Coronavirus Life Cycle Research Articles

Development of a Fluorescent Assay and Imidazole-Containing Inhibitors by Targeting SARS-CoV-2 Nsp13 Helicase.

Nsp13, a non-structural protein belonging to the coronavirus family 1B (SF1B) helicase, exhibits 5'-3' polarity-dependent DNA or RNA unwinding using NTPs. Crucially, it serves as a key component of the viral replication-transcription complex (RTC), playing an indispensable role in the coronavirus life cycle and thereby making it a promising target for broad-spectrum antiviral therapies. The imidazole scaffold, known for its antiviral potential, has been proposed as a potential scaffold. In this study, a fluorescence-based assay was designed by labeling dsDNA substrates with a commercial fluorophore and monitoring signal changes upon Nsp13 helicase activity. Optimization and high-throughput screening validated the feasibility of this approach. In accordance with the structural characteristics of ADP, we employed a structural-based design strategy to synthesize three classes of imidazole-based compounds through substitution reaction. Through in vitro activity research, pharmacokinetic parameter analysis, and molecular docking simulation, we identified compounds A16 (IC50 = 1.25 μM) and B3 (IC50 = 0.98 μM) as potential lead antiviral compounds for further targeted drug research.

Endoproteolysis of Oligopeptide-Based Coacervates for Enzymatic Modeling.

Better insights into the fate of membraneless organelles could strengthen the understanding of the transition from prebiotic components to multicellular organisms. Compartmentalized enzyme reactions in a synthetic coacervate have been investigated, yet there remains a gap in understanding the enzyme interactions with coacervate as a substrate hub. Here, we study how the molecularly crowded nature of the coacervate affects the interactions of the embedded substrate with a protease. We design oligopeptide-based coacervates that comprise an anionic Asp-peptide (D10) and a cationic Arg-peptide (R5R5) with a proteolytic cleavage site. The coacervates dissolve in the presence of the main protease (Mpro) implicated in the coronavirus lifecycle. We capitalize on the condensed structure, introduce a self-quenching mechanism, and model the enzyme kinetics by using Cy5.5-labeled peptides. The determined specificity constant (kcat/KM) is 5817 M-1 s-1 and is similar to that of the free substrate. We further show that the enzyme kinetics depend on the type and quantity of dye incorporated into the coacervates. Our work presents a simple design for enzyme-responsive coacervates and provides insights into the interactions between the enzyme and coacervates as a whole.

Membrane activity of viral proteins.

COVID-19 infection, caused by the SARS-CoV-2 coronavirus, has led to the largest pandemic since the Spanish flu in 1918. In view of this, the development of vaccines and antiviral drugs that can stop the spread of this infection has become an acute issue. Currently, in the search for antiviral drugs against COVID-19, much attention is paid to the study of the structure of the receptor-binding domain of the surface protein S. However, the emergence of new SARS-CoV-2 coronavirus strains indicates its high variability, which reduces the effectiveness of vaccines and antiviral drugs. At the same time, the envelope protein E of this virus is membrane active and shows a rather high conservatism. Despite the critical importance of this protein in the coronavirus life cycle, the physicochemical mechanisms of its interaction with cell membranes still remain unclear. So, we investigated the membrane activity of protein E of the SARS-CoV-2 coronavirus on models of giant unilamellar vesicles and lipid nanotubes. As a result, it was found that the protein forms pores in the lipid bilayer, i.e., performs the main function of viroporin. In addition, protein E is able to deform lipid membranes and form double-membrane vesicles depending on the concentration.

Dynamical Nonequilibrium Molecular Dynamics Simulations Identify Allosteric Sites and Positions Associated with Drug Resistance in the SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro) plays an essential role in the coronavirus lifecycle by catalyzing hydrolysis of the viral polyproteins at specific sites. Mpro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how Mpro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of Mpro to the presence and absence of a substrate. The results highlight communication between the Mpro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behavior of Mpro. More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.

Crystal structures of main proteases of SARS-CoV-2 variants bound to a benzothiazole-based inhibitor.

Main protease (M pro) serves as an indispensable factor in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as its constantly emerging variants and is therefore considered an attractive target for antiviral drug development. Benzothiazole-based inhibitors targeting M pro have recently been investigated by several groups and proven to be promising leads for coronaviral drug development. In the present study, we determine the crystal structures of a benzothiazole-based inhibitor, YH-53, bound to M pro mutants from SARS-CoV-2 variants of concern (VOCs) or variants of interest (VOIs), including K90R (Beta, B.1.351), G15S (Lambda, C.37), Y54C (Delta, AY.4), M49I (Omicron, BA.5) and P132H (Omicron, B.1.1.529). The structures show that the benzothiazole group in YH-53 forms a C-S covalent bond with the sulfur atom of catalytic residue Cys145 in SARS-CoV-2 M pro mutants. Structural analysis reveals the key molecular determinants necessary for interaction and illustrates the binding mode of YH-53 to these mutant M pros. In conclusion, structural insights from this study offer more information to develop benzothiazole-based drugs that are broader spectrum, more effective and safer.

Discovery of 3CLpro Inhibitor of SARS-CoV-2 Main Protease

Coronavirus main protease (3CLpro), a special cysteine protease in coronavirus family, is highly desirable in the life cycle of coronavirus. Here, molecular docking, ADMET pharmacokinetic profiles and molecular dynamics (MD) simulation were performed to develop specific 3CLpro inhibitor. The results showed that the 137 compounds originated from Chinese herbal have good binding affinity to 3CLpro. Among these, Cleomiscosin C, (+)-Norchelidonine, Protopine, Turkiyenine, Isochelidonine and Mallotucin A possessed prominent drug-likeness properties. Cleomiscosin C and Turkiyenine exhibited excellent pharmacokinetic profiles. Furthermore, the complex of Cleomiscosin C with SARS-CoV-2 main protease presented high stability. The findings in this work indicated that Cleomiscosin C is highly promising as a potential 3CLpro inhibitor, thus facilitating the development of effective drugs for COVID-19.

Biochemical analysis of SARS-CoV-2 Nsp13 helicase implicated in COVID-19 and factors that regulate its catalytic functions

Replication of the 30 kilobase genome of SARS-CoV-2, responsible for COVID-19, is a key step in the coronavirus life cycle that requires a set of virally encoded nonstructural proteins such as the highly conserved Nsp13 helicase. However, the features that contribute to catalytic properties of Nsp13 are not well established. Here, we biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein, focusing on its catalytic functions, nucleic acid substrate specificity, nucleotide/metal cofactor requirements, and displacement of proteins from RNA molecules proposed to be important for its proofreading role during coronavirus replication. We determined that Nsp13 preferentially interacts with single-stranded DNA compared to single-stranded RNA to unwind a partial duplex helicase substrate. We present evidence for functional cooperativity as a function of Nsp13 concentration, which suggests that oligomerization is important for optimal activity. In addition, under single-turnover conditions, Nsp13 unwound partial duplex RNA substrates of increasing double-stranded regions (16 - 30 base pairs) with similar efficiency, suggesting the enzyme unwinds processively in this range. We also show Nsp13-catalyzed RNA unwinding is abolished by a site-specific neutralizing linkage in the sugar-phosphate backbone, demonstrating continuity in the helicase-translocating strand is essential for unwinding the partial duplex substrate. Taken together, we demonstrate for the first time that coronavirus helicase Nsp13 disrupts a high affinity RNA-protein interaction in a uni-directional and ATP-dependent manner. Furthermore, sensitivity of Nsp13 catalytic functions to Mg2+ concentration suggests a regulatory mechanism for ATP hydrolysis, duplex unwinding, and RNA protein remodeling, processes implicated in SARS-CoV-2 replication and proofreading.

Herbal Medicines Used for the Management of COVID-19

Abstract: A worldwide outbreak of respiratory illnesses has been caused by coronavirus disease (COVID-19). Traditional healers have used herbs and dietary plants for centuries to treat various conditions. This review discusses the prevention of COVID-19, multiple herbs used in the treatment of COVID-19, and their future perspectives. Various databases, such as PubMed, Web of Science, Scopus, Medline, and Google Scholar, were searched for articles related to herbal products' antiviral effects using different keywords: herbal, SARS-CoV-2, plant-derived drugs, COVID-19, coronavirus, etc. Herbal treatment has been used as a contemporary alternative medicine for COVID-19. By inhibiting the replication and entry of SARS-CoV-2 into host cells, herbs can inhibit the pathogenesis of COVID-19. This article discusses COVID-19 infection, its salient features, spread, the life cycle of coronavirus, active response to coronavirus, proposed treatment, and herbal drugs used in the prevention and treatment of COVID-19.

A Review of Covid-19 Detailed Study of History, Life Cycle, Diagnosis and Prevention of Corona Virus

Coronavirus Disease-2019 is a new life-threatening, quickly-spreadable pandemic disease. It is a huge family of viruses known to cause sickness from breathing trouble, fever, fatigue, cough, sore throat, breathlessness, and common cold to the continuation of acute respiratory tract infection and the severity of the infection sometimes visible as pneumonia, acute respiratory syndrome and even death. The disease is commonly known as COVID-19. Since December 2019, Covid-19 emerged in Hunan seafood market at Wuhan in South China and rapidly spreading throughout the world, the virus epidemic has been proclaimed a public health emergency of International concern by World Health Organization (WHO). An exceedingly infectious potential for spreading resulted in the universal coronavirus disease 2019 (COVID-19) pandemic in 2020. Though some specialists cast doubts that the virus is transmitted from animals to humans, there are mixed results on the origin of coronavirus. In this review, we go through the history of covid-19, virus life cycle, diagnosis, and prevention with reference to the historical coronavirus pandemic in 2002.

Main and papain-like proteases as prospective targets for pharmacological treatment of coronavirus SARS-CoV-2.

The pandemic caused by the coronavirus SARS-CoV-2 led to a global crisis in the world healthcare system. Despite some progress in the creation of antiviral vaccines and mass vaccination of the population, the number of patients continues to grow because of the spread of new SARS-CoV-2 mutations. There is an urgent need for direct-acting drugs capable of suppressing or stopping the main mechanisms of reproduction of the coronavirus SARS-CoV-2. Several studies have shown that the successful replication of the virus in the cell requires proteolytic cleavage of the protein structures of the virus. Two proteases are crucial in replicating SARS-CoV-2 and other coronaviruses: the main protease (Mpro) and the papain-like protease (PLpro). In this review, we summarize the essential viral proteins of SARS-CoV-2 required for its viral life cycle as targets for chemotherapy of coronavirus infection and provide a critical summary of the development of drugs against COVID-19 from the drug repurposing strategy up to the molecular design of novel covalent and non-covalent agents capable of inhibiting virus replication. We overview the main antiviral strategy and the choice of SARS-CoV-2 Mpro and PLpro proteases as promising targets for pharmacological impact on the coronavirus life cycle.

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro

The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid-liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi-pi/cation-pi interactions. The set of antivirals included fleximers, 5'-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5'-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.

Antiviral drug design based on structural insights into the N-terminal domain and C-terminal domain of the SARS-CoV-2 nucleocapsid protein

Nucleocapsid (N) protein plays crucial roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including the formation of ribonucleoprotein (RNP) complex with the viral RNA. Here we reported the crystal structures of the N-terminal domain (NTD) and C-terminal domain (CTD) of the N protein and an NTD-RNA complex. Our structures reveal a unique tetramer organization of NTD and identify a distinct RNA binding mode in the NTD-RNA complex, which could contribute to the formation of the RNP complex. We also screened small molecule inhibitors of N-NTD and N-CTD and discovered that ceftriaxone sodium, an antibiotic, can block the binding of RNA to NTD and inhibit the formation of the RNP complex. These results together could facilitate the further research of antiviral drug design targeting N protein.

Two Ligand-Binding Sites on SARS-CoV-2 Non-Structural Protein 1 Revealed by Fragment-Based X-ray Screening.

The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 by suppressing its replication or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against the N-terminal domain of SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.99 Å). Binding affinities of hits against nsp1 and potential stabilisation were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal shift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medically relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1.

Recent Drug Development and Medicinal Chemistry Approaches for the Treatment of SARS-CoV-2 Infection and COVID-19.

COVID‐19, caused by SARS‐CoV‐2 infection, continues to be a major public health crisis around the globe. Development of vaccines and the first cluster of antiviral drugs has brought promise and hope for prevention and treatment of severe coronavirus disease. However, continued development of newer, safer, and more effective antiviral drugs are critically important to combat COVID‐19 and counter the looming pathogenic variants. Studies of the coronavirus life cycle revealed several important biochemical targets for drug development. In the present review, we focus on recent drug design and medicinal chemistry efforts on small molecule drug discovery including the development of nirmatrelvir that targets viral protein synthesis and remdesivir and molnupiravir that target viral RdRp. These are recent FDA approved drugs for the treatment of COVID‐19.

Small GTPase-A Key Role in Host Cell for Coronavirus Infection and a Potential Target for Coronavirus Vaccine Adjuvant Discovery.

Small GTPases are signaling molecules in regulating key cellular processes (e.g., cell differentiation, proliferation, and motility) as well as subcellular events (e.g., vesicle trafficking), making them key participants, especially in a great array of coronavirus infection processes. In this review, we discuss the role of small GTPases in the coronavirus life cycle, especially pre-entry, endocytosis, intracellular traffic, replication, and egress from the host cell. Furthermore, we also suggest the molecules that have potent adjuvant activity by targeting small GTPases. These studies provide deep insights and references to understand the pathogenesis of coronavirus as well as to propose the potential of small GTPases as targets for adjuvant development.

Targeting two potential sites of SARS-CoV-2 main protease through computational drug repurposing

Before the rise of SARS-CoV-2, emergence of different coronaviruses such as SARS-CoV and MERS-CoV has been reported that indicates possibility of the future novel pathogen from the coronavirus family at a pandemic level. In this context, explicit studies on identifying inhibitors focused on the coronavirus life cycle, are immensely important. The main protease is critical for the life cycle of coronaviruses. Majority of the work done on the inhibitor studies on the catalytically active dimeric SARS-CoV-2 main protease (Mpro), primarily focussed on the catalytic site of a single protomer, with a few targeting the dimeric site. In this study, we have exploited the FDA-approved drugs, for a computational drug repurposing study against the Mpro. A virtual screening approach was employed with docking and molecular dynamics (MD) methods. Out of 1576, FDA-approved compounds, our study suggests three compounds: netupitant, paliperidone and vilazodone as possible inhibitors with a potential to inhibit both sites (monomeric and dimeric) of the Mpro. These compounds were found to be stable during the MD simulations and their post simulation binding energies were also correlated for both the targeted sites, suggesting equal binding capacity. This unique efficiency of the reported compounds might support further experimental studies on developing inhibitors against SARS-CoV-2 main protease. Communicated by Ramaswamy H. Sarma

Molecular and structural insights of β-boswellic acid and glycyrrhizic acid as potent SARS-CoV-2 Envelope protein inhibitors

BackgroundOver million people have been infected with SARS-CoV-2 virus worldwide, with around 3% reported deaths till date. A few conventional antiviral treatments have been tried to mitigate the coronavirus. However, many alternative therapeutics are being evaluated worldwide. In the present study, we investigated traditional Indian medicinal compounds antiviral potencies as an effective drug for targeting SARS-CoV-2E. SARS-CoV-2 E protein plays a key role in coronavirus life cycle and is an interesting target for the development of anti-SARS-CoV-2 E drugs. MethodsMolecular docking studies of medicinal compounds possessing wide range of pharmacological and antiviral activities against enveloped viruses were evaluated with the computer-aided drug design screening software; PyRx. Twelve medicinal compounds isolated from plants were screened and visualized on Biovia Discovery-Studio. Moreover, SARS-CoV-2 E protein's secondary structural insights were deciphered using Swiss Model and ProFunc web server. ResultsGlycyrrhizic acid, triterpene glycoside isolated from plants of Glycyrrhiza (licorice) showed interactions with envelope protein at chain A: Arg 61, chain B: Phe 23, chain B: Tyr 57, and chain C: Val 25. β- boswellic acid, an ayurvedic herb (pentacyclic terpenoid are produced by Boswellia) represented direct interactions and indirect binding with chain C. Their pharmacological aspects and drug-likeness properties were deduced by DruLiTo. Toxicological assessment, along with their ADME profiling, was validated using vNNADMET. The findings showed that ligands, β-boswellic acid, and glycyrrhizic acid possessed the best bindings, with the target having binding affinity (-9.1 kcal/mol) amongst compounds tested against SARS-CoV-2 E. In-vitro studies reveals the promising effect as potent SARS-CoV-2 E inhibitors. Functionality loss and structural disruptions with ∼90% were observed by UV-spectra and fluorescent based analyses. ConclusionThe study demonstrated that β-boswellic acid, and glycyrrhizic acid are strong SARS-CoV-2 E protein inhibitors. In addition, the work linked GA antiviral activity to its effect on SARS-CoV- 2 E protein that can pave the way for designing antiviral therapeutics.

Artecanin of Laurus nobilis is a novel inhibitor of SARS-CoV-2 main protease with highly desirable druglikeness

Main protease (Mpro) is a critical enzyme in the life cycle of severe acute respiratory syndrome Coronavirus −2 (SARS-CoV-2). Due to its essential role in the maturation of the polyproteins, the necessity to inhibit Mpro is one of the essential means to prevent the outbreak of COVID-19. In this context, this study was conducted on the natural compounds of medicinal plants that are commonly available in the Middle East to find out the most potent one to inhibit Mpro with the best bioavailability and druglikeness properties. A total of 3392 compounds of sixty-six medicinal plants were retrieved from PubChem database and docked against Mpro. Thirty compounds with the highest docking scores with Mpro were chosen for further virtual screening. Variable druglikeness and toxicity potentials of these compounds were evaluated using SwissADME and Protox servers respectively. Out of these virtually screened compounds, artecanin was predicted to exhibit the most favourable druglikeness potentials, accompanied by no predicted hepatoxicity, carcinogenicity, mutagenicity, and cytotoxicity. Molecular dynamics (MD) simulations showed that Mpro-artecanin complex exhibited comparable stability with that observed in the ligand-free Mpro. This study revealed for the first time that artecanin from Laurus nobilis provided a novel static and dynamic inhibition for Mpro with excellent safety, oral bioavailability, and pharmacokinetic profile. This study suggested the ability of artecanin to be used as a potential natural inhibitor that can be used to block or at least counteract the SARS-CoV-2 invasion. Communicated by Ramaswamy H. Sarma

The order of events on Avian coronavirus life cycle shapes the order of quasispecies evolution during host switches.

To understand the population genetics events during coronavirus host switches, the Beaudette strain of Avian coronavirus (AvCoV) adapted to BHK-21 cells was passaged 15 times in VERO cells, the virus load and the variants at each passage being determined by RT-qPCR and genome-length deep sequencing. From BHK-21 P2 to VERO P3, a trend for the extinction of variants was followed by stability up to VERO P11 and both the emergence and the rise in frequency in some variants, while the virus loads were stable up to VERO P12. At the spillover from BHK-21 to VERO cells, variants that both emerged, showed a rise in frequency or were extinguished were detected on the spike, while variants at the M gene showed the same pattern only at VERO passage 13. Furthermore, nsps 3-5, 9 and 15 variants were detected at lower passages compared to the consensus sequences, with those at nsp3 being detected in the spectra also at higher passages. This suggests that quasispecies coronavirus evolution in spillovers follows the virus life cycle, starting with the evolution of the receptor binding proteins, followed by the replicase and then proteins involved in virion assembly, keeping the general fitness of the mutant spectrum stable.

Elucidation of SARS-Cov-2 Budding Mechanisms through Molecular Dynamics Simulations of M and E Protein Complexes.

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory’s refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein–lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics that inhibit coronavirus budding.