Exploring the Binding Mechanism of PF-07321332 SARS-CoV-2 Protease Inhibitor through Molecular Dynamics and Binding Free Energy Simulations

The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CL^pro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CL^pro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CL^pro–PF-07321332 and 3CL^pro–α-ketoamide complexes remained stable compared with 3CL^pro–ritonavir and 3CL^pro–lopinavir. Investigating the dynamic behavior of ligand–protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41–Cys145 of 3CL^pro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CL^pro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.     

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.     

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.     

De novo ssRNA Aptamers against the SARS-CoV-2 Main Protease: In Silico Design and Molecular Dynamics Simulation

Herein, we have generated ssRNA aptamers to inhibit SARS-CoV-2 M^pro, a protease necessary for the SARS-CoV-2 coronavirus replication. Because there is no aptamer 3D structure currently available in the databanks for this protein, first, we modeled an ssRNA aptamer using an entropic fragment-based strategy. We refined the initial sequence and 3D structure by using two sequential approaches, consisting of an elitist genetic algorithm and an RNA inverse process. We identified three specific aptamers against SARS-CoV-2 M^pro, called MApta^pro, MApta^pro-IR1, and MApta^pro-IR2, with similar 3D conformations and that fall in the dimerization region of the SARS-CoV-2 M^pro necessary for the enzymatic activity. Through the molecular dynamic simulation and binding free energy calculation, the interaction between the MApta^pro-IR1 aptamer and the SARS-CoV-2 M^pro enzyme resulted in the strongest and the highest stable complex; therefore, the ssRNA MApta^pro-IR1 aptamer was selected as the best potential candidate for the inhibition of SARS-CoV-2 M^pro and a perspective therapeutic drug for the COVID-19 disease.     

Human Superantibodies to 3CLpro Inhibit Replication of SARS-CoV-2 across Variants

Broadly effective and safe anti-coronavirus agent is existentially needed. Major protease (3CL^pro) is a highly conserved enzyme of betacoronaviruses. The enzyme plays pivotal role in the virus replication cycle. Thus, it is a good target of a broadly effective anti-Betacoronavirus agent. In this study, human single-chain antibodies (HuscFvs) of the SARS-CoV-2 3CL^pro were generated using phage display technology. The 3CL^pro-bound phages were used to infect Escherichia coli host for the production the 3CL^pro-bound HuscFvs. Computerized simulation was used to guide the selection of the phage infected-E. coli clones that produced HuscFvs with the 3CL^pro inhibitory potential. HuscFvs of three phage infected-E. coli clones were predicted to form contact interface with residues for 3CL^pro catalytic activity, substrate binding, and homodimerization. These HuscFvs were linked to a cell-penetrating peptide to make them cell-penetrable, i.e., became superantibodies. The superantibodies blocked the 3CL^pro activity in vitro, were not toxic to human cells, traversed across membrane of 3CL^pro-expressing cells to co-localize with the intracellular 3CL^pro and most of all, they inhibited replication of authentic SARS-CoV-2 Wuhan wild type and α, β, δ, and Omicron variants that were tested. The superantibodies should be investigated further towards clinical application as a safe and broadly effective anti-Betacoronavirus agent.     

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.     

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.     

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.     

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.     

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.     

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors**

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly “druggable” or relatively easy-to-drug target is the coronavirus Main Protease (3CL^pro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.     

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.     

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.     

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.     

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.     

Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS-CoV-2 Papain-Like Protease

The two SARS-CoV-2 proteases, i. e. the main protease (M^pro) and the papain-like protease (PL^pro), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL^pro catalysis in vitro. The assay was applied to investigate the effect of reported small-molecule PL^pro inhibitors and selected M^pro inhibitors on PL^pro catalysis. The results reveal that some, but not all, PL^pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M^pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL^pro. Less selective M^pro inhibitors, e. g. auranofin, inhibit PL^pro, highlighting the potential for dual PL^pro/M^pro inhibition. MS-based PL^pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.     

Identification of Entry Inhibitors against Delta and Omicron Variants of SARS-CoV-2

Entry inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to control the outbreak of coronavirus disease 2019 (COVID-19). This study developed a robust and straightforward assay that detected the molecular interaction between the receptor-binding domain (RBD) of viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor in just 10 min. A drug library of 1068 approved compounds was used to screen for SARS-CoV2 entry inhibition, and 9 active drugs were identified as specific pseudovirus entry inhibitors. A plaque reduction neutralization test using authentic SARS-CoV-2 virus in Vero E6 cells confirmed that 2 of these drugs (Etravirine and Dolutegravir) significantly inhibited the infection of SARS-CoV-2. With molecular docking, we showed that both Etravirine and Dolutegravir are preferentially bound to primary ACE2-interacting residues on the RBD domain, implying that these two drug blocks may prohibit the viral attachment of SARS-CoV-2. We compared the neutralizing activities of these entry inhibitors against different pseudoviruses carrying spike proteins from alpha, beta, gamma, and delta variants. Both Etravirine and Dolutegravir showed similar neutralizing activities against different variants, with EC50 values between 4.5 to 5.8 nM for Etravirine and 10.2 to 22.9 nM for Dolutegravir. These data implied that Etravirine and Dolutegravir may serve as general spike inhibitors against dominant viral variants of SARS-CoV-2.     

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.