Predictions in Clinical Efficiency of SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp) Inhibitors by Molecular Docking

This study utilizes the enzyme-substrate complex theory to predict the clinical efficacy of COVID-19 treatments at the biological systems level, using molecular docking stability indicators. Experimental data from the Protein Data Bank and molecular structures generated by AlphaFold 3 were used to create macromolecular complex templates. Six templates were developed, including the holo nsp7-nsp8-nsp12 (RNA-dependent RNA polymerase) complex with dsRNA primers (holo-RdRp-RNA). The study evaluated several ligands—Favipiravir-RTP, Remdesivir, Abacavir, Ribavirin, and Oseltamivir—as potential viral RNA polymerase inhibitors. Notably, the first four of these ligands have been clinically employed in the treatment of COVID-19, allowing for comparative analysis. Molecular docking simulations were performed using AutoDock 4, and statistical differences were assessed through t-tests and Mann-Whitney U tests. A review of the literature on COVID-19 treatment outcomes and inhibitors targeting RNA polymerase enzymes was conducted, and the inhibitors were ranked according to their clinical efficacy: Remdesivir > Favipiravir-RTP > Oseltamivir. Docking results obtained from the second and third templates aligned with clinical observations. Furthermore, Abacavir demonstrated a predicted efficacy comparable to Favipiravir-RTP, while Ribavirin exhibited a predicted efficacy similar to that of Remdesivir. This research, focused on inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase, establishes a framework for screening AI-generated drug templates based on clinical outcomes. Additionally, it develops a drug screening platform based on molecular docking binding energy, enabling the evaluation of novel or repurposed drugs and potentially accelerating the drug development process.

Analysis of RNA-Dependent RNA Polymerase Sequence of Infectious Flacherie Virus Isolated in China and Its Expression in BmN Cells

Full gene sequence of RNA-dependent RNA polymerase （RdRp） from Bombyx mori infectious flacherie virus isolated in Zhejiang Province, China （Zhejiang01/CHN/2002） was cloned. The sequence was 1 920 nucleotides in length coding 639 amino acid residues. Sequences comparison of RdRp showed Zhejiang01/CHN/2002 was 99.7% nucleotide sequence and 99.1% amino acids sequence homology with Japanese strain. The RdRp sequence was aligned with 8 representative picorna（-like） viruses and 8 highly conserved regions were detected. The result indicated their relevance function. Phylogenetic tree of 14 picorna（-like） viruses which RdRp presumed protein sequences revealed that the viruses from Iflavirus genus formed an independent clade. The RdRp was successfully expressed in BmN cells using Bac-to-Bac expression system.

Crystal structures of RNA-dependent RNA polymerases from Jingmen tick virus and Alongshan virus

Jingmenviruses are a group of flavi-like viruses with segmented genome and have been found in various types of hosts,including humans,cattle,monkeys,bats,rodents,sheep,ticks,mosquitoes and nematodes.Jingmenviruses,including the Jingmen tick virus(JMTV)and Alongshan virus(ALSV),have been associated with febrile illness and flu-like symptoms in humans.Viral polymerase plays critical roles in genome replication and transcription and is an ideal target for antiviral drugs.Here,we determined the crystal structures of RNA-dependent RNA polymerase(RdRp)domains of JMTV and ALSV at 2.6Åand 3.2Åresolutions,respectively.The overall structures of JMTV and ALSV RdRp domains are similar to those from the typical unsegmented viruses in Flaviviridae family,especially the Flavivirus genus.JMTV and ALSV RdRps can be divided into three subdomains and the catalytical Motif A-G are conserved like the typical flaviviruses,whereas the zinc-binding pockets are absent from the JMTV and ALSV RdRps.The 50-ends of jingmenvirus genomes are varied in length and sequence,and a highly conserved 8-nucleotide element located on the tip of stem loop A was identified and shown to be required for binding with RdRp and performing de novo replication activity.These findings provide important structural insights into RdRp of segmented flavivirus and reveal the key region of virus genome responsible for replication initiation,which would promote molecular understanding of segmented flavivirus replication and the structure-based design of antiviral drugs against flaviviruses.

Future of the current anticoronaviral agents: A viewpoint on thevalidation for the next COVIDs and pandemics

Despite the global decline in the severity of the coronavirus disease 2019 (COVID-19) cases, the disease stillrepresents a major concern to the relevant scientific and medical communities. The primary concern of drug scientists,virologists, and other concerned specialists in this respect is to find ready-to-use suitable and potent anticoronaviraltherapies that are broadly effective against the different species/strains of the coronaviruses in general, not only againstthe current and previous coronaviruses (e.g., the recently-appeared severe acute respiratory syndrome coronavirus 2“SARS-CoV-2”), i.e., effective antiviral agents for treatment and/or prophylaxis of any coronaviral infections, includingthose of the coming ones from the next species and strains (if any). As an expert in this field, I tried, in this up-to-dateperspective “viewpoint” article, to evaluate the suitability and applicability of using the currently-availableanticoronaviral agents for the next coronavirus diseases (COVIDs) and coronaviral pandemics, highlighting the mostimportant general guidelines that should be considered in the next pandemics from the therapeutic points of view.

Neuroprotective role of edaravone and the effects of endoplasmic reticulum stress in an adult rat model of focal cerebral ischemia/reperfusion

BACKGROUND： Endoplasmic reticulum （ER） stress impairs ER functions and leads to the accumulation of unfolded or misfolded proteins in the ER lumen. ER stress-induced cell death plays an important role in cerebral ischemia. Edaravon （3-methyl-1-phenyl-2-pyrazolin-5-one） is a potent and novel scavenger of free radicals that inhibit delayed neuronal death, as demonstrated by in vitro and animal studies. However, its effect on ER stress and induced neuronal apoptosis in a rat model of brief middle cerebral artery occlusion remains unclear. OBJECTIVE： To explore the effects of edaravone on the expression of ER stress-related factors and neuronal apoptosis, based on the hypothesis that edaravone influences ER stress in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of Department of Neurology, Xiangya Hospital and the Department of Laboratory Animals, Xiangya Medical College, Central South University in China from June 2005 to May 2006. MATERIALS： Edaravone was purchased from Simcere Pharmaceutical Group, China. METHODS： A total of 216 adult, male, Sprague Dawley rats were randomly assigned to sham-surgery, model and edaravone groups, with 72 rats in each group, Brief middle cerebral artery occlusion was established in the model and edaravone groups. In addition, the edaravone group rats were injected with 3 mg/kg edaravone through the tail vein. MAIN OUTCOME MEASURES： RNA-dependent protein kinase-like endoplasmic reticulum eukaryotic translation initiation factor 2a kinase （PERK） and C/EBP homology protein （CHOP） mRNA expression in the ischemic parietal cortex was determined by reverse transcriptionpolymerase chain reaction; phosphorylated PERK and CHOP protein expression was detected by immunohistochemistry; neuronal apoptosis was detected by TdT-mediated-dUTP nick end labeling. RESULTS： Neurological deficit scores were significantly reduced in the edaravone group compared to the model group at 12, 24, and 72 hours following reperfusion （P〈 0.05）. In addition, PERK and CHOP mRNA as well as phosphorylated PERK and CHOP protein expression were significantly reduced in the edaravone group compared to the model group at 1,3, and 6 hours following reperfusion （P 〈 0.05, P 〈 0.01）. CHOP mRNA expression was decreased in the edaravone group compared to the model group at 3, 6, 12, and 24 hours following reperfusion （P〈 0.01）, while CHOP protein expression was less than the model group at 6, 12, and 24 hours following reperfusion （P 〈 0.05）. CONCLUSION： Edaravone treatment resulted in decreased PERK and CHOP expression following ischemia/reperfusion, as well as reduced neuronal apoptosis. Edaravone exhibited a neuroprotective role by inhibiting endoplasmic reticulum stress.

Alternative Role of Motif B in Template Dependent Polymerase Inhibition

Severe acute respiratory syndrome coronavirus 2(SARS-Co V-2) relies on the central molecular machine RNA-dependent RNA polymerase(Rd Rp) for the viral replication and transcription. Remdesivir at the template strand has been shown to effectively inhibit the RNA synthesis in SARS-Co V-2 Rd Rp by deactivating not only the complementary UTP incorporation but also the next nucleotide addition. However, the underlying molecular mechanism of the second inhibitory point remains unclear. In this work, we have performed molecular dynamics simulations and demonstrated that such inhibition has not directly acted on the nucleotide addition at the active site. Instead, the translocation of Remdesivir from +1 to-1 site is hindered thermodynamically as the posttranslocation state is less stable than the pre-translocation state due to the motif B residue G683. Moreover, another conserved residue S682 on motif B further hinders the dynamic translocation of Remdesivir due to the steric clash with the 1′-cyano substitution. Overall,our study has unveiled an alternative role of motif B in mediating the translocation when Remdesivir is present in the template strand and complemented our understanding about the inhibitory mechanisms exerted by Remdesivir on the RNA synthesis in SARS-Co V-2 Rd Rp.

Molecules against COVID-19:An in Silico Approach for Drug Development

A large number of deaths have been caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)worldwide,turning it into a serious and momentous threat to public health.This study tends to contribute to the development of effective treatment strategies through a computational approach,investigating the mechanisms in relation to the binding and subsequent inhibition of SARS-CoV-2 ribonucleic acid(RNA)-dependent RNA polymerase(RdRp).Molecular docking was performed to screen six naturally occurring molecules with antineoplastic properties(Ellipticine,Ecteinascidin,Homoharringtonine,Dolastatin 10,Halichondrin,and Plicamycin).Absorption,distribution,metabolism,and excretion(ADME)investigation was also conducted to analyze the druglike properties of these compounds.The docked results have clearly shown binding of ligands to the SARS-CoV-2 RdRp protein.Interestingly,all ligands were found to obey Lipinski’s rule of five.These results provide a basis for repurposing and using molecules,derived from plants and animals,as a potential treatment for the coronavirus disease 2019(COVID-19)infection as they could be effective therapeutics for the same.

Unusual substructure conformations observed in crystal structures of a dicistrovirus RNA-dependent RNA polymerase suggest contribution of the N-terminal extension in proper folding

The Dicistroviridae is a virus family that includes many insect pathogens.These viruses contain a positive-sense RNA genome that is replicated by the virally encoded RNA-dependent RNA polymerase(RdRP)also named 3D^(pol).Compared with the Picornaviridae RdRPs such as poliovirus(PV)3D^(pol),the Dicistroviridae representative Israeli acute paralysis virus(IAPV)3D^(pol) has an additional N-terminal extension(NE)region that is about 40-residue in length.To date,both the structure and catalytic mechanism of the Dicistroviridae RdRP have remain elusive.Here we reported crystal structures of two truncated forms of IAPV 3D^(pol),namelyΔ85 andΔ40,both missing the NE region,and the 3D^(pol) protein in these structures exhibited three conformational states.The palm and thumb domains of these IAPV 3D^(pol) structures are largely consistent with those of the PV 3D^(pol) structures.However,in all structures,the RdRP fingers domain is partially disordered,while different conformations of RdRP substructures and interactions between them are also present.In particular,a large-scale conformational change occurred in the motif B-middle finger region in one protein chain of theΔ40 structure,while a previously documented alternative conformation of motif A was observed in all IAPV structures.These experimental data on one hand show intrinsic conformational variances of RdRP substructures,and on the other hand suggest possible contribution of the NE region in proper RdRP folding in IAPV.

Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China

Enterovirus 71(EV71),one of the major causative agents for hand-foot-and-mouth disease(HFMD),has caused more than 100 deaths among Chinese children since March 2008.The EV71 genome encodes an RNAdependent RNA polymerase(RdRp),denoted 3D^(pol),which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics.Here we report the crystal structures of EV71 RdRp(3D^(pol))and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4Åresolution.The structure of EV71 RdRp(3D^(pol))has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp.And the EV71 RdRp(3D^(pol))complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding.The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus(FMDV)3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase.These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.

Crystal structure of the coxsackievirus A16 RNA-dependent RNA polymerase elongation complex reveals novel features in motif A dynamics

Dear Editor, Coxsackievirus A16 （CV A16） and enterovirus 71 （EV71） are currently the two primary causative agents of hand- foot-and-mouth disease （HFMD） （Solomon et al., 2010; Mao et al., 2014）, threatening health of children world- wide. They both belong to the Enterovirus genus of the Picornaviridae family, and have single-stranded positive- sense RNA genomes of about 7.5 kilobases （kb） in length. As with other positive-strand RNA viruses, the genome rep- lication process ofCV A16 is carried out by a membrane- associated replication complex with the virally encoded RNA-dependent RNA polymerase （RdRP） as the essential catalytic enzyme.

RNA-DEPENDENT RNA POLYMERASE ASSOCIATED WITH WHEAT ROSETTE STUNT VIRIONS

Wheat rosette stunt virus (WRSV) contains an RNA-dependent RNA polymerase. The RNA polymerase activity is associated with the viral nucleocapsid (NP).In vitro transcription of purified NP required all of the four nucleoside triphosphates and Mg^(2+).There was a need of a proper salt concentration and some reducing reagents in the system for increasing the RNA polymerase activity.The optimum temperature for in vitro transcription was around 25℃.Within the first 90 min of in vitro reaction, incorporation rose linearly with the time course of incubation. The experiments of ribonuclease and deoxyribonuclease treatments showed that single-stranded RNAs were synthesized in vitro by the RNA polymerase.Two fractions of WRSV-NP could be separated by SDS-dissociation and ultracentrifugation.The supernatant fraction contained three structural proteins of NP: L, N and NS;and the pellet fraction contained the viral RNA.When the supernatant proteins and the viral RNA were mixed together, RNA polymerase activity could be reconstituted.When the ratio between the amounts of the supernatant proteins and the viral RNA in the mixture was about 100:7.7, the reconstituted RNA polymerase activity reached the maximum.

Identifying potential compounds from Bacopa monnieri(brahmi)against coxsackievirus A16 RdRp targeting HFM disease(tomato flu)

Hand,foot,and mouth disease(HFMD),primarily instigated by Coxsackievirus A16(CVA16),poses a serious health concern,necessitating effective therapeutic interventions.The RNA-dependent RNA polymerase(RdRp)of CVA16 emerges as a promising drug target for HFMD treatment.This study presents an in-silico pipeline for the identification of potential RdRp inhibitors against CVA16.A library of 91 natural compounds derived from Bacopa monnieri(brahmi)was virtually screened against the CVA16 RdRp.Here,Bacobitacin D emerged as a promising hit molecule,forming 8 hydrogen bonds including key catalytic site residues(Asp^(238)and Asp^(329))within the RdRp active site.Further,molecular dynamics(MD)simulations and MM/GBSA binding free energy calculations was applied on the top three hits that were selected based on exhaustive docking scores(≤-9.55 kcal/mol).Bacobitacin D exhibited sustainable stability,as evidenced by minimal deviation(RMSD=0.75±0.02 nm)during a 100 ns MD simulation.Importantly,Bacopaside IV exhibited the lowestΔGTOTAL binding free energy(-23.70 kcal/mol),while Bacobitacin D displayed a comparableΔGTOTAL of19.14 kcal/mol.Structural interpretation of the most populated cluster derived from MD simulations showed direct interactions of Bacobitacin D with pivotal catalytic residues,including Asp^(238)and Ser^(289).This comprehensive study confirmed Bacobitacin D as a potent inhibitor of CVA16 RdRp,offering a potential avenue for therapeutic intervention against HFMD.Experimental validation is required to confirm the inhibitory action of Bacobitacin D against HFMD.

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp, nonstructural proteins p16 and p23, playing a role in virus replication

Rabbit hemorrhagic disease virus(RHDV)is a member of the Caliciviridae family and cannot be propagated in vitro,which has impeded the progress of investigating its replication mechanism.Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells.Here,aided by this replicon system and using two-step affinity purification,we purified the RHDV replicase and identified its associated host factors.We identified rabbit nucleolin(NCL)as a physical link,which mediating the interaction between other RNA-dependent RNA polymerase(Rd Rp)-related host proteins and the viral replicase Rd Rp.We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells,respectively.NCL was identified to directly interact with RHDV Rd Rp,p16,and p23.Furthermore,NCL knockdown severely impaired the binding of Rd Rp to Rd Rp-related host factors.Collectively,these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.

Endoplasmic reticulum stress and liver diseases

Endoplasmic reticulum(ER)stress occurs when ER homeostasis is perturbed with accumulation of unfolded/misfolded protein or calcium depletion.The unfolded protein response(UPR),comprising of inositol-requiring enzyme 1 a(IRE1 a),double-stranded RNA-dependent protein kinase(PKR)-like ER kinase(PERK)and activating transcription factor 6(ATF6)signaling pathways,is a protective cellular response activated by ER stress.However,UPR activation can also induce cell death upon persistent ER stress.The liver is susceptible to ER stress given its synthetic and other biological functions.Numerous studies from human liver samples and animal disease models have indicated a crucial role of ER stress and the UPR signaling pathways in the pathogenesis of liver diseases,including non-alcoholic fatty liver disease(NAFLD),alcoholic liver disease(ALD),alpha-1 antitrypsin(AAT)deficiency(AATD),cholestatic liver disease,drug-induced liver injury,ischemia/reperfusion(I/R)injury,viral hepatitis and hepatocel-lular carcinoma(HCC).Extensive investigations have demonstrated the potential underlying mechanisms of the induction of ER stress and the contribution of the UPR pathways during the development of the diseases.Moreover,ER stress and the UPR proteins and genes have become emerging therapeutic targets to treat liver diseases.

The R Protein of SARS-CoV: Analyses of Structure and Function Based on Four Complete Genome Sequences of Isolates BJ01-BJ04

The R (replicase) protein is the uniquely defined non-structural protein (NSP) responsible for RNA replication, mutation rate or fidelity, regulation of transcription in coronaviruses and many other ssRNA viruses. Based on our complete genome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China, we analyzed the structure and predicted functions of the R protein in comparison with 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF (open-reading frame) encodes for two major enzyme activities, RNA-dependent RNA polymerase (RdRp) and proteinase activities. The R polyprotein undergoes a complex proteolytic process to produce 15 function-related peptides. A hydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identified within NSP1. The substitution rate of the R protein is close to the average of the SARS-CoV genome. The functional domains in all NSPs of the R protein give different phylogenetic results that suggest their different mutation rate under selective pressure. Eleven highly conserved regions in RdRp and twelve cleavage sites by 3CLP (chymotrypsin-like protein) have been identified as potential drug targets. Findings suggest that it is possible to obtain information about the phy-logeny of SARS-CoV, as well as potential tools for drug design, genotyping and diagnostics of SARS.