Computational Analysis Reveals Monomethylated Triazolopyrimidine as a Novel Inhibitor of SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp)

The human population is still facing appalling conditions due to several outbreaks of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus. The absence of specific drugs, appropriate vaccines for mutants, and knowledge of potential therapeutic agents makes this situation more difficult. Several 1, 2, 4-triazolo [1, 5-a] pyrimidine (TP)-derivative compounds were comprehensively studied for antiviral activities against RNA polymerase of HIV, HCV, and influenza viruses, and showed immense pharmacological interest. Therefore, TP-derivative compounds can be repurposed against the RNA-dependent RNA polymerase (RdRp) protein of SARS-CoV-2. In this study, a meta-analysis was performed to ensure the genomic variability and stability of the SARS-CoV-2 RdRp protein. The molecular docking of natural and synthetic TP compounds to RdRp and molecular dynamic (MD) simulations were performed to analyse the dynamic behaviour of TP compounds at the active site of the RdRp protein. TP compounds were also docked against other non-structural proteins (NSP1, NSP2, NSP3, NSP5, NSP8, NSP13, and NSP15) of SARS-CoV-2. Furthermore, the inhibition potential of TP compounds was compared with Remdesivir and Favipiravir drugs as a positive control. Additionally, TP compounds were analysed for inhibitory activity against SARS-CoV RdRp protein. This study demonstrates that TP analogues (monomethylated triazolopyrimidine and essramycin) represent potential lead molecules for designing an effective inhibitor to control viral replication. Furthermore, in vitro and in vivo studies will strengthen the use of these inhibitors as suitable drug candidates against SARS-CoV-2.     

Electron Density Analysis of SARS-CoV-2 RNA-Dependent RNA Polymerase Complexes

The work is devoted to the study of the complementarity of the electronic structures of the ligands and SARS-CoV-2 RNA-dependent RNA polymerase. The research methodology was based on determining of 3D maps of electron densities of complexes using an original quantum free-orbital AlteQ approach. We observed a positive relationship between the parameters of the electronic structure of the enzyme and ligands. A complementarity factor of the enzyme-ligand complexes has been proposed. The console applications of the AlteQ complementarity assessment for Windows and Linux (alteq_map_enzyme_ligand_4_win.exe and alteq_map_enzyme_ligand_4_linux) are available for free at the ChemoSophia webpage.     

Computational Study of SARS-CoV-2 RNA Dependent RNA Polymerase Allosteric Site Inhibition

The COVID-19 pandemic has caused millions of fatalities since 2019. Despite the availability of vaccines for this disease, new strains are causing rapid ailment and are a continuous threat to vaccine efficacy. Here, molecular docking and simulations identify strong inhibitors of the allosteric site of the SARS-CoV-2 virus RNA dependent RNA polymerase (RdRp). More than one hundred different flavonoids were docked with the SARS-CoV-2 RdRp allosteric site through computational screening. The three top hits were Naringoside, Myricetin and Aureusidin 4,6-diglucoside. Simulation analyses confirmed that they are in constant contact during the simulation time course and have strong association with the enzyme’s allosteric site. Absorption, distribution, metabolism, excretion and toxicity (ADMET) data provided medicinal information of these top three hits. They had good human intestinal absorption (HIA) concentrations and were non-toxic. Due to high mutation rates in the active sites of the viral enzyme, these new allosteric site inhibitors offer opportunities to drug SARS-CoV-2 RdRp. These results provide new information for the design of novel allosteric inhibitors against SARS-CoV-2 RdRp.     

Computational Screening of Phenylamino-Phenoxy-Quinoline Derivatives against the Main Protease of SARS-CoV-2 Using Molecular Docking and the ONIOM Method

In the search for new anti-HIV-1 agents, two forms of phenylamino-phenoxy-quinoline derivatives have been synthesized, namely, 2-phenylamino-4-phenoxy-quinoline and 6-phenylamino-4-phenoxy-quinoline. In this study, the binding interactions of phenylamino-phenoxy-quinoline derivatives and six commercially available drugs (hydroxychloroquine, ritonavir, remdesivir, S-217622, N3, and PF-07321332) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro) were investigated using molecular docking and the ONIOM method. The molecular docking showed the hydrogen bonding and hydrophobic interactions of all the compounds in the pocket of SARS-CoV-2 main protease (M^pro), which plays an important role for the division and proliferation of the virus into the cell. The binding free energy values between the ligands and M^pro ranged from −7.06 to −10.61 kcal/mol. The molecular docking and ONIOM results suggested that 4-(2′,6′-dimethyl-4′-cyanophenoxy)-2-(4″-cyanophenyl)-aminoquinoline and 4-(4′-cyanophenoxy)-2-(4″-cyanophenyl)-aminoquinoline have low binding energy values and appropriate molecular properties; moreover, both compounds could bind to M^pro via hydrogen bonding and Pi-Pi stacking interactions with amino acid residues, namely, HIS41, GLU166, and GLN192. These amino acids are related to the proteolytic cleavage process of the catalytic triad mechanisms. Therefore, this study provides important information for further studies on synthetic quinoline derivatives as antiviral candidates in the treatment of SARS-CoV-2.     

Screening of Natural Products Inhibitors of SARS-CoV-2 Entry

The COVID-19 pandemic has led to the search for new molecules with antiviral activity against SARS-CoV-2. The entry of the virus into the cell is one of the main targets for inhibiting SARS-CoV-2 infection. Natural products are an important source of new therapeutic alternatives against diseases. Pseudotyped viruses allow the study of SARS-CoV-2 viral entry inhibitors, and due to their simplicity, they allow the screening of a large number of antiviral candidates in Biosafety Level 2 facilities. We used pseudotyped HIV-1 with the D614G SARS-CoV-2 spike glycoprotein to test its ability to infect ACE2-expressing HEK 293T cells in the presence of diverse natural products, including 21 plant extracts, 7 essential oils, and 13 compounds from plants and fungi. The 50% cytotoxic concentration (CC₅₀) was evaluated using the resazurin method. From these analyses, we determined the inhibitory activity of the extract of Stachytarpheta cayennensis, which had a half-maximal inhibitory concentration (IC₅₀) of 91.65 µg/mL, a CC₅₀ of 693.5 µg/mL, and a selectivity index (SI) of 7.57, indicating its potential use as an inhibitor of SARS-CoV-2 entry. Moreover, our work indicates the usefulness of the pseudotyped-virus system in the screening of SARS-CoV-2 entry inhibitors.     

l-Arginine Improves Solubility and ANTI SARS-CoV-2 Mpro Activity of Rutin but Not the Antiviral Activity in Cells

The COVID-19 pandemic outbreak prompts an urgent need for efficient therapeutics, and repurposing of known drugs has been extensively used in an attempt to get to anti-SARS-CoV-2 agents in the shortest possible time. The glycoside rutin shows manifold pharmacological activities and, despite its use being limited by its poor solubility in water, it is the active principle of many pharmaceutical preparations. We herein report our in silico and experimental investigations of rutin as a SARS-CoV-2 Mpro inhibitor and of its water solubility improvement obtained by mixing it with l-arginine. Tests of the rutin/l-arginine mixture in a cellular model of SARS-CoV-2 infection highlighted that the mixture still suffers from unfavorable pharmacokinetic properties, but nonetheless, the results of this study suggest that rutin might be a good starting point for hit optimization.     

Phytochemical Discrimination,Biological Activity and Molecular Docking of Water-Soluble Inhibitors from Saussurea costus Herb against Main Protease of SARS-CoV-2

Siddha medicine is one of the oldest medical systems in the world and is believed to have originated more than 10,000 years ago and is prevalent across ancient Tamil land. It is undeniable that inhibitor preferences rise with increasing solubility in water due to the considerations pertaining to the bioavailability and the ease of which unabsorbed residues can be disposed of. In this study, we showed the phytochemical discrimination of Saussurea costus extracted with water at room temperature as a green extraction procedure. A total of 48 compounds were identified using gas chromatography-mass spectrometry (GC-MS). The fatty acids had a high phytochemical abundance at 73.8%, followed by tannins at 8.2%, carbohydrates at 6.9%, terpenoids at 4.3%, carboxylic acids at 2.5%, hydrocarbons at 2.4%, phenolic compounds at 0.2%, and sterols at 1.5%. Of these compounds, 22 were docked on the active side and on the catalytic dyad of His41 and Cys145 of the main protease of SARS-CoV-2 (M^pro). Eight active inhibitors were carbohydrates, five were fatty acids, three were terpenoids, two were carboxylic acids, one was a tannin, one was a phenolic compound, and one was a sterol. The best inhibitors were 4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl), Andrographolide, and delta.4-Androstene-3.beta.,17.beta.-diol, with a binding affinity that ranged from −6.1 kcal/mol to −6.5 kcal/mol. The inhibitory effect of Saussurea costus of SARS-CoV-2 entry into the cell was studied using a pseudovirus with Spike proteins from the D614G variant and the VOC variants Gamma and Delta. Based on the viral cycle of SARS-CoV-2, our results suggest that the Saussurea costus aqueous extract has no virucidal effect and inhibits the virus in the events after cell entry. Furthermore, the biological activity of the aqueous extract was investigated against HSV-1 virus and two bacterial strains, namely Staphylococcus aureus ATCC BAA 1026 and Escherichia coli ATCC 9637. According to this study, an enormous number of water-soluble inhibitors were identified from Saussurea costus against the M^pro, and this is unprecedented as far as we know.     

Novel Inhibitors of 2′-O-Methyltransferase of the SARS-CoV-2 Coronavirus

The COVID-19 pandemic is still affecting many people worldwide and causing a heavy burden to global health. To eliminate the disease, SARS-CoV-2, the virus responsible for the pandemic, can be targeted in several ways. One of them is to inhibit the 2′-O-methyltransferase (nsp16) enzyme that is crucial for effective translation of viral RNA and virus replication. For methylation of substrates, nsp16 utilizes S-adenosyl methionine (SAM). Binding of a small molecule in the protein site where SAM binds can disrupt the synthesis of viral proteins and, as a result, the replication of the virus. Here, we performed high-throughput docking into the SAM-binding site of nsp16 for almost 40 thousand structures, prepared for compounds from three libraries: Enamine Coronavirus Library, Enamine Nucleoside Mimetics Library, and Chemdiv Nucleoside Analogue Library. For the top scoring ligands, semi-empirical quantum-chemical calculations were performed, to better estimate protein–ligand binding enthalpy. Relying upon the calculated binding energies and predicted docking poses, we selected 21 compounds for experimental testing.     

Targeting non-structural proteins and 3CLpro 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 non-structural-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.     

Antiviral Properties of the NSAID Drug Naproxen Targeting the Nucleoprotein of SARS-CoV-2 Coronavirus

There is an urgent need for specific antiviral treatments directed against SARS-CoV-2 to prevent the most severe forms of COVID-19. By drug repurposing, affordable therapeutics could be supplied worldwide in the present pandemic context. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus could be a strategy to impede viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, a non-steroidal anti-inflammatory drug (NSAID) that was previously demonstrated to be active against Influenza A virus, were evaluated against SARS-CoV-2. Intrinsic fluorescence spectroscopy, fluorescence anisotropy, and dynamic light scattering assays demonstrated naproxen binding to the nucleoprotein of SARS-Cov-2 as predicted by molecular modeling. Naproxen impeded recombinant N oligomerization and inhibited viral replication in infected cells. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen specifically inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2-induced damage. No inhibition of viral replication was observed with paracetamol or the COX-2 inhibitor celecoxib. Thus, among the NSAID tested, only naproxen combined antiviral and anti-inflammatory properties. Naproxen addition to the standard of care could be beneficial in a clinical setting, as tested in an ongoing clinical study.     

In-Silico Screening and Molecular Dynamics Simulation of Drug Bank Experimental Compounds against SARS-CoV-2

For the last few years, the world has been going through a difficult time, and the reason behind this is severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), one of the significant members of the Coronaviridae family. The major research groups have shifted their focus towards finding a vaccine and drugs against SARS-CoV-2 to reduce the infection rate and save the life of human beings. Even the WHO has permitted using certain vaccines for an emergency attempt to cut the infection curve down. However, the virus has a great sense of mutation, and the vaccine’s effectiveness remains questionable. No natural medicine is available at the community level to cure the patients for now. In this study, we have screened the vast library of experimental drugs of Drug Bank with Schrodinger’s maestro by using three algorithms: high-throughput virtual screening (HTVS), standard precision, and extra precise docking followed by Molecular Mechanics/Generalized Born Surface Area (MMGBSA). We have identified 3-(7-diaminomethyl-naphthalen-2-YL)-propionic acid ethyl ester and Thymidine-5′-thiophosphate as potent inhibitors against the SARS-CoV-2, and both drugs performed impeccably and showed stability during the 100 ns molecular dynamics simulation. Both of the drugs are among the category of small molecules and have an acceptable range of ADME properties. They can be used after their validation in in-vitro and in-vivo conditions.     

Gold Metallodrugs to Target Coronavirus Proteins: Inhibitory Effects on the Spike-ACE2 Interaction and on PLpro Protease Activity by Auranofin and Gold Organometallics**

Gold complexes have a long tradition in medicine and for many examples antirheumatic, anticancer or anti-infective effects have been confirmed. Herein, we evaluated the lead compound Auranofin and five selected gold organometallics as inhibitors of two relevant drug targets of severe acute respiratory syndrome coronaviruses (SARS-CoV). The gold metallodrugs were effective inhibitors of the interaction of the SARS-CoV-2 spike protein with the angiotensin converting enzyme 2 (ACE2) host receptor and might thus interfere with the viral entry process. The gold metallodrugs were also efficient inhibitors of the papain-like protease (PLpro) of SARS-CoV-1 and SARS-CoV-2, which is a key enzyme in the viral replication. Regarding PLpro from SARS-CoV-2, the here reported inhibitors are among the very first experimentally confirmed examples with activity against this target enzyme. Importantly, the activity of the complexes against both PLpro enzymes correlated with the ability of the inhibitors to remove zinc ions from the labile zinc center of the enzyme. Taken together, the results of this pilot study suggest further evaluation of gold complexes as SARS-CoV antiviral drugs.     

Identification of Novel Influenza Polymerase PB2 Inhibitors Using a Cascade Docking Virtual Screening Approach

To discover novel inhibitors that target the influenza polymerase basic protein 2 (PB2) cap-binding domain (CBD), commercial ChemBridge compound libraries containing 384,796 compounds were screened using a cascade docking of LibDock–LigandFit–GOLD, and 60 compounds were selected for testing with cytopathic effect (CPE) inhibition assays and surface plasmon resonance (SPR) assay. Ten compounds were identified to rescue cells from H1N1 virus-mediated death at non-cytotoxic concentrations with EC₅₀ values ranging from 0.30 to 67.65 μM and could bind to the PB2 CBD of H1N1 with Kd values ranging from 0.21 to 6.77 μM. Among these, four compounds (11D4, 12C5, 21A5, and 21B1) showed inhibition of a broad spectrum of influenza virus strains, including oseltamivir-resistant ones, the PR/8-R292K mutant (H1N1, recombinant oseltamivir-resistant strain), the PR/8-I38T mutant (H1N1, recombinant baloxavir-resistant strain), and the influenza B/Lee/40 virus strain. These compounds have novel chemical scaffolds and relatively small molecular weights and are suitable for optimization as lead compounds. Based on sequence and structure comparisons of PB2 CBDs of various influenza virus subtypes, we propose that the Phe323/Gln325, Asn429/Ser431, and Arg355/Gly357 mutations, particularly the Arg355/Gly357 mutation, have a marked impact on the selectivities of PB2 CBD-targeted inhibitors of influenza A and influenza B.     

Molecular Dynamics and MM-PBSA Analysis of the SARS-CoV-2 Gamma Variant in Complex with the hACE-2 Receptor

The SARS-CoV-2 virus, since its appearance in 2019, has caused millions of cases and deaths. To date, there is no effective treatment or a vaccine that is fully protective. Despite the efforts made by governments and health institutions around the globe to control its propagation, the evolution of the virus has accelerated, diverging into hundreds of variants. However, not all of them are variants of concern (VoC’s). VoC’s have appeared in different regions and throughout the two years of the pandemic they have spread around the world. Specifically, in South America, the gamma variant (previously known as P.1) appeared in early 2021, bringing with it a second wave of infections. This variant contains the N501Y, E484K and K417T mutations in the receptor binding domain (RBD) of the spike protein. Although these mutations have been described experimentally, there is still no clarity regarding their role in the stabilization of the complex with the human angiotensin converting enzyme 2 (hACE-2) receptor. In this article we dissect the influence of mutations on the interaction with the hACE-2 receptor using molecular dynamics and estimations of binding affinity through a screened version of the molecular mechanics Poisson Boltzmann surface area (MM-PBSA) and interaction entropy. Our results indicate that mutations E484K and K417T compensate each other in terms of binding affinity, while the mutation N501Y promotes a more convoluted effect. This effect consists in the adoption of a cis configuration in the backbone of residue Y495 within the RBD, which in turn promotes polar interactions with the hACE-2 receptor. These results not only correlate with experimental observations and complement previous knowledge, but also expose new features associated with the specific contribution of concerned mutations. Additionally, we propose a recipe to assess the residue-specific contribution to the interaction entropy.     

Chemiluminescent Imaging Assay of SARS-CoV-2 Protein with Target-Induced Enzyme Activity Regulation

Simple but robust testing assays are essential for screening and diagnosis of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in COVID-19 pandemic. Here, we described a chemiluminescent imaging assay (CLIA) for sensitive and convenient detection of SARS-CoV-2 nucleocapsid protein (NP) by a target-induced enzyme activity regulation (T-EAR) strategy. The T-EAR used a pair of antibody-DNA probes to recognize SARS-CoV-2 NP and proximity-induce rolling circle amplification for mass-production of pyrophosphate to coordinate with Cu²⁺, which prevented the reduction of Cu²⁺ to Cu⁺ by sodium ascorbate as well as the Cu⁺-caused inactivation of horseradish peroxidase (HRP). The activity retention of HRP produced strong CL signal for the detection of SARS-CoV-2 NP by catalyzing the oxidation of luminol by H₂O₂. The T-EAR based CLIA showed a wide detection range from 1 pg/mL to 100 ng/mL (13 fM to 1.3 nM) with the requirement of only 0.75 μL of sample. This CLIA had advantages of good sensitivity, simple wash-free operation, acceptable accuracy, and high-throughput imaging detection, displaying potential applicability in screening assay of COVID-19 infection.     

Virtual Screening and Molecular Dynamics Simulation Study of Influenza Polymerase PB2 Inhibitors

Influenza A virus is the main cause of worldwide epidemics and annual influenza outbreaks in humans. In this study, a virtual screen was performed to identify compounds that interact with the PB2 cap-binding domain (CBD) of influenza A polymerase. A virtual screening workflow based on Glide docking was used to screen an internal database containing 8417 molecules, and then the output compounds were selected based on solubility, absorbance, and structural fingerprints. Of the 16 compounds selected for biological evaluation, six compounds were identified that rescued cells from H1N1 virus-mediated death at non-cytotoxic concentrations, with EC50 values ranging from 2.5–55.43 μM, and that could bind to the PB2 CBD of H1N1, with Kd values ranging from 0.081–1.53 μM. Molecular dynamics (MD) simulations of the docking complexes of our active compounds revealed that each compound had its own binding characteristics that differed from those of VX-787. Our active compounds have novel structures and unique binding modes with PB2 proteins, and are suitable to serve as lead compounds for the development of PB2 inhibitors. An analysis of the MD simulation also helped us to identify the dominant amino acid residues that play a key role in binding the ligand to PB2, suggesting that we should focus on increasing and enhancing the interaction between inhibitors and these major amino acids during lead compound optimization to obtain more active PB2 inhibitors.     

Synthesis,Structure–Activity Relationships,and Antiviral Profiling of 1-Heteroaryl-2-Alkoxyphenyl Analogs as Inhibitors of SARS-CoV-2 Replication

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure–activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity.     

新型冠状病毒检测以及相关药物研发中的基础药学知识

Abundant basic pharmaceutical knowledge is involved in the detection of SARS-CoV-2 and the discovery and development of antiviral drugs. This paper gives detailed summary and provides rich materials for case teaching.