Multi-ligand molecular docking, simulation, free energy calculations and wavelet analysis of the synergistic effects between natural compounds baicalein and cubebin for the inhibition of the main protease of SARS-CoV-2.

Combination drugs have been used for several diseases for many years since they produce better therapeutic effects. However, it is still a challenge to discover candidates to form a combination drug. This study aimed to investigate whether using a comprehensive in silico approach to identify novel combination drugs from a Chinese herbal formula is an appropriate and creative strategy. We, therefore, used Toujie Quwen Granules for the main protease (Mpro) of SARS-CoV-2 as an example. We first used molecular docking to identify molecular components of the formula which may inhibit Mpro. Baicalein (HQA004) is the most favorable inhibitory ligand. We also identified a ligand from the other component, cubebin (CHA008), which may act to support the proposed HQA004 inhibitor. Molecular dynamics simulations were then performed to further elucidate the possible mechanism of inhibition by HQA004 and synergistic bioactivity conferred by CHA008. HQA004 bound strongly at the active site and that CHA008 enhanced the contacts between HQA004 and Mpro. However, CHA008 also dynamically interacted at multiple sites, and continued to enhance the stability of HQA004 despite diffusion to a distant site. We proposed that HQA004 acted as a possible inhibitor, and CHA008 served to enhance its effects via allosteric effects at two sites. Additionally, our novel wavelet analysis showed that as a result of CHA008 binding, the dynamics and structure of Mpro were observed to have more subtle changes, demonstrating that the inter-residue contacts within Mpro were disrupted by the synergistic ligand. This work highlighted the molecular mechanism of synergistic effects between different herbs as a result of allosteric crosstalk between two ligands at a protein target, as well as revealed that using the multi-ligand molecular docking, simulation, free energy calculations and wavelet analysis to discover novel combination drugs from a Chinese herbal remedy is an innovative pathway.

Glycyrrhizin as a promising kryptonite against SARS-CoV-2: Clinical, experimental, and theoretical evidences.

COVID-19 is the most devastating disease in recent times affecting most people globally. The higher rate of transmissibility and mutations of SARS-CoV-2 along with the lack of potential therapeutics has made it a global crisis. Potential molecules from natural sources could be a fruitful remedy to combat COVID-19. This systematic review highlights the detailed therapeutic implication of naturally occurring glycyrrhizin and its related derivatives against COVID-19. Glycyrrhizin has already been established for blocking different biomolecular targets related to the SARS-CoV-2 replication cycle. In this article, several experimental and theoretical evidences of glycyrrhizin and related derivatives have been discussed in detail to evaluate their potential as a promising therapeutic strategy against COVID-19. Moreover, the implication of glycyrrhizin in traditional Chinese medicines for alleviating the symptoms of COVID-19 has been reviewed. The potential role of glycyrrhizin and related compounds in affecting various stages of the SARS-CoV-2 life cycle has also been discussed in detail. Derivatization of glycyrrhizin for designing potential lead compounds along with combination therapy with other anti-SARS-CoV-2 agents followed by extensive evaluation may assist in the formulation of novel anti-coronaviral therapy for better treatment to combat COVID-19.

Identification of alkaloids from Justicia adhatoda as potent SARS CoV-2 main protease inhibitors: An in silico perspective.

The COVID-19 pandemic, caused by SARS CoV-2, is responsible for millions of death worldwide. No approved/proper therapeutics is currently available which can effectively combat this outbreak. Several attempts have been undertaken in the search of effective drugs to control the spread of SARS CoV-2 infection. The main protease (Mpro), key component for the cleavage of the viral polyprotein, is considered to be one of the important drug targets for treating COVID-19. Various phytochemicals, including polyphenols and alkaloids, have been proposed as potent inhibitors of Mpro. The alkaloids from leaf extracts of Justicia adhatoda have also been reported to possess anti-viral activity. But whether these alkaloids exhibit any inhibitory effect on SARS CoV-2 Mpro is far from clear. To explore this in detail, we have adopted computational approaches. Justicia adhatoda alkaloids possessing proper drug-likeness properties and two anti-HIV drugs (lopinavir and darunavir; having binding affinity -7.3 to -7.4 kcal/mol) were docked against SARS CoV-2 Mpro to study their binding properties. Only one alkaloid (anisotine) had interaction with both the catalytic residues (His41 and Cys145) of Mpro and exhibited good binding affinity (-7.9 kcal/mol). Molecular dynamic simulations (100 ns) revealed that Mpro-anisotine complex is more stable, conformationally less fluctuated; slightly less compact and marginally expanded than Mpro-darunavir/lopinavir complex. Even the number of intermolecular H-bonds and MM-GBSA analysis suggested that anisotine is a more potent Mpro inhibitor than the two previously recommended antiviral drugs (lopinavir and darunavir) and may evolve as a promising anti-COVID-19 drug if proven in animal experiments and on patients.

Benchmarked molecular docking integrated molecular dynamics stability analysis for prediction of SARS-CoV-2 papain-like protease inhibition by olive secoiridoids.

Objectives: We performed a virtual screening of olive secoiridoids of the OliveNetTM library to predict SARS-CoV-2 PLpro inhibition. Benchmarked molecular docking protocol that evaluated the performance of two docking programs was applied to execute virtual screening. Molecular dynamics stability analysis of the top-ranked olive secoiridoid docked to PLpro was also carried out. Methods: Benchmarking virtual screening used two freely available docking programs, AutoDock Vina 1.1.2. and AutoDock 4.2.1. for molecular docking of olive secoiridoids to a single PLpro structure. Screening also included benchmark structures of known active and decoy molecules from the DEKOIS 2.0 library. Based on the predicted binding energies, the docking programs ranked the screened molecules. We applied the usual performance evaluation metrices to evaluate the docking programs using the predicted ranks. Molecular dynamics of the top-ranked olive secoiridoid bound to PLpro and computation of MM-GBSA energy using three iterations during the last 50 ps of the analysis of the dynamics in Desmond supported the stability prediction. Results and discussions: Predictiveness curves suggested that AutoDock Vina has a better predictive ability than AutoDock, although there was a moderate correlation between the active molecules rankings (Kendall's correlation of rank (τ) = 0.581). Interestingly, two same molecules, Demethyloleuropein aglycone, and Oleuroside enriched the top 1 % ranked olive secoiridoids predicted by both programs. Demethyloleuropein aglycone bound to PLpro obtained by docking in AutoDock Vina when analyzed for stability by molecular dynamics simulation for 50 ns displayed an RMSD, RMSF<2 Å, and MM-GBSA energy of -94.54 ± 6.05 kcal/mol indicating good stability. Molecular dynamics also revealed the interactions of Demethyloleuropein aglycone with binding sites 2 and 3 of PLpro, suggesting a potent inhibition. In addition, for 98 % of the simulation time, two phenolic hydroxy groups of Demethyloleuropein aglycone maintained two hydrogen bonds with Asp302 of PLpro, specifying the significance of the groups in receptor binding. Conclusion: AutoDock Vina retrieved the active molecules accurately and predicted Demethyloleuropein aglycone as the best inhibitor of PLpro. The Arabian diet consisting of olive products rich in secoiridoids benefits from the PLpro inhibition property and reduces the risk of viral infection.

Pharmacophore model-aided virtual screening combined with comparative molecular docking and molecular dynamics for identification of marine natural products as SARS-CoV-2 papain-like protease inhibitors.

Targeting SARS-CoV-2 papain-like protease using inhibitors is a suitable approach for inhibition of virus replication and dysregulation of host anti-viral immunity. Engaging all five binding sites far from the catalytic site of PLpro is essential for developing a potent inhibitor. We developed and validated a structure-based pharmacophore model with 9 features of a potent PLpro inhibitor. The pharmacophore model-aided virtual screening of the comprehensive marine natural product database predicted 66 initial hits. This hit library was downsized by filtration through a molecular weight filter of ≤ 500 g/mol. The 50 resultant hits were screened by comparative molecular docking using AutoDock and AutoDock Vina. Comparative molecular docking enables benchmarking docking and relieves the disparities in the search and scoring functions of docking engines. Both docking engines retrieved 3 same compounds at different positions in the top 1 % rank, hence consensus scoring was applied, through which CMNPD28766, aspergillipeptide F emerged as the best PLpro inhibitor. Aspergillipeptide F topped the 50-hit library with a pharmacophore-fit score of 75.916. Favorable binding interactions were predicted between aspergillipeptide F and PLpro similar to the native ligand XR8-24. Aspergillipeptide F was able to engage all the 5 binding sites including the newly discovered BL2 groove, site V. Molecular dynamics for quantification of Cα-atom movements of PLpro after ligand binding indicated that it exhibits highly correlated domain movements contributing to the low free energy of binding and a stable conformation. Thus, aspergillipeptide F is a promising candidate for pharmaceutical and clinical development as a potent SARS-CoV-2 PLpro inhibitor.

Elucidating the enhanced binding affinity of a double mutant SP-D with trimannose on the influenza A virus using molecular dynamics.

Surfactant protein D (SP-D) is an essential component of the human pulmonary surfactant system, which is crucial in the innate immune response against glycan-containing pathogens, including Influenza A viruses (IAV) and SARS-CoV-2. Previous studies have shown that wild-type (WT) SP-D can bind IAV but exhibits poor antiviral activities. However, a double mutant (DM) SP-D consisting of two point mutations (Asp325Ala and Arg343Val) inhibits IAV more potently. Presently, the structural mechanisms behind the point mutations' effects on SP-D's binding affinity with viral surface glycans are not fully understood. Here we use microsecond-scale, full-atomistic molecular dynamics (MD) simulations to understand the molecular mechanism of mutation-induced SP-D's higher antiviral activity. We find that the Asp325Ala mutation promotes a trimannose conformational change to a more stable state. Arg343Val increases the binding with trimannose by increasing the hydrogen bonding interaction with Glu333. Free energy perturbation (FEP) binding free energy calculations indicate that the Arg343Val mutation contributes more to the increase of SP-D's binding affinity with trimannose than Asp325Ala. This study provides a molecular-level exploration of how the two mutations increase SP-D binding affinity with trimannose, which is vital for further developing preventative strategies for related diseases.

Unsymmetrical aromatic disulfides as SARS-CoV-2 Mpro inhibitors: Molecular docking, molecular dynamics, and ADME scoring investigations.

COVID-19 pandemic caused by very severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) agent is an ongoing major global health concern. The disease has caused more than 452 million affected cases and more than 6 million death worldwide. Hence, there is an urgency to search for possible medications and drug treatments. There are no approved drugs available to treat COVID-19 yet, although several vaccine candidates are already available and some of them are listed for emergency use by the world health organization (WHO). Identifying a potential drug candidate may make a significant contribution to control the expansion of COVID-19. The in vitro biological activity of asymmetric disulfides against coronavirus through the inhibition of SARS-CoV-2 main protease (Mpro) protein was reported. Due to the lack of convincing evidence those asymmetric disulfides have favorable pharmacological properties for the clinical treatment of Coronavirus, in silico evaluation should be performed to assess the potential of these compounds to inhibit the SARS-CoV-2 Mpro. In this context, we report herein the molecular docking for a series of 40 unsymmetrical aromatic disulfides as SARS-CoV-2 Mpro inhibitor. The optimal binding features of disulfides within the binding pocket of SARS-CoV-2 endoribonuclease protein (Protein Data Bank [PDB]: 6LU7) was described. Studied compounds were ranked for potential effectiveness, and those have shown high molecular docking scores were proposed as novel drug candidates against SARS-CoV-2. Moreover, the outcomes of drug similarity and ADME (Absorption, Distribution, Metabolism, and Excretion) analyses have may have the effectiveness of acting as medicines, and would be of interest as promising starting point for designing compounds against SARS-CoV-2. Finally, the stability of these three compounds in the complex with Mpro was validated through molecular dynamics (MD) simulation, in which they displayed stable trajectory and molecular properties with a consistent interaction profile.

Nature's therapy for COVID-19: Targeting the vital non-structural proteins (NSP) from SARS-CoV-2 with phytochemicals from Indian medicinal plants.

Background: Containing COVID-19 is still a global challenge. It has affected the "normal" world by targeting its economy and health sector. The effect is shifting of focus of research from life threatening diseases like cancer. Thus, we need to develop a medical solution at the earliest. The purpose of this present work was to understand the efficacy of 22 rationally screened phytochemicals from Indian medicinal plants obtained from our previous work, following drug-likeness properties, against 6 non-structural-proteins (NSP) from SARS-CoV-2. Methods: 100 ns molecular dynamics simulations were performed, and relative binding free energies were computed by MM/PBSA. Further, principal component analysis, dynamic cross correlation and hydrogen bond occupancy were analyzed to characterize protein-ligand interactions. Biological pathway enrichment analysis was also carried out to elucidate the therapeutic targets of the phytochemicals in comparison to SARS-CoV-2. Results: The potential binding modes and favourable molecular interaction profile of 9 phytochemicals, majorly from Withania somnifera with lowest free binding energies, against the SARS-CoV-2 NSP targets were identified. It was understood that phytochemicals and 2 repurposed drugs with steroidal moieties in their chemical structures formed stable interactions with the NSPs. Additionally, human target pathway analysis for SARS-CoV-2 and phytochemicals showed that cytokine mediated pathway and phosphorylation pathways were with the most significant p-value. Conclusions: To summarize this work, we suggest a global approach of targeting multiple proteins of SARS-CoV-2 with phytochemicals as a natural alternative therapy for COVID-19. We also suggest that these phytochemicals need to be tested experimentally to confirm their efficacy.

The fatty acid site is coupled to functional motifs in the SARS-CoV-2 spike protein and modulates spike allosteric behaviour.

The SARS-CoV-2 spike protein is the first contact point between the SARS-CoV-2 virus and host cells and mediates membrane fusion. Recently, a fatty acid binding site was identified in the spike (Toelzer et al. Science 2020). The presence of linoleic acid at this site modulates binding of the spike to the human ACE2 receptor, stabilizing a locked conformation of the protein. Here, dynamical-nonequilibrium molecular dynamics simulations reveal that this fatty acid site is coupled to functionally relevant regions of the spike, some of them far from the fatty acid binding pocket. Removal of a ligand from the fatty acid binding site significantly affects the dynamics of distant, functionally important regions of the spike, including the receptor-binding motif, furin cleavage site and fusion-peptide-adjacent regions. Simulations of the D614G mutant show differences in behaviour between these clinical variants of the spike: the D614G mutant shows a significantly different conformational response for some structural motifs relevant for binding and fusion. The simulations identify structural networks through which changes at the fatty acid binding site are transmitted within the protein. These communication networks significantly involve positions that are prone to mutation, indicating that observed genetic variation in the spike may alter its response to linoleate binding and associated allosteric communication.

Mechanism of the enhancing effect of glycyrrhizin on nifedipine penetration through a lipid membrane.

The saponin glycyrrhizin from liquorice root shows the ability to enhance the therapeutic activity of other drugs when used as a drug delivery system. Due to its amphiphilic properties, glycyrrhizin can form self-associates (dimers, micelles) and supramolecular complexes with a wide range of hydrophobic drugs, which leads to an increase in their solubility, stability and bioavailability. That is why the mechanism of the biological activity of glycyrrhizin is of considerable interest and has been the subject of intensive physical and chemical research in the last decade. Two mechanisms have been proposed to explain the effect of glycyrrhizin on drug bioavailability, namely, the increase in drug solubility in water and enhancement of the membrane permeability. Interest in the membrane-modifying ability of glycyrrhizic acid (GA) is also growing at present due to its recently discovered antiviral activity against SARS-CoV-2 Bailly and Vergoten (2020) [1]. In the present study, the passive permeability of the DOPC lipid membrane for the calcium channel blocker nifedipine was elucidated by parallel artificial membrane permeability assay (PAMPA) and full atomistic molecular dynamics (MD) simulation with free energy calculations. PAMPA experiments show a remarkable increase in the amount of nifedipine (NF) permeated with glycyrrhizin compared to free NF. In previous studies, we have shown using MD techniques that glycyrrhizin molecules can integrate into the lipid bilayer. In this study, MD simulation demonstrates a significant decrease in the energy barrier of NF penetration through the lipid bilayer in the presence of glycyrrhizin both in the pure DOPC membrane and in the membrane with cholesterol. This effect can be explained by the formation of hydrogen bonds between NF and GA in the middle of the bilayer.

Co-crystallization and structure determination: An effective direction for anti-SARS-CoV-2 drug discovery.

Safer and more-effective drugs are urgently needed to counter infections with the highly pathogenic SARS-CoV-2, cause of the COVID-19 pandemic. Identification of efficient inhibitors to treat and prevent SARS-CoV-2 infection is a predominant focus. Encouragingly, using X-ray crystal structures of therapeutically relevant drug targets (PLpro, Mpro, RdRp, and S glycoprotein) offers a valuable direction for anti-SARS-CoV-2 drug discovery and lead optimization through direct visualization of interactions. Computational analyses based primarily on MMPBSA calculations have also been proposed for assessing the binding stability of biomolecular structures involving the ligand and receptor. In this study, we focused on state-of-the-art X-ray co-crystal structures of the abovementioned targets complexed with newly identified small-molecule inhibitors (natural products, FDA-approved drugs, candidate drugs, and their analogues) with the assistance of computational analyses to support the precision design and screening of anti-SARS-CoV-2 drugs.

In search of SARS CoV-2 replication inhibitors: Virtual screening, molecular dynamics simulations and ADMET analysis.

Severe acute respiratory syndrome has relapsed recently as novel coronavirus causing a life threat to the entire world in the absence of an effective therapy. To hamper the replication of the deadly SARS CoV-2 inside the host cells, systematic in silico virtual screening of total 267,324 ligands from Asinex EliteSynergy and BioDesign libraries has been performed using AutoDock Vina against RdRp. The molecular modeling studies revealed the identification of twenty-one macrocyclic hits (2-22) with better binding energy than remdesivir (1), marketed SARS CoV-2 inhibitor. Further, the analysis using rules for drug-likeness and their ADMET profile revealed the candidature of these hits due to superior oral bioavailability and druggability. Further, the MD simulation studies of top two hits (2 and 3) performed using GROMACS 2020.1 for 10 ns revealed their stability into the docked complexes. These results provide an important breakthrough in the design of macrocyclic hits as SARS CoV-2 RNA replicase inhibitor.

Phytoconstituents from ten natural herbs as potent inhibitors of main protease enzyme of SARS-COV-2: In silico study.

Background: Lack of treatment of novel Coronavirus disease led to the search of specific antivirals that are capable to inhibit the replication of the virus. The plant kingdom has demonstrated to be an important source of new molecules with antiviral potential. Purpose: The present study aims to utilize various computational tools to identify the most eligible drug candidate that have capabilities to halt the replication of SARS-COV-2 virus by inhibiting Main protease (Mpro) enzyme. Methods: We have selected plants whose extracts have inhibitory potential against previously discovered coronaviruses. Their phytoconstituents were surveyed and a library of 100 molecules was prepared. Then, computational tools such as molecular docking, ADMET and molecular dynamic simulations were utilized to screen the compounds and evaluate them against Mpro enzyme. Results: All the phytoconstituents showed good binding affinities towards Mpro enzyme. Among them laurolitsine possesses the highest binding affinity i.e. -294.1533 kcal/mol. On ADMET analysis of best three ligands were simulated for 1.2 ns, then the stable ligand among them was further simulated for 20 ns. Results revealed that no conformational changes were observed in the laurolitsine w.r.t. protein residues and low RMSD value suggested that the Laurolitsine-protein complex was stable for 20 ns. Conclusion: Laurolitsine, an active constituent of roots of Lindera aggregata, was found to be having good ADMET profile and have capabilities to halt the activity of the enzyme. Therefore, this makes laurolitsine a good drug candidate for the treatment of COVID-19.

Screening of inhibitors against SARS-CoV-2 spike protein and their capability to block the viral entry mechanism: A viroinformatics study.

SARS-CoV-2, previously named 2019 novel coronavirus (2019-nCoV), has been associated with the global pandemic of acute respiratory distress syndrome. First reported in December 2019 in the Wuhan province of China, this new RNA virus has several folds higher transmission among humans than its other family member (SARS-CoV and MERS-CoV). The SARS-CoV-2 spike receptor-binding domain (RBD) is the region mediating the binding of the virus to host cells via Angiotensin-converting enzyme 2 (ACE2), a critical step of viral. Here in this study, we have utilized in silico approach for the virtual screening of antiviral library extracted from the Asinex database against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein. Further, the molecules were ranked based on their binding affinity against RBD, and the top 15 molecules were selected. The affinity of these selected molecules to interrupt the ACE2-Spike interaction was also studied. It was found that the chosen molecules were demonstrating excellent binding affinity against spike protein, and these molecules were also very effectively interrupting the ACE2-RBD interaction. Furthermore, molecular dynamics (MD) simulation studies were utilized to investigate the top 3 selected molecules' stability in the ACE2-RBD complexes. To the best of our knowledge, this is the first study where molecules' inhibitory potential against the Receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike glycoprotein and their inhibitory potential against the ACE2-Spike has been studied. We believe that these compounds can be further tested as a potential therapeutic option against COVID-19.

Nicotinic cholinergic system and COVID-19: In silico evaluation of nicotinic acetylcholine receptor agonists as potential therapeutic interventions.

SARS-CoV-2 infection was announced as a pandemic in March 2020. Since then, several scientists have focused on the low prevalence of smokers among hospitalized COVID-19 patients. These findings led to our hypothesis that the Nicotinic Cholinergic System (NCS) plays a crucial role in the manifestation of COVID-19 and its severe symptoms. Molecular modeling revealed that the SARS-CoV-2 Spike glycoprotein might bind to nicotinic acetylcholine receptors (nAChRs) through a cryptic epitope homologous to snake toxins, substrates well documented and known for their affinity to the nAChRs. This binding model could provide logical explanations for the acute inflammatory disorder in patients with COVID-19, which may be linked to severe dysregulation of NCS. In this study, we present a series of complexes with cholinergic agonists that can potentially prevent SARS-CoV-2 Spike glycoprotein from binding to nAChRs, avoiding dysregulation of the NCS and moderating the symptoms and clinical manifestations of COVID-19. If our hypothesis is verified by in vitro and in vivo studies, repurposing agents currently approved for smoking cessation and neurological conditions could provide the scientific community with a therapeutic option in severe COVID-19.

Computational and network pharmacology analysis of bioflavonoids as possible natural antiviral compounds in COVID-19.

Bioflavonoids are the largest group of plant-derived polyphenolic compounds with diverse biological potential and have also been proven efficacious in the treatment of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). The present investigation validates molecular docking, simulation, and MM-PBSA studies of fifteen bioactive bioflavonoids derived from plants as a plausible potential antiviral in the treatment of COVID-19. Molecular docking studies for 15 flavonoids on the three SARS CoV-2 proteins, non-structural protein-15 Endoribonuclease (NSP15), the receptor-binding domain of spike protein (RBD of S protein), and main protease (Mpro/3CLpro) were performed and selected protein-ligand complexes were subjected to Molecular Dynamics simulations. The molecular dynamics trajectories were subjected to free energy calculation by the MM-PBSA method. All flavonoids were further assessed for their effectiveness as adjuvant therapy by network pharmacology analysis on the target proteins. The network pharmacology analysis suggests the involvement of selected bioflavonoids in the modulation of multiple signaling pathways like p53, FoxO, MAPK, Wnt, Rap1, TNF, adipocytokine, and leukocyte transendothelial migration which plays a significant role in immunomodulation, minimizing the oxidative stress and inflammation. Molecular docking and molecular dynamics simulation studies illustrated the potential of glycyrrhizic acid, amentoflavone, and mulberroside in inhibiting key SARS-CoV-2 proteins and these results could be exploited further in designing future ligands from natural sources.