Seleno-Functionalization of Quercetin Improves the Non-Covalent Inhibition of Mpro and Its Antiviral Activity in Cells against SARS-CoV-2

The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (M^pro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent M^pro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC₅₀ of 192 μM and 8 μM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for M^pro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher M^pro activity of 2d and, as a result, its better antiviral profile.     

Action of Dipeptidyl Peptidase-4 Inhibitors on SARS-CoV-2 Main Protease

In a recent publication, Eleftheriou et al. proposed that inhibitors of dipeptidyl peptidase-4 (DPP-4) are functional inhibitors of the main protease (M^pro) of SARS-CoV-2. Their predictions prompted the authors to suggest linagliptin, a DPP-4 inhibitor and approved anti-diabetes drug, as a repurposed drug candidate against the ongoing COVID-19 pandemic. We used an enzymatic assay measuring the inhibition of M^pro catalytic activity in the presence of four different commercially available gliptins (linagliptin, sitagliptin, alogliptin and saxagliptin) and several structural analogues of linagliptin to study the binding of DPP-4 inhibitors to M^pro and their functional activity. We show here that DPP-4 inhibitors like linagliptin, other gliptins and structural analogues are inactive against M^pro.     

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease

As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain-like protease or PL^Pro. It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS-CoV-2 infection. To repurpose drugs for PL^Pro, we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PL^Pro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PL^Pro at 200 μM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC₅₀ value below 40 μM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC₅₀ value below 10 μM. Among four inhibitors with an IC₅₀ value below 10 μM, SJB2-043 is the most unique in that it does not fully inhibit PL^Pro but has a noteworthy IC₅₀ value of 0.56 μM. SJB2-043 likely binds to an allosteric site of PL^Pro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PL^Pro holds promise, but in-depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.     

Structure-Activity Relationships of Benzamides and Isoindolines Designed as SARS-CoV Protease Inhibitors Effective against SARS-CoV-2

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PL^pro) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide ( 2 b ), which is known to bind into the S3 and S4 pockets of the SARS-CoV PL^pro. Moreover, we report the discovery of isoindolines as a new class of potent PL^pro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PL^pro are valuable starting points for the development of new pan-coronaviral inhibitors.     

In silico allicin induced S-thioallylation of SARS-CoV-2 main protease

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused due to new coronavirus infection with ³716075 deaths across the world as reported by the World Health Organization (WHO). SARS-CoV-2 main protease (M^pro) plays a vital role in the replication of coronavirus and thus an attractive target for the screening of inhibitors for the therapy of COVID-19. The preclinical drugs ebselen and PX-12 are potent inhibitors of SARS-CoV-2 M^pro and covalently modifies the active site Cys-145 residue of M^pro through selenosulfide/disulfide. In the current report, using virtual screening methods, reactive sulfur species allicin is subjecting for covalent docking at the active site of SARS-CoV-2 M^pro using PX-12 as a benchmark reference compound. The results indicate that allicin induces dual S-thioallylation of Cys-145 and Cys-85/ Cys-156 residues of SARS-CoV-2 M^pro. Using density functional theory (DFT), Gibbs free energy change (DG) is calculated for the putative reactions between N-acetylcysteine amide thiol and allicin/allyl sulfenic acid. The overall reaction is exergonic and allyl disulfide of Cys-145 residue of M^pro is involved in a sulfur mediated hydrogen bond. The results indicate that allicin causes dual S-thioallylation of SARS-CoV-2 M^pro which may be of interest for treatment and attenuation of ongoing coronavirus infection.     

A Multistage In Silico Study of Natural Potential Inhibitors Targeting SARS-CoV-2 Main Protease

Among a group of 310 natural antiviral natural metabolites, our team identified three compounds as the most potent natural inhibitors against the SARS-CoV-2 main protease (PDB ID: 5R84), M^pro. The identified compounds are sattazolin and caprolactin A and B. A validated multistage in silico study was conducted using several techniques. First, the molecular structures of the selected metabolites were compared with that of GWS, the co-crystallized ligand of M^pro, in a structural similarity study. The aim of this study was to determine the thirty most similar metabolites (10%) that may bind to the M^pro similar to GWS. Then, molecular docking against M^pro and pharmacophore studies led to the choice of five metabolites that exhibited good binding modes against the M^pro and good fit values against the generated pharmacophore model. Among them, three metabolites were chosen according to ADMET studies. The most promising M^pro inhibitor was determined by toxicity and DFT studies to be caprolactin A (292). Finally, molecular dynamics (MD) simulation studies were performed for caprolactin A to confirm the obtained results and understand the thermodynamic characteristics of the binding. It is hoped that the accomplished results could represent a positive step in the battle against COVID-19 through further in vitro and in vivo studies on the selected compounds.     

In Silico Characterization of Masitinib Interaction with SARS-CoV-2 Main Protease

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a global health problem. Despite the current implementation of COVID-19 vaccination schedules, identifying effective antiviral drug treatments for this disease continues to be a priority. A recent study showed that masitinib (MST), a tyrosine kinase inhibitor, blocks the proteolytic activity of SARS-CoV-2 main protease (M^pro). Although MST is a potential candidate for COVID-19 treatment, a comprehensive analysis of its interaction with M^pro has not been done. In this work, we performed molecular dynamics simulations of the MST-M^pro complex crystal structure. The effect of the protonation states of M^pro H163 residue and MST titratable groups were studied. Furthermore, we identified the MST substituents and M^pro mutations that affect the stability of the complex. Our results provide valuable insights into the design of new MST analogs as potential treatments for COVID-19.     

In Silico Drug Repositioning to Target the SARS-CoV-2 Main Protease as Covalent Inhibitors Employing a Combined Structure-Based Virtual Screening Strategy of Pharmacophore Models and Covalent Docking

The epidemic caused by the SARS-CoV-2 coronavirus, which has spread rapidly throughout the world, requires urgent and effective treatments considering that the appearance of viral variants limits the efficacy of vaccines. The main protease of SARS-CoV-2 (M^pro) is a highly conserved cysteine proteinase, fundamental for the replication of the coronavirus and with a specific cleavage mechanism that positions it as an attractive therapeutic target for the proposal of irreversible inhibitors. A structure-based strategy combining 3D pharmacophoric modeling, virtual screening, and covalent docking was employed to identify the interactions required for molecular recognition, as well as the spatial orientation of the electrophilic warhead, of various drugs, to achieve a covalent interaction with Cys145 of M^pro. The virtual screening on the structure-based pharmacophoric map of the SARS-CoV-2 M^pro in complex with an inhibitor N3 (reference compound) provided high efficiency by identifying 53 drugs (FDA and DrugBank databases) with probabilities of covalent binding, including N3 (Michael acceptor) and others with a variety of electrophilic warheads. Adding the energy contributions of affinity for non-covalent and covalent docking, 16 promising drugs were obtained. Our findings suggest that the FDA-approved drugs Vaborbactam, Cimetidine, Ixazomib, Scopolamine, and Bicalutamide, as well as the other investigational peptide-like drugs (DB04234, DB03456, DB07224, DB7252, and CMX-2043) are potential covalent inhibitors of SARS-CoV-2 M^pro.     

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors**

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M^Pro) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M^Pro), we have designed and synthesized a series of SC2M^Pro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M^Pro active-site cysteine C145. All inhibitors display high potency with K_i values at or below 100 nM. The most potent compound, MPI3, has as a K_i value of 8.3 nM. Crystallographic analyses of SC2M^Pro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5–5 μM and A549/ACE2 cells at 0.16–0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M^Pro inhibitors with ultra-high antiviral potency.     

Structure-Activity Studies Reveal Scope for Optimisation of Ebselen-Type Inhibition of SARS-CoV-2 Main Protease

The reactive organoselenium compound ebselen is being investigated for treatment of coronavirus disease 2019 (COVID-19) and other diseases. We report structure-activity studies on sulfur analogues of ebselen with the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro), employing turnover and protein-observed mass spectrometry-based assays. The results reveal scope for optimisation of ebselen/ebselen derivative- mediated inhibition of M^pro, particularly with respect to improved selectivity.     

Computational Selectivity Assessment of Protease Inhibitors against SARS-CoV-2

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious global health threat. Since no specific therapeutics are available, researchers around the world screened compounds to inhibit various molecular targets of SARS-CoV-2 including its main protease (M^pro) essential for viral replication. Due to the high urgency of these discovery efforts, off-target binding, which is one of the major reasons for drug-induced toxicity and safety-related drug attrition, was neglected. Here, we used molecular docking, toxicity profiling, and multiple molecular dynamics (MD) protocols to assess the selectivity of 33 reported non-covalent inhibitors of SARS-CoV-2 M^pro against eight proteases and 16 anti-targets. The panel of proteases included SARS-CoV M^pro, cathepsin G, caspase-3, ubiquitin carboxy-terminal hydrolase L1 (UCHL1), thrombin, factor Xa, chymase, and prostasin. Several of the assessed compounds presented considerable off-target binding towards the panel of proteases, as well as the selected anti-targets. Our results further suggest a high risk of off-target binding to chymase and cathepsin G. Thus, in future discovery projects, experimental selectivity assessment should be directed toward these proteases. A systematic selectivity assessment of SARS-CoV-2 M^pro inhibitors, as we report it, was not previously conducted.     

A Structural Comparison of SARS-CoV-2 Main Protease and Animal Coronaviral Main Protease Reveals Species-Specific Ligand Binding and Dimerization Mechanism

Animal coronaviruses (CoVs) have been identified to be the origin of Severe Acute Respiratory Syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and probably SARS-CoV-2 that cause severe to fatal diseases in humans. Variations of zoonotic coronaviruses pose potential threats to global human beings. To overcome this problem, we focused on the main protease (M^pro), which is an evolutionary conserved viral protein among different coronaviruses. The broad-spectrum anti-coronaviral drug, GC376, was repurposed to target canine coronavirus (CCoV), which causes gastrointestinal infections in dogs. We found that GC376 can efficiently block the protease activity of CCoV M^pro and can thermodynamically stabilize its folding. The structure of CCoV M^pro in complex with GC376 was subsequently determined at 2.75 Å. GC376 reacts with the catalytic residue C144 of CCoV M^pro and forms an (R)- or (S)-configuration of hemithioacetal. A structural comparison of CCoV M^pro and other animal CoV M^pros with SARS-CoV-2 M^pro revealed three important structural determinants in a substrate-binding pocket that dictate entry and release of substrates. As compared with the conserved A141 of the S1 site and P188 of the S4 site in animal coronaviral M^pros, SARS-CoV-2 M^pro contains N142 and Q189 at equivalent positions which are considered to be more catalytically compatible. Furthermore, the conserved loop with residues 46–49 in animal coronaviral M^pros has been replaced by a stable α-helix in SARS-CoV-2 M^pro. In addition, the species-specific dimerization interface also influences the catalytic efficiency of CoV M^pros. Conclusively, the structural information of this study provides mechanistic insights into the ligand binding and dimerization of CoV M^pros among different species.     

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

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

The Se–S Bond Formation in the Covalent Inhibition Mechanism of SARS-CoV-2 Main Protease by Ebselen-like Inhibitors: A Computational Study

The inhibition mechanism of the main protease (M^pro) of SARS-CoV-2 by ebselen (EBS) and its analog with a hydroxyl group at position 2 of the benzisoselenazol-3(2H)-one ring (EBS-OH) was studied by using a density functional level of theory. Preliminary molecular dynamics simulations on the apo form of M^pro were performed taking into account both the hydrogen donor and acceptor natures of the Nδ and Nε of His41, a member of the catalytic dyad. The potential energy surfaces for the formation of the Se–S covalent bond mediated by EBS and EBS-OH on M^pro are discussed in detail. The EBS-OH shows a distinctive behavior with respect to EBS in the formation of the noncovalent complex. Due to the presence of canonical H-bonds and noncanonical ones involving less electronegative atoms, such as sulfur and selenium, the influence on the energy barriers and reaction energy of the Minnesota hybrid meta-GGA functionals M06, M06-2X and M08HX, and the more recent range-separated hybrid functional wB97X were also considered. The knowledge of the inhibition mechanism of M^pro by the small protease inhibitors EBS or EBS-OH can enlarge the possibilities for designing more potent and selective inhibitor-based drugs to be used in combination with other antiviral therapies.     

Peptide Derivatives of the Zonulin Inhibitor Larazotide (AT1001) as Potential Anti SARS-CoV-2: Molecular Modelling,Synthesis and Bioactivity Evaluation

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the pathogen responsible for the outbreak of a severe, rapidly developing pneumonia (Coronavirus disease 2019, COVID-19). The virus enzyme, called 3CLpro or main protease (M^pro), is essential for viral replication, making it a most promising target for antiviral drug development. Recently, we adopted the drug repurposing as appropriate strategy to give fast response to global COVID-19 epidemic, by demonstrating that the zonulin octapeptide inhibitor AT1001 (Larazotide acetate) binds M^pro catalytic domain. Thus, in the present study we tried to investigate the antiviral activity of AT1001, along with five derivatives, by cell-based assays. Our results provide with the identification of AT1001 peptide molecular framework for lead optimization step to develop new generations of antiviral agents of SARS-CoV-2 with an improved biological activity, expanding the chance for success in clinical trials.     

Multi-Step In Silico Discovery of Natural Drugs against COVID-19 Targeting Main Protease

In continuation of our antecedent work against COVID-19, three natural compounds, namely, Luteoside C (130), Kahalalide E (184), and Streptovaricin B (278) were determined as the most promising SARS-CoV-2 main protease (M^pro) inhibitors among 310 naturally originated antiviral compounds. This was performed via a multi-step in silico method. At first, a molecular structure similarity study was done with PRD_002214, the co-crystallized ligand of M^pro (PDB ID: 6LU7), and favored thirty compounds. Subsequently, the fingerprint study performed with respect to PRD_002214 resulted in the election of sixteen compounds (7, 128, 130, 156, 157, 158, 180, 184, 203, 204, 210, 237, 264, 276, 277, and 278). Then, results of molecular docking versus M^pro PDB ID: 6LU7 favored eight compounds (128, 130, 156, 180, 184, 203, 204, and 278) based on their binding affinities. Then, in silico toxicity studies were performed for the promising compounds and revealed that all of them have good toxicity profiles. Finally, molecular dynamic (MD) simulation experiments were carried out for compounds 130, 184, and 278, which exhibited the best binding modes against M^pro. MD tests revealed that luteoside C (130) has the greatest potential to inhibit SARS-CoV-2 main protease.     

Discovery of SARS-CoV-2 3CLPro Peptidomimetic Inhibitors through the Catalytic Dyad Histidine-Specific Protein–Ligand Interactions

As the etiological agent for the coronavirus disease 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenges the ongoing efforts of vaccine development and drug design. Due to the accumulating cases of breakthrough infections, there are urgent needs for broad-spectrum antiviral medicines. Here, we designed and examined five new tetrapeptidomimetic anti-SARS-CoV-2 inhibitors targeting the 3C-Like protease (3CL^Pro), which is highly conserved among coronaviruses and essential for viral replications. We significantly improved the efficacy of a ketoamide lead compound based on high-resolution co-crystal structures, all-atom simulations, and binding energy calculations. The inhibitors successfully engaged the catalytic dyad histidine residue (H41) of 3CLPro as designed, and they exhibited nanomolar inhibitory capacity as well as mitigated the viral loads of SARS-CoV-2 in cellular assays. As a widely applicable design principle, our results revealed that the potencies of 3CL^Pro-specific drug candidates were determined by the interplay between 3CL^Pro H41 residue and the peptidomimetic inhibitors.     

Development of Effective Therapeutic Molecule from Natural Sources against Coronavirus Protease

The SARS-CoV-2 main protease (M^pro) is one of the molecular targets for drug design. Effective vaccines have been identified as a long-term solution but the rate at which they are being administered is slow in several countries, and mutations of SARS-CoV-2 could render them less effective. Moreover, remdesivir seems to work only with some types of COVID-19 patients. Hence, the continuous investigation of new treatments for this disease is pivotal. This study investigated the inhibitory role of natural products against SARS-CoV-2 M^pro as repurposable agents in the treatment of coronavirus disease 2019 (COVID-19). Through in silico approach, selected flavonoids were docked into the active site of M^pro. The free energies of the ligands complexed with M^pro were computationally estimated using the molecular mechanics-generalized Born surface area (MM/GBSA) method. In addition, the inhibition process of SARS-CoV-2 Mpro with these ligands was simulated at 100 ns in order to uncover the dynamic behavior and complex stability. The docking results showed that the selected flavonoids exhibited good poses in the binding domain of M^pro. The amino acid residues involved in the binding of the selected ligands correlated well with the residues involved with the mechanism-based inhibitor (N3) and the docking score of Quercetin-3-O-Neohesperidoside (−16.8 Kcal/mol) ranked efficiently with this inhibitor (−16.5 Kcal/mol). In addition, single-structure MM/GBSA rescoring method showed that Quercetin-3-O-Neohesperidoside (−87.60 Kcal/mol) is more energetically favored than N3 (−80.88 Kcal/mol) and other ligands (Myricetin 3-Rutinoside (−87.50 Kcal/mol), Quercetin 3-Rhamnoside (−80.17 Kcal/mol), Rutin (−58.98 Kcal/mol), and Myricitrin (−49.22 Kcal/mol). The molecular dynamics simulation (MDs) pinpointed the stability of these complexes over the course of 100 ns with reduced RMSD and RMSF. Based on the docking results and energy calculation, together with the RMSD of 1.98 ± 0.19 Å and RMSF of 1.00 ± 0.51 Å, Quercetin-3-O-Neohesperidoside is a better inhibitor of M^pro compared to N3 and other selected ligands and can be repurposed as a drug candidate for the treatment of COVID-19. In addition, this study demonstrated that in silico docking, free energy calculations, and MDs, respectively, are applicable to estimating the interaction, energetics, and dynamic behavior of molecular targets by natural products and can be used to direct the development of novel target function modulators.