/mL, followed by catechin and gallic acid (IC50 26.2, 30.35 µg/mL, respectively), while the direct antiviral inhibition effect of naringin confirmed the potency with an EC50 of 0.0169 µM. To show the molecular interaction, in situ molecular docking was carried out using a COVID-19 protease enzyme. Both biological effects and docking studies showed the hydrophobic interactions with Gln 189 or Glu 166, per the predicated binding pose of the isolated naringin.

Targeting non-structural proteins and 3CL(pro) in SARS-CoV-2 virus using phytochemicals from medicinal plants- In-silico approach

In the present study, the main protease 3CL(pro) and non-structural protein (NSP-12 with co-factors 7 and 8) trimer complex are used to study the protein-drug interactions with the phytochemicals from Ocimum Sanctum, Tinospora Cordifolia, Glycyrrhiza Glabra, and Azadirachta Indica. Which can give insight to be used as potent antiviral drugs against SARS-CoV-2. Twenty phytochemicals, five from each plant species, known for their wide range of biological activities were chosen from the literature. The in-silico study was carried out using virtual screening tools and the top five, which showed the least binding energies, were selected. Molecular docking tools revealed that gedunin and epoxy azadiradione proved to be excellent inhibitors for 3CL(pro) and so did Tinosporide for nonstructural-protein complex. Further, the best-hit phytochemicals with respect to structure similarities with FDA drugs and investigatory drugs, were considered for comparative study. Molecular docking was done to check the drug-protein interactions and to check the inhibitory responses of these drugs against the viral protein. The analyses showed that the phytochemicals had similar responses on the protein complex but with exceptionally higher inhibitory responses hence which may be taken for further clinical study.

Molecular docking and machine learning affinity prediction of compounds identified upon softwood bark extraction to the main protease of the SARS-CoV-2 virus.

Molecular docking of 234 unique compounds identified in the softwood bark (W set) is presented with a focus on their inhibition potential to the main protease of the SARS-CoV-2 virus 3CLpro (6WQF). The docking results are compared with the docking results of 866 COVID19-related compounds (S set). Furthermore, machine learning (ML) prediction of docking scores of the W set is presented using the S set trained TensorFlow, XGBoost, and SchNetPack ML approaches. Docking scores are evaluated with the Autodock 4.2.6 software. Four compounds in the W set achieve a docking score below -13 kcal/mol, with (+)-lariciresinol 9'-p-coumarate (CID 11497085) achieving the best docking score (-15 kcal/mol) within the W and S sets. In addition, 50% of W set docking scores are found below -8 kcal/mol and 25% below -10 kcal/mol. Therefore, the compounds identified in the softwood bark, show potential for antiviral activity upon extraction or further derivatization. The W set molecular docking studies are validated by means of molecular dynamics (five best compounds). The solubility (Log S, ESOL) and druglikeness of the best docking compounds in S and W sets are compared to evaluate the pharmacological potential of compounds identified in softwood bark.

Bioactive prenylated phenolic compounds from the aerial parts of Glycyrrhiza uralensis.

In this work, a bioassay-guided fractionation strategy was used to isolate 26 phenolic compounds from the ethyl acetate partition of an ethanol extract of the aerial parts of Glycyrrhiza uralensis Fisch. ex DC. Among them, 8 prenylated phenolic compounds (glycyuralins Q-X) were described for the first time. The two enantiomers of glycyuralin Q were purified and their absolute configurations were established by ECD spectral calculations. (1″R, 2″S)-glycyuralin Q and (1″S, 2″R)-glycyuralin Q showed significant inhibitory activities against SARS-CoV-2 virus proteases 3CLpro with IC50 values of 1.5 ± 1.0 and 4.0 ± 0.3 µM, and PLpro with IC50 values of 2.4 ± 0.2 and 1.9 ± 0.1 µM, respectively. Four compounds showed potent cytotoxic activities against A549, Huh-7, and HepG2 human cancer cells with IC50 values ranging from 0.5 to 2.5 µM.

Computational investigation of benzalacetophenone derivatives against SARS-CoV-2 as potential multi-target bioactive compounds.

Benzalacetophenones, precursors of flavonoids are aromatic ketones and enones and possess the immunostimulant as well as antiviral activities. Thus, benzalacetophenones were screened against the COVID-19 that could be lethal in patients with compromised immunity. We considered ChEBI recorded benzalacetophenone derivative(s) and evaluated their activity against 3C-like protease (3CLpro), papain-like protease (PLpro), and spike protein of SARS-Cov-2 to elucidate their possible role as antiviral agents. The probable targets for each compound were retrieved from DIGEP-Pred at 0.5 pharmacological activity and all the modulated proteins were enriched to identify the probably regulated pathways, biological processes, cellular components, and molecular functions. In addition, molecular docking was performed using AutoDock 4 and the best-identified hits were subjected to all-atom molecular dynamics simulation and binding energy calculations using molecular mechanics Poisson-Boltzmann surface area (MMPBSA). The compound 4-hydroxycordoin showed the highest druglikeness score and regulated nine proteins of which five were down-regulated and four were upregulated. Similarly, enrichment analysis identified the modulation of multiple pathways concerned with the immune system as well as pathways related to infectious and non-infectious diseases. Likewise, 3'-(3-methyl-2-butenyl)-4'-O-ß-d-glucopyranosyl-4,2'-dihydroxychalcone with 3CLpro, 4-hydroxycordoin with PLpro and mallotophilippen D with spike protein receptor-binding domain showed highest binding affinity, revealed stable interactions during the simulation, and scored binding free energy of -26.09 kcal/mol, -16.28 kcal/mol, and -39.2 kcal/mol, respectively. Predicted anti-SARS-CoV-2 activities of the benzalacetophenones reflected the requirement of wet lab studies to develop novel antiviral candidates.

Discovery of 9,10-dihydrophenanthrene derivatives as SARS-CoV-2 3CLpro inhibitors for treating COVID-19.

The epidemic coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread worldwide and efficacious therapeutics are urgently needed. 3-Chymotrypsin-like cysteine protease (3CLpro) is an indispensable protein in viral replication and represents an attractive drug target for fighting COVID-19. Herein, we report the discovery of 9,10-dihydrophenanthrene derivatives as non-peptidomimetic and non-covalent inhibitors of the SARS-CoV-2 3CLpro. The structure-activity relationships of 9,10-dihydrophenanthrenes as SARS-CoV-2 3CLpro inhibitors have carefully been investigated and discussed in this study. Among all tested 9,10-dihydrophenanthrene derivatives, C1 and C2 display the most potent SARS-CoV-2 3CLpro inhibition activity, with IC50 values of 1.55 ± 0.21 µM and 1.81 ± 0.17 µM, respectively. Further enzyme kinetics assays show that these two compounds dose-dependently inhibit SARS-CoV-2 3CLprovia a mixed-inhibition manner. Molecular docking simulations reveal the binding modes of C1 in the dimer interface and substrate-binding pocket of the target. In addition, C1 shows outstanding metabolic stability in the gastrointestinal tract, human plasma, and human liver microsome, suggesting that this agent has the potential to be developed as an orally administrated SARS-CoV-2 3CLpro inhibitor.

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

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

Inhibition of the SARS-CoV-2 3CLpro main protease by plant polyphenols.

The abundance of polyphenols in edible plants makes them an important component of human nutrition. Considering the ongoing COVID-19 pandemic, a number of studies have investigated polyphenols as bioactive constituents. We applied in-silico molecular docking as well as molecular dynamics supported by in-vitro assays to determine the inhibitory potential of various plant polyphenols against an important SARS-CoV-2 therapeutic target, the protease 3CLpro. Of the polyphenols in initial in-vitro screening, quercetin, ellagic acid, curcumin, epigallocatechin gallate and resveratrol showed IC50 values of 11.8 µM to 23.4 µM. In-silico molecular dynamics simulations indicated stable interactions with the 3CLpro active site over 100 ns production runs. Moreover, surface plasmon resonance spectroscopy was used to measure the binding of polyphenols to 3CLpro in real time. Therefore, we provide evidence for inhibition of SARS-CoV-2 3CLpro by natural plant polyphenols, and suggest further research into the development of these novel 3CLpro inhibitors or biochemical probes.

A Review of the Current Landscape of SARS-CoV-2 Main Protease Inhibitors: Have We Hit the Bullseye Yet?

In this review, we collected 1765 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M-pro inhibitors from the bibliography and other sources, such as the COVID Moonshot project and the ChEMBL database. This set of inhibitors includes only those compounds whose inhibitory capacity, mainly expressed as the half-maximal inhibitory concentration (IC50) value, against M-pro from SARS-CoV-2 has been determined. Several covalent warheads are used to treat covalent and non-covalent inhibitors separately. Chemical space, the variation of the IC50 inhibitory activity when measured by different methods or laboratories, and the influence of 1,4-dithiothreitol (DTT) are discussed. When available, we have collected the values of inhibition of viral replication measured with a cellular antiviral assay and expressed as half maximal effective concentration (EC50) values, and their possible relationship to inhibitory potency against M-pro is analyzed. Finally, the most potent covalent and non-covalent inhibitors that simultaneously inhibit the SARS-CoV-2 M-pro and the virus replication in vitro are discussed.

In silico screening of the active compounds of Syzygium cumini (L.) Skeels. as anti-coronavirus

Coronavirus disease-19 (Covid-19) has been pandemic since 2019 and the world is still trying to cope with it. Even though there is a new hope with the invention of the vaccine, the virus has rapid mutation rate. Therefore alternative solutions are necessary and one of them is using the herbs with their active compounds. Syzygium cumini (L.) Skeels. is a species of Myrtaceae containing various phytochemical compounds with medicinal activity, such as anti-oxidants, anti-inflammatory, anti-cancer, anti-tumour, anti-diabetes and anti-microbial. Previous studies showed that several compound contained in S. cumini had the potential of having anti-coronavirus activities. This study aimed to determine the phytochemical compounds of S. cumini and to screen their potential as an anti-coronavirus. The method used in this research were literature study and molecular docking. The results showed that S. cumini contained the active compounds of anti-coronavirus, namely betulinic acid, kaempferol, malvidin, myricetin and quercetin. Those compounds are contained in the bark of S. cumini.