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Since the emergence of SARS‐CoV‐2, mutations in all subunits of the RNA‐dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano‐differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS‐CoV‐2 subvariants. [ABSTRACT FROM AUTHOR]

Coronaviruses have a major impact on human and animal health. The SARS-CoV-2, a beta coronavirus responsible for the COVID-19 pandemic, is a clear example. It continues circulating and causes human deaths, and its high replication rate results in numerous variants. Coronaviruses adapt to birds and mammals and constitute a serious threat, and new viruses are likely to emerge. Urban pigeons (Columbiformes) are synanthropic birds of great interest from a 'One Health' perspective, due to their interaction with humans and other animals. Aware that they may act as viral reservoirs and contribute to their spread, we aimed to investigate the possible presence of SARS-CoV-2 and other coronaviruses in Columbiformes in the city of Logroño, Spain. Oropharyngeal and cloacal swabs were tested using real-time (N1 and E genes from SARS-CoV-2) and conventional PCR assays (RdRp gene from all coronaviruses). SARS-CoV-2 was not detected. A total of 13.3% of pigeons harbored coronaviruses closely related to Gamma coronavirus (Igacovirus) from Columbiformes in Finland, Poland and China. Monitoring the emergence of a new variant of SARS-CoV-2 capable of infecting Columbiformes should continue. SARS-CoV-2 is still circulating, the viral RNA of this virus has been detected in avian species (Phasianidae and Anatidae), and other coronaviruses are associated with animals that are in close contact with humans. The presence of Gamma coronavirus in urban pigeons must be considered for the risk of surveillance of human infections. [ABSTRACT FROM AUTHOR]

Abstract Background RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production. Methods and results BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp′)-OmpA′, SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34. Conclusion The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form.

The Covid-19 pandemic has emerged as a global health crisis, with no definitive end in sight. In this study, molecular docking was employed to screen compounds derived from Eclipta prostrata for their potential inhibitory effects on the RNA-dependent RNA polymerase (RdRp) enzyme of the Sars-CoV-2 virus. RdRp plays a crucial role in the replication of the virus. A library consisting of 55 natural compounds from E. prostrata was evaluated for their anti-RdRp activity. Among them, five potential compounds exhibited the ability to interact primarily with RdRp through hydrophobic interactions with amino acid groups located in the active site region of RdRp. These compounds include echinocystis acid (T21), eclalbasaponin I (T22), ecliptasaponin A (T23), oleanolic acid (T41), and ursolic acid (T52). These interactions have the potential to render the RdRp enzyme inactive, thereby impeding viral replication. This research holds promise for the development of Covid-19 therapeutics using natural compounds.

Background: RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production. Methods and results: BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp′)-OmpA′, SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34. Conclusion: The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form. [ABSTRACT FROM AUTHOR]

In our previous report, the unique architecture of the catalytic chamber of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), which harbours two distinctive binding sites, was fully characterized at molecular level. The significant differences in the two binding sites BS1 and BS2 in terms of binding pockets motif, as well as the preferential affinities of eight anti-viral drugs to each of the two binding sites were described. Recent Cryogenic Electron Microscopy (Cryo-EM) studies on the RdRp revealed that two suramin molecules, a SARS-CoV-2 inhibitor, bind to RdRp in two different sites with distinctive interaction landscape. Here, we provide the first account of investigating the combined inhibitor binding to both binding sites, and whether the binding of two inhibitors molecules concurrently is "Cooperative binding" or not. It should be noted that the binding of inhibitors to different sites do not necessary constitute mutually independent events, therefore, we investigated two scenarios to better understand cooperativity: simultaneous binding and sequential binding. It has been demonstrated by binding free energy calculations (MM/PBSA) and piecewise linear potential (PLP) interaction energy analysis that the co-binding of two suramin molecules is not cooperative in nature; rather, when compared to individual binding, both molecules adversely affect one another's binding affinities. This observation appeared to be primarily due to RdRp's rigidity, which prevented both ligands from fitting comfortably within the catalytic chamber. Instead, the suramin molecules showed a tendency to change their orientation within the binding pockets in order to maintain their binding to the protein, but at the expense of the ligand internal energies. Although co-binding resulted in the loss of several important key interactions, a few interactions were conserved, and these appear to be crucial in preserving the binding of ligands in the active site. The structural and mechanistic details of this study will be useful for future research on creating and developing RdRp inhibitors against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Coronaviruses have a major impact on human and animal health. The SARS-CoV-2, a beta coronavirus responsible for the COVID-19 pandemic, is a clear example. It continues circulating and causes human deaths, and its high replication rate results in numerous variants. Coronaviruses adapt to birds and mammals and constitute a serious threat, and new viruses are likely to emerge. Urban pigeons (Columbiformes) are synanthropic birds of great interest from a ‘One Health’ perspective, due to their interaction with humans and other animals. Aware that they may act as viral reservoirs and contribute to their spread, we aimed to investigate the possible presence of SARS-CoV-2 and other coronaviruses in Columbiformes in the city of Logroño, Spain. Oropharyngeal and cloacal swabs were tested using real-time (N1 and E genes from SARS-CoV-2) and conventional PCR assays (RdRp gene from all coronaviruses). SARS-CoV-2 was not detected. A total of 13.3% of pigeons harbored coronaviruses closely related to Gamma coronavirus (Igacovirus) from Columbiformes in Finland, Poland and China. Monitoring the emergence of a new variant of SARS-CoV-2 capable of infecting Columbiformes should continue. SARS-CoV-2 is still circulating, the viral RNA of this virus has been detected in avian species (Phasianidae and Anatidae), and other coronaviruses are associated with animals that are in close contact with humans. The presence of Gamma coronavirus in urban pigeons must be considered for the risk of surveillance of human infections.

Dysregulation of mRNA processing results in diseases such as cancer. Although RNA editing technologies attract attention as gene therapy for repairing aberrant mRNA, substantial sequence defects arising from mis-splicing cannot be corrected by existing techniques using adenosine deaminase acting on RNA (ADAR) due to the limitation of adenosine-to-inosine point conversion. Here, we report an RNA editing technology called "RNA overwriting" that overwrites the sequence downstream of a designated site on the target RNA by utilizing the RNA-dependent RNA polymerase (RdRp) of the influenza A virus. To enable RNA overwriting within living cells, we developed a modified RdRp by introducing H357A and E361A mutations in the polymerase basic 2 of RdRp and fusing the C-terminus with catalytically inactive Cas13b (dCas13b). The modified RdRp knocked down 46% of the target mRNA and further overwrote 21% of the mRNA. RNA overwriting is a versatile editing technique that can perform various modifications, including addition, deletion, and mutation introduction, and thus allow for repair of the aberrant mRNA produced by dysregulation of mRNA processing, such as mis-splicing. [ABSTRACT FROM AUTHOR]

Respiratory Syncytial Virus (RSV) is the top cause of infant hospitalization globally, with no effective treatments available. Researchers have sought small molecules to target the RNA-dependent RNA Polymerase (RdRP) of RSV, which is essential for replication and transcription. Based on the cryo-EM structure of the RSV polymerase, in silico computational analysis including molecular docking and the protein-ligand simulation of a database, including 6554 molecules, is currently undergoing phases 1–4 of clinical trials and has resulted in the top ten repurposed compound candidates against the RSV polymerase, including Micafungin, Totrombopag, and Verubecestat. We performed the same procedure to evaluate 18 small molecules from previous studies and chose the top four compounds for comparison. Among the top identified repurposed compounds, Micafungin, an antifungal medication, showed significant inhibition and binding affinity improvements over current inhibitors such as ALS-8112 and Ribavirin. We also validated Micafungin's inhibition of the RSV RdRP using an in vitro transcription assay. These findings contribute to RSV drug development and hold promise for broad-spectrum antivirals targeting the non-segmented negative-sense (NNS) RNA viral polymerases, including those of rabies (RABV) and Ebola (EBOV). [ABSTRACT FROM AUTHOR]

We have previously published research on the anti-viral properties of an alkaloid mixture extracted from Nuphar lutea, the major components of the partially purified mixture found by NMR analysis. These are mostly dimeric sesquiterpene thioalkaloids called thiobinupharidines and thiobinuphlutidines against the negative strand RNA measles virus (MV). We have previously reported that this extract inhibits the MV as well as its ability to downregulate several MV proteins in persistently MV-infected cells, especially the P (phospho)-protein. Based on our observation that the Nuphar extract is effective in vitro against the MV, and the immediate need that the coronavirus disease 2019 (COVID-19) pandemic created, we tested here the ability of 6,6′-dihydroxythiobinupharidine DTBN, an active small molecule, isolated from the Nuphar lutea extract, on COVID-19. As shown here, DTBN effectively inhibits SARS-CoV-2 production in Vero E6 cells at non-cytotoxic concentrations. The short-term daily administration of DTBN to infected mice delayed the occurrence of severe clinical outcomes, lowered virus levels in the lungs and improved survival with minimal changes in lung histology. The viral load on lungs was significantly reduced in the treated mice. DTBN is a pleiotropic small molecule with multiple targets. Its anti-inflammatory properties affect a variety of pathogens including SARS-CoV-2 as shown here. Its activity appears to target both pathogen specific (as suggested by docking analysis) as well as cellular proteins, such as NF-κB, PKCs, cathepsins and topoisomerase 2, that we have previously identified in our work. Thus, this combined double action of virus inhibition and anti-inflammatory activity may enhance the overall effectivity of DTBN. The promising results from this proof-of-concept in vitro and in vivo preclinical study should encourage future studies to optimize the use of DTBN and/or its molecular derivatives against this and other related viruses. [ABSTRACT FROM AUTHOR]

Introduction: Autophagy not only plays an antiviral role but also can be utilized by viruses to facilitate virus infection. However, the underlying mechanism of potato virus Y (PVY) infection against plant autophagy remains unclear. BI-1, localizing to the endoplasmic reticulum (ER), is a multifunctional protein and may affect the virus infection. Methods: In this study, Y2H, BiFC, qRT-PCR, RNA-Seq, WB and so on were used for research. Results: P3 and P3N-PIPO of PVY can interact with the Bax inhibitor 1 (BI-1) of N. benthamiana. However, BI-1 knockout mutant showed better growth and development ability. In addition, when the BI-1 gene was knocked out or knocked down in N. benthamiana, the PVY-infected mutant showed milder symptoms and lower virus accumulation. Analysis of transcriptome data showed that the deletion of NbBI-1 weakened the gene expression regulation induced by PVY infection and NbBI-1 may reduce the mRNA level of NbATG6 by regulated IRE1-dependent decay (RIDD) in PVY-infected N. benthamiana. The expression level of the ATG6 gene of PVY-infected WT was significantly down-regulated, relative to the PVY-infected mutant. Further results showed that ATG6 of N. benthamiana can degrade NIb, the RNA-dependent RNA polymerase (RdRp) of PVY. NbATG6 has a higher mRNA level in PVY-infected BI-1 knockout mutants than in PVY-infected WT. Conclussion: The interaction of P3 and/or P3N-PIPO of PVY with BI-1 decrease the expression of the ATG6 gene might be mediated by RIDD, which inhibits the degradation of viral NIb and enhances viral replication. [ABSTRACT FROM AUTHOR]

The development of potent non-nucleoside inhibitors (NNIs) could be an alternate strategy to combating infectious bovine viral diarrhea virus (BVDV), other than the traditional vaccination. RNA-dependent RNA polymerase (RdRp) is an essential enzyme for viral replication; therefore, it is one of the primary targets for countermeasures against infectious diseases. The reported NNIs, belonging to the classes of quinolines (2h: imidazo[4,5-g]quinolines and 5m: pyrido[2,3-g] quinoxalines), displayed activity in cell-based and enzyme-based assays. Nevertheless, the RdRp binding site and microscopic mechanistic action are still elusive, and can be explored at a molecular level. Here, we employed a varied computational arsenal, including conventional and accelerated methods, to identify quinoline compounds' most likely binding sites. Our study revealed A392 and I261 as the mutations that can render RdRp resistant against quinoline compounds. In particular, for ligand 2h, mutation of A392E is the most probable mutation. The loop L1 and linker of the fingertip is recognized as a pivotal structural determinant for the stability and escape of quinoline compounds. Overall, this work demonstrates that the quinoline inhibitors bind at the template entrance channel, which is governed by conformational dynamics of interactions with loops and linker residues, and reveals structural and mechanistic insights into inhibition phenomena, for the discovery of improved antivirals. [ABSTRACT FROM AUTHOR]

The emergence of fast-spreading SARS-CoV-2 mutants has sparked a new phase of COVID-19 pandemic. There is a dire necessity for antivirals targeting highly conserved genomic domains on SARS-CoV-2 that are less prone to mutation. The nsp12, also known as the RNA-dependent RNA-polymerase (RdRp), the core component of 'SARS-CoV-2 replication-transcription complex', is a potential well-conserved druggable antiviral target. Several FDA-approved RdRp 'nucleotide analog inhibitors (NAIs)' such as remdesivir, have been repurposed to treat COVID-19 infections. The NAIs target RdRp protein translation and competitively block the nucleotide insertion into the RNA chain, resulting in the inhibition of viral replication. However, the replication proofreading function of nsp14-ExoN could provide resistance to SARS-CoV-2 against many NAIs. Conversely, the 'non-nucleoside analog inhibitors (NNAIs)' bind to allosteric sites on viral polymerase surface, change the redox state; thereby, exert antiviral activity by altering interactions between the enzyme substrate and active core catalytic site of the RdRp. NNAIs neither require metabolic activation (unlike NAIs) nor compete with intracellular pool of nucleotide triphosphates (NTPs) for anti-RdRp activity. The NNAIs from phytonutrient origin are potential antiviral candidates compared to their synthetic counterparts. Several in-silico studies reported the antiviral spectrum of natural phytonutrient-NNAIs such as Suramin, Silibinin (flavonolignan), Theaflavin (tea polyphenol), Baicalein (5,6,7-trihydroxyflavone), Corilagin (gallotannin), Hesperidin (citrus bioflavonoid), Lycorine (pyrrolidine alkaloid), with superior redox characteristics (free binding energy, hydrogen-bonds, etc.) than antiviral drugs (i.e. remdesivir, favipiravir). These phytonutrient-NNAIs also exert anti-inflammatory, antioxidant, immunomodulatory and cardioprotective functions, with multifunctional therapeutic benefits in the clinical management of COVID-19. [ABSTRACT FROM AUTHOR]

Mysterious evolution of a new strain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the Omicron variant, led to a new challenge in the persistent coronavirus disease 2019 (COVID-19) battle. Objecting the conserved SARS-CoV-2 enzymes RNA-dependent RNA polymerase (RdRp) and 3′-to-5′ exoribonuclease (ExoN) together using one ligand is a successful new tactic to stop SARS-CoV-2 multiplication and COVID-19 progression. The current comprehensive study investigated most nucleoside analogs (NAs) libraries, searching for the most ideal drug candidates expectedly able to act through this double tactic. Gradual computational filtration afforded six different promising NAs, riboprine/forodesine/tecadenoson/nelarabine/vidarabine/maribavir. Further biological assessment proved that riboprine and forodesine are able to powerfully inhibit the replication of the new virulent strains of SARS-CoV-2 with extremely minute in vitro anti-RdRp and anti-SARS-CoV-2 EC50 values of about 0.21 and 0.45 μM for riboprine and about 0.23 and 0.70 μM for forodesine, respectively, surpassing both remdesivir and the new anti-COVID-19 drug molnupiravir. These biochemical findings were supported by the prior in silico data. Additionally, the ideal pharmacophoric features of riboprine and forodesine molecules render them typical dual-action inhibitors of SARS-CoV-2 replication and proofreading. These findings suggest that riboprine and forodesine could serve as prospective lead compounds against COVID-19. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease virus (FMDV), an economically important pathogen of cloven-hoofed livestock, is a positive-sense, single-stranded RNA virus classified in the Picornaviridae family. RNA-dependent RNA polymerase (RdRp) of RNA viruses is highly conserved. Compounds that bind to the RdRp active site can block viral replication. Herein, we combined double virtual screenings and cell-based antiviral approaches to screen and identify potential inhibitors targeting FMDV RdRp (3Dpol). From 5596 compounds, the blind- followed by focus-docking filtered 21 candidates fitting in the 3Dpol active sites. Using the BHK-21 cell-based assay, we found that four compounds—NSC217697 (quinoline), NSC670283 (spiro compound), NSC292567 (nigericin), and NSC65850—demonstrated dose-dependent antiviral actions in vitro with the EC50 ranging from 0.78 to 3.49 µM. These compounds could significantly block FMDV 3Dpol activity in the cell-based 3Dpol inhibition assay with small IC50 values ranging from 0.8 nM to 0.22 µM without an effect on FMDV's main protease, 3Cpro. The 3Dpol inhibition activities of the compounds were consistent with the decreased viral load and negative-stranded RNA production in a dose-dependent manner. Conclusively, we have identified potential FMDV 3Dpol inhibitors that bound within the enzyme active sites and blocked viral replication. These compounds might be beneficial for FMDV or other picornavirus treatment. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a significant threat to infants and elderly individuals globally. Currently, there are no effective therapies or treatments for RSV infection because of an insufficient understanding of the RSV viral machinery. In this study, we investigated the effects of the template variations on RNA synthesis by the RSV polymerase through in vitro RNA synthesis assays. We confirmed the previously reported back-priming activity of the RSV polymerase, which is likely due to the secondary structure of the RNA template. We found that the expansion of the hairpin loop size of the RNA template abolishes the RSV polymerase back-priming activity. At the same time, it seemingly does not affect the de novo RNA synthesis activities of the RSV polymerase. Interestingly, our results show that the RSV polymerase also has a new primer-based terminal extension activity that adds nucleotides to the template and primer in a nonspecific manner. We also mapped the impact of the RNA 5′ chemical group on its mobility in a urea-denaturing RNA gel shift assay. Overall, these results enhance our knowledge about the RNA synthesis processes of the RSV polymerase and may guide future therapeutic efforts to develop effective antiviral drugs for RSV treatment. [ABSTRACT FROM AUTHOR]

Respiratory Syncytial Virus (RSV) causes severe respiratory infections and concomitant disease resulting in significant morbidity and mortality in infants, elderly, and immunocompromised adults. Vaccines, monoclonal antibodies, and small-molecule antivirals are now either available or in development to prevent and treat RSV infections. Although rodent and non-rodent preclinical animal models have been used to evaluate these emerging agents, there is still a need to improve our understanding of the pharmacokinetic (PK)-pharmacodynamic (PD) relationships within and between animal models to enable better design of human challenge studies and clinical trials. Herein, we report a PKPD evaluation of MRK-1, a novel small molecule non-nucleoside inhibitor of the RSV L polymerase protein, in the semi-permissive cotton rat and African green monkey models of RSV infection. These studies demonstrate a strong relationship between in vitro activity, in vivo drug exposure, and pharmacodynamic efficacy as well as revealing limitations of the cotton rat RSV model. Additionally, we report unexpected horizontal transmission of human RSV between co-housed African green monkeys, as well as a lack of drug specific resistant mutant generation. Taken together these studies further our understanding of these semi-permissive animal models and offer the potential for expansion of their preclinical utility in evaluating novel RSV therapeutic agents. [Display omitted] • Efficacy of an oral non-nucleoside inhibitor of the respiratory syncytial virus (RSV) in rodents and monkeys. • Experimentally demonstrated horizontal transmission of human RSV from infected to uninfected monkeys. • No evidence of drug-resistant mutants generated in vivo following discontinuation of the compound. • These studies show advantages of evaluating treatment paradigms against respiratory viruses in non-human primates. [ABSTRACT FROM AUTHOR]

Vaccines are used as one of the major weapons for the eradication of pandemic. However, the rise of different variants of the SARS-CoV-2 virus is creating doubts regarding the end of the pandemic. Hence, there is an urgent need to develop more drug candidates which can be useful for the treatment of COVID-19. In the present research for the scientific hypothesis, emphasis was given on the direct antiviral therapy available for the treatment of COVID-19. In lieu of this, the available molecular targets which include Severe Acute Respiratory Syndrome Chymotrypsin-like Protease (SARS-3CLpro), Papain-Like Cysteine Protease (PLpro), and RNA-Dependent RNA Polymerase (RdRp) were explored. As per the current scientific reports and literature, among all the available molecular targets, RNA-Dependent RNA Polymerase (RdRp) was found to be a crucial molecular target for the treatment of COVID-19. Most of the inhibitors which are reported against this target consisted of the free amine group and carbonyl group which might be playing an important role in the binding interaction with the RdRp protein. Among all the reported RdRp inhibitors, remdesivir, favipiravir, and molnupiravir were found to be the most promising drugs against COVID-19. Overall, the structural features of this RNA-Dependent RNA Polymerase (RdRp) inhibitors proved the importance of pyrrolo-triazine and pyrimidine scaffolds. Previous computational models of these drug molecules indicated that substitution with the polar functional group, hydrogen bond donor, and electronegative atoms on these scaffolds may increase the activity against the RdRp protein. Hence, in line with the proposed hypothesis, in the present research work for the evaluation of the hypothesis, new molecules were designed from the pyrrolo-triazine and pyrimidine scaffolds. Further, molecular docking and MD simulation studies were performed with these designed molecules. All these designed molecules (DM-1, DM-2, and DM-3) showed the results as per the proposed hypothesis. Among all the designed molecules, DM-1 showed promising results against the RdRp protein of SARS-CoV-2. In the future, these structural features can be used for the development of new RdRp inhibitors with improved activity. Also, in the future lead compound DM-1 can be explored against the RdRp protein for the treatment of COVID-19. [ABSTRACT FROM AUTHOR]

Given the rapid progression of the coronavirus disease 2019 (COVID‐19) pandemic, an ultrafast response was urgently required to handle this major public crisis. To contain the pandemic, investments are required to develop diagnostic tests, prophylactic vaccines, and novel therapies. Lately, nucleoside analog (NA) antivirals topped the scene as top options for the treatment of COVID‐19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections. Meanwhile, the continuous generation of new lineages of the SARS‐CoV‐2 Omicron variant caused a new challenge in the persistent COVID‐19 battle. Hitting the two crucial SARS‐CoV‐2 enzymes RNA‐dependent RNA polymerase (RdRp) and 3'‐to‐5' exoribonuclease (ExoN) collectively together using only one single ligand is a very successful new approach to stop SARS‐CoV‐2 multiplication and combat COVID‐19 irrespective of the SARS‐CoV‐2 variant type because RdRps and ExoNs are broadly conserved among all SARS‐CoV‐2 strains. Herein, the current comprehensive study investigated most NAs libraries, searching for the most ideal drug candidates expectedly able to perfectly act through this double tactic. Gradual computational filtration gave rise to six different promising NAs, which are riboprine, forodesine, tecadenoson, nelarabine, vidarabine, and maribavir, respectively. Further biological assessment proved for the first time, using the in vitro anti‐RdRp/ExoN and anti‐SARS‐CoV‐2 bioassays, that riboprine and forodesine, among all the six tested NAs, are able to powerfully inhibit the replication of the new virulent strains of SARS‐CoV‐2 with extremely minute in vitro anti‐RdRp and anti‐SARS‐CoV‐2 EC50 values of about 0.22 and 0.49 μM for riboprine and about 0.25 and 0.73 μM for forodesine, respectively, surpassing both remdesivir and the new anti‐COVID‐19 drug molnupiravir. The prior in silico data supported these biochemical findings, suggesting that riboprine and forodesine molecules strongly hit the key catalytic pockets of the SARS‐CoV‐2 (Omicron variant) RdRp′s and ExoN′s main active sites. Additionally, the ideal pharmacophoric features of riboprine and forodesine molecules render them typical dual‐action inhibitors of SARS‐CoV‐2 replication and proofreading, with their relatively flexible structures open for diverse types of chemical derivatization. In Brief, the current important results of this comprehensive study revealed the interesting repurposing potentials of, mainly, the two nucleosides riboprine and forodesine to effectively shut down the polymerase/exoribonuclease‐RNA nucleotides interactions of the SARS‐CoV‐2 Omicron variant and consequently treat COVID‐19 infections, motivating us to rapidly begin the two drugs′ broad preclinical/clinical anti‐COVID‐19 bioevaluations, hoping to combine both drugs soon in the COVID‐19 treatment protocols. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 triggered a worldwide medical crisis, affecting the world’s social, emotional, physical, and economic equilibrium. However, treatment choices and targets for finding a solution to COVID-19’s threat are becoming limited. A viable approach to combating the threat of COVID-19 is by unraveling newer pharmacological and therapeutic targets pertinent in the viral survival and adaptive mechanisms within the host biological milieu which in turn provides the opportunity to discover promising inhibitors against COVID-19. Therefore, using high-throughput virtual screening, manually curated compounds library from some medicinal plants were screened against four main drivers of SARS-CoV-2 (spike glycoprotein, PLpro, 3CLpro, and RdRp). In addition, molecular docking, Prime MM/GBSA (molecular mechanics/generalized Born surface area) analysis, molecular dynamics (MD) simulation, and drug-likeness screening were performed to identify potential phytodrugs candidates for COVID-19 treatment. In support of these approaches, we used a series of computational modeling approaches to develop therapeutic agents against COVID-19. Out of the screened compounds against the selected SARS-CoV-2 therapeutic targets, only compounds with no violations of Lipinski’s rule of five and high binding affinity were considered as potential anti-COVID-19 drugs. However, lonchocarpol A, diplacol, and broussonol E (lead compounds) were recorded as the best compounds that satisfied this requirement, and they demonstrated their highest binding affinity against 3CLpro. Therefore, the 3CLpro target and the three lead compounds were selected for further analysis. Through protein–ligand mapping and interaction profiling, the three lead compounds formed essential interactions such as hydrogen bonds and hydrophobic interactions with amino acid residues at the binding pocket of 3CLpro. The key amino acid residues at the 3CLpro active site participating in the hydrophobic and polar inter/intra molecular interaction were TYR54, PRO52, CYS44, MET49, MET165, CYS145, HIS41, THR26, THR25, GLN189, and THR190. The compounds demonstrated stable protein–ligand complexes in the active site of the target (3CLpro) over a 100 ns simulation period with stable protein–ligand trajectories. Drug-likeness screening shows that the compounds are druggable molecules, and the toxicity descriptors established that the compounds demonstrated a good biosafety profile. Furthermore, the compounds were chemically reactive with promising molecular electron potential properties. Collectively, we propose that the discovered lead compounds may open the way for establishing phytodrugs to manage COVID-19 pandemics and new chemical libraries to prevent COVID-19 entry into the host based on the findings of this computational investigation.

Viruses have multiple mutation rates that are higher than any other member of the kingdom of life. This gives them the ability to evolve, even within the course of a single infection, and to evade multiple host defenses, thereby impacting pathogenesis. Additionally, there are also interplays between mutation and recombination and the high multiplicity of infection (MOI) that enhance viral adaptability and increase levels of recombination leading to complex and conflicting effects on genome selection, and the net results is difficult to predict. Recently, the outbreak of COVID-19 virus represents a pandemic threat that has been declared a public health emergency of international concern. Up to present, however, due to the high mutation rate of COVID-19 virus, there are no effective procedures to contain the spread of this virus across the globe. For such a purpose, there is then an urgent need to explore new approaches. As an opinion, the present approach emphasizes on (a) the use of a nonspecific way of blocking the entry of COVID-19 virus as well as its variants into the cells via a therapeutic biocompatible compound (ideally, "in a pill") targeting its spike (S) glycoprotein; and (b) the construction of expression vectors via the glycosyl-phosphatidylinositol, GPI, anchor for studying intermolecular interactions between the spike S of COVID-19 virus as well as its variants and the angiotensin-converting enzyme 2 (ACE2) of its host receptor for checking the efficacy of any therapeutic biocompatible compound of the nonspecific way of blocking. Such antiviral drug would be safer than the ACE1 and ACE2 inhibitors/angiotensin receptor blockers, and recombinant human ACE2 as well as nucleoside analogs or protease inhibitors used for fighting the spread of the virus inside the cells, and it would also be used as a universal one for any eventual future pandemic related to viruses, especially the RNA viruses with high mutation rates. [ABSTRACT FROM AUTHOR]

• (Re)emerging RNA viruses are responsible for highly fatal viral diseases. • Ribonucleoside analogs for clinical use in the treatment of RNA viruses. • Targeting viral RNA-dependent RNA polymerase. • Structure-activity relationship for RdRp inhibitory nucleosides. • Challenges in the design of new nucleosides. With several US Food and Drug Administration (FDA)-approved drugs and high barriers to resistance, nucleoside and nucleotide analogs remain the cornerstone of antiviral therapies for not only herpesviruses, but also HIV and hepatitis viruses (B and C); however, with the exception of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which vaccines have been developed at unprecedented speed, there are no vaccines or small antivirals yet available for (re)emerging viruses, which are primarily RNA viruses. Thus, herein, we present an overview of ribonucleoside analogs recently developed and acting as inhibitors of the viral RNA-dependent RNA polymerase (RdRp). They are new lead structures that will be exploited for the discovery of new antiviral nucleosides. [ABSTRACT FROM AUTHOR]

Respiratory Syncytial Virus (RSV) is the top cause of infant hospitalization globally, with no effective treatments available. Researchers have sought small molecules to target the RNA-dependent RNA Polymerase (RdRP) of RSV, which is essential for replication and transcription. Based on the cryo-EM structure of the RSV polymerase, in silico computational analysis including molecular docking and the protein-ligand simulation of a database, including 6554 molecules, is currently undergoing phases 1–4 of clinical trials and has resulted in the top ten repurposed compound candidates against the RSV polymerase, including Micafungin, Totrombopag, and Verubecestat. We performed the same procedure to evaluate 18 small molecules from previous studies and chose the top four compounds for comparison. Among the top identified repurposed compounds, Micafungin, an antifungal medication, showed significant inhibition and binding affinity improvements over current inhibitors such as ALS-8112 and Ribavirin. We also validated Micafungin’s inhibition of the RSV RdRP using an in vitro transcription assay. These findings contribute to RSV drug development and hold promise for broad-spectrum antivirals targeting the non-segmented negative-sense (NNS) RNA viral polymerases, including those of rabies (RABV) and Ebola (EBOV).

In an effort to identify functional-energetic correlations leading to the development of efficient anti-SARS-CoV-2 therapeutic agents, we have designed synthetic analogs of aurintricarboxylic acid (ATA), a heterogeneous polymeric mixture of structurally related linear homologs known to exhibit a host of biological properties, including antiviral activity. These derivatives are evaluated for their ability to interact with a plasma transporter protein (human serum albumin), eukaryotic (yeast) ribosomes, and a SARS-CoV-2 target, the RNA-dependent RNA polymerase (RdRp). The resultant data are critical for characterizing drug distribution, bioavailability, and effective inhibition of host and viral targets. Promising lead compounds are selected on the basis of their binding energetics which have been characterized and correlated with functional activities as assessed by inhibition of RNA replication and protein synthesis. Our results reveal that the activity of heterogeneous ATA is mimicked by linear compounds of defined molecular weight, with a dichlorohexamer salicylic-acid derivative exhibiting the highest potency. These findings are instrumental for optimizing the design of structurally defined ATA analogs that fulfill the requirements of an antiviral drug with respect to bioavailability, homogeneity, and potency, thereby expanding the arsenal of therapeutic regimens that are currently available to address the urgent need for effective SARS-CoV-2 treatment strategies. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 (severe acute respiratory syndrome coronavirus‐2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA virus that relies on RNA‐dependent RNA polymerase (RdRp) activity in viral transcription and replication. Due to its high sequence and structural conservation in coronavirus and new SARS-CoV-2 variants, RdRp has been recognized as the key therapeutic target to design novel antiviral strategies. Nucleotide analogs (NAs), such as remdesivir, is the most promising class of RdRp inhibitors to be used in the treatment of COVID-19. However, the presence of exonucleases in SARS-CoV-2 caused a great challenge to NAs; the excision of incorporated NAs will lead to viral resistance to this group of inhibitors. Here, we expressed active RdRp protein in both a eukaryotic expression system of baculovirus-infected insect cells and a prokaryotic expression system of Escherichia coli cells. Nsp7 and nsp8 of the functional RdRp holoenzyme were generated in E. coli. An in vitro RdRp activity assay has been established with a reconstituted nsp12/nsp7/nsp8 complex and biotin-labeled self-priming RNAs, and the activity of the RdRp complex was determined by detecting binding and extension of RNAs. Moreover, to meet the needs of high-throughput drug screening, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex, which is also suitable for testing in 96-well plates. We demonstrated that the active triphosphate form of remdesivir (RTP) and several reported non-nucleotide analog viral polymerase inhibitors blocked the RdRp in the in vitro RdRp activity assay and high-throughput screening model. This high-throughput screening model has been applied to a custom synthetic chemical and natural product library of thousands of compounds for screening SARS-CoV-2 RdRp inhibitors. Our efficient RdRp inhibitor discovery system provides a powerful platform for the screening, validation, and evaluation of novel antiviral molecules targeting SARS-CoV-2 RdRp, particularly for non-nucleotide antivirals drugs (NNAs). [ABSTRACT FROM AUTHOR]

SARS-CoV-2 (severe acute respiratory syndrome coronavirus‐2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA virus that relies on RNA‐dependent RNA polymerase (RdRp) activity in viral transcription and replication. Due to its high sequence and structural conservation in coronavirus and new SARS-CoV-2 variants, RdRp has been recognized as the key therapeutic target to design novel antiviral strategies. Nucleotide analogs (NAs), such as remdesivir, is the most promising class of RdRp inhibitors to be used in the treatment of COVID-19. However, the presence of exonucleases in SARS-CoV-2 caused a great challenge to NAs; the excision of incorporated NAs will lead to viral resistance to this group of inhibitors. Here, we expressed active RdRp protein in both a eukaryotic expression system of baculovirus-infected insect cells and a prokaryotic expression system of Escherichia coli cells. Nsp7 and nsp8 of the functional RdRp holoenzyme were generated in E. coli. An in vitro RdRp activity assay has been established with a reconstituted nsp12/nsp7/nsp8 complex and biotin-labeled self-priming RNAs, and the activity of the RdRp complex was determined by detecting binding and extension of RNAs. Moreover, to meet the needs of high-throughput drug screening, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex, which is also suitable for testing in 96-well plates. We demonstrated that the active triphosphate form of remdesivir (RTP) and several reported non-nucleotide analog viral polymerase inhibitors blocked the RdRp in the in vitro RdRp activity assay and high-throughput screening model. This high-throughput screening model has been applied to a custom synthetic chemical and natural product library of thousands of compounds for screening SARS-CoV-2 RdRp inhibitors. Our efficient RdRp inhibitor discovery system provides a powerful platform for the screening, validation, and evaluation of novel antiviral molecules targeting SARS-CoV-2 RdRp, particularly for non-nucleotide antivirals drugs (NNAs).

Objectives: the COVID-19 pandemic triggering acute respiratory syndrome has become a major global health concern. After one year into this pandemic, special therapies for COVID-19 remain an unprecedented challenge to mankind and finding drugs to treat this disease is extremely urgent. The SARS-CoV-2 RNAdependent RNA polymerase (RdRp) enzyme that regulates viral replication has been examined as a potential therapeutic target for the inhibition of SARS-CoV-2 infection. In this study, the authors evaluated the ability of RNA-dependent RNA polymerase drug inhibitors by using an in silico molecular docking model. Methods: the 3D structure of RdRp enzyme (PDB ID:6M71, resolution of 2.90 Å) was derived from the Protein Data Bank RCSB. The ligand structures were collected from DrugBank for the RdRp target. Molecular docking was done by AutoDock Vina software. Lipinski’s rule of five is used to compare compounds with drug-like and nondrug-like properties. Pharmacokinetic parameters of potential compounds were evaluated using the pkCSM tool. Results: based on the DrugBank database, we collected 192 antiviral molecules and compared them to remdesivir, which has inhibitory activity with this protein target. Results showed that 26 out of 192 compounds have a higher ability to inhibit the SARS-CoV-2 RdRp enzyme than remdesivir. Next, 6 drugs were selected by visually inspecting the docking results with focus on the main interaction between crucial residues at the binding site of the SARS-CoV-2 RdRp enzyme. For the visual inspection, the existence of polar interactions with ASP760 and ASP761 were utilised as the preference criterion. Finally, Lipinski’s rule of 5 criteria and absorption, distribution, metabolism, excretion and toxicity (ADMET) profile analysis suggested five drugs that have good pharmacokinetic properties. Conclusions: these drugs were dihydroergotamine, sofosbuvir, nilotinib, tipranavir, and darunavir and may be used as anti-SARS-CoV-2 agents.

The development of potent non-nucleoside inhibitors (NNIs) could be an alternate strategy to combating infectious bovine viral diarrhea virus (BVDV), other than the traditional vaccination. RNA-dependent RNA polymerase (RdRp) is an essential enzyme for viral replication; therefore, it is one of the primary targets for countermeasures against infectious diseases. The reported NNIs, belonging to the classes of quinolines (2h: imidazo[4,5-g]quinolines and 5m: pyrido[2,3-g] quinoxalines), displayed activity in cell-based and enzyme-based assays. Nevertheless, the RdRp binding site and microscopic mechanistic action are still elusive, and can be explored at a molecular level. Here, we employed a varied computational arsenal, including conventional and accelerated methods, to identify quinoline compounds’ most likely binding sites. Our study revealed A392 and I261 as the mutations that can render RdRp resistant against quinoline compounds. In particular, for ligand 2h, mutation of A392E is the most probable mutation. The loop L1 and linker of the fingertip is recognized as a pivotal structural determinant for the stability and escape of quinoline compounds. Overall, this work demonstrates that the quinoline inhibitors bind at the template entrance channel, which is governed by conformational dynamics of interactions with loops and linker residues, and reveals structural and mechanistic insights into inhibition phenomena, for the discovery of improved antivirals.

Since the emergence of SARS-CoV-2, mutations in all subunits of the RNA-dependent RNA polymerase (RdRp) of the virus have been repeatedly reported. Although RdRp represents a primary target for antiviral drugs, experimental studies exploring the phenotypic effect of these mutations have been limited. This study focuses on the phenotypic effects of substitutions in the three RdRp subunits: nsp7, nsp8, and nsp12, selected based on their occurrence rate and potential impact. We employed nano-differential scanning fluorimetry and microscale thermophoresis to examine the impact of these mutations on protein stability and RdRp complex assembly. We observed diverse impacts; notably, a single mutation in nsp8 significantly increased its stability as evidenced by a 13°C increase in melting temperature, whereas certain mutations in nsp7 and nsp8 reduced their binding affinity to nsp12 during RdRp complex formation. Using a fluorometric enzymatic assay, we assessed the overall effect on RNA polymerase activity. We found that most of the examined mutations altered the polymerase activity, often as a direct result of changes in stability or affinity to the other components of the RdRp complex. Intriguingly, a combination of nsp8 A21V and nsp12 P323L mutations resulted in a 50% increase in polymerase activity. To our knowledge, this is the first biochemical study to demonstrate the impact of amino acid mutations across all components constituting the RdRp complex in emerging SARS-CoV-2 subvariants., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Summary: Exportin 1/XPO1 is an important nuclear export receptor that binds directly to cargo proteins and translocates the cargo proteins to the cytoplasm. To understand XPO1 protein functions during potyvirus infections, we investigated the nuclear export of the NIb protein encoding the RNA‐dependent RNA polymerase (RdRp) of turnip mosaic virus (TuMV).Previously, we found that NIb is transported to the nucleus after translation and sumoylated by the sumoylation (small ubiquitin‐like modifier) pathway to support viral infection. Here, we report that XPO1 interacts with NIb to facilitate translocation from the nucleus to the viral replication complexes (VRCs) that accumulate in the perinuclear regions of TuMV‐infected cells. XPO1 contains two NIb‐binding domains that recognize and interact with NIb in the nucleus and in the perinuclear regions, respectively, which facilitates TuMV replication.Moreover, XPO1 is involved in nuclear export of the sumoylated NIb and host factors tagged with SUMO3 that is essential for suppression of plant immunity in the nucleus. Deficiencies of XPO1 in Arabidopsis and Nicotiana benthamiana plants inhibit TuMV replication and infection.These data demonstrate that XPO1 functions as a host factor in TuMV infection by regulating NIb nucleocytoplasmic transport and plant immunity. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a significant threat to infants and elderly individuals globally. Currently, there are no effective therapies or treatments for RSV infection because of an insufficient understanding of the RSV viral machinery. In this study, we investigated the effects of the template variations on RNA synthesis by the RSV polymerase through in vitro RNA synthesis assays. We confirmed the previously reported back-priming activity of the RSV polymerase, which is likely due to the secondary structure of the RNA template. We found that the expansion of the hairpin loop size of the RNA template abolishes the RSV polymerase back-priming activity. At the same time, it seemingly does not affect the de novo RNA synthesis activities of the RSV polymerase. Interestingly, our results show that the RSV polymerase also has a new primer-based terminal extension activity that adds nucleotides to the template and primer in a nonspecific manner. We also mapped the impact of the RNA 5′ chemical group on its mobility in a urea-denaturing RNA gel shift assay. Overall, these results enhance our knowledge about the RNA synthesis processes of the RSV polymerase and may guide future therapeutic efforts to develop effective antiviral drugs for RSV treatment.

Foot-and-mouth disease virus (FMDV), an economically important pathogen of cloven-hoofed livestock, is a positive-sense, single-stranded RNA virus classified in the Picornaviridae family. RNA-dependent RNA polymerase (RdRp) of RNA viruses is highly conserved. Compounds that bind to the RdRp active site can block viral replication. Herein, we combined double virtual screenings and cell-based antiviral approaches to screen and identify potential inhibitors targeting FMDV RdRp (3Dpol). From 5596 compounds, the blind- followed by focus-docking filtered 21 candidates fitting in the 3Dpol active sites. Using the BHK-21 cell-based assay, we found that four compounds—NSC217697 (quinoline), NSC670283 (spiro compound), NSC292567 (nigericin), and NSC65850—demonstrated dose-dependent antiviral actions in vitro with the EC50 ranging from 0.78 to 3.49 µM. These compounds could significantly block FMDV 3Dpol activity in the cell-based 3Dpol inhibition assay with small IC50 values ranging from 0.8 nM to 0.22 µM without an effect on FMDV’s main protease, 3Cpro. The 3Dpol inhibition activities of the compounds were consistent with the decreased viral load and negative-stranded RNA production in a dose-dependent manner. Conclusively, we have identified potential FMDV 3Dpol inhibitors that bound within the enzyme active sites and blocked viral replication. These compounds might be beneficial for FMDV or other picornavirus treatment.

Designing anticoronavirus disease 2019 (anti-COVID-19) agents is the primary concern of medicinal chemists/drug designers nowadays. Repurposing of known active compounds against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new effective and time-saving trend in anti-COVID-19 drug discovery. Thorough inhibition of the coronaviral-2 proteins (i.e., multitarget inhibition) is a possible powerful favorable strategy for developing effectively potent drugs for COVID-19. In this new research study, I succeeded to repurpose the two antioxidant polyhydroxy-1,3,4-oxadiazole compounds CoViTris2020 and ChloViD2020 as the first multitarget coronaviral protein blockers with extremely higher potencies (reach about 65 and 304 times, for CoViTris2020, and 20 and 93 times, for ChloViD2020, more potent than remdesivir and favipiravir, respectively). These two 2,5-disubstituted-1,3,4-oxadiazoles were computationally studied (through molecular docking in almost all SARS-CoV-2 proteins) and biologically assessed (through a newly established robust in vitro anti-COVID-19 assay) for their anticoronaviral-2 bioactivities. The data obtained from the docking investigation showed that both ligands promisingly exhibited very strong inhibitory binding affinities with almost all docked enzymes (e.g., they displayed extremely lower binding energies of − 12.00 and − 9.60 kcal/mol, respectively, with the SARS-CoV-2 RNA-dependent RNA polymerase "RdRp"). The results of the biological assay revealed that CoViTris2020 and ChloViD2020 significantly displayed very high anti-COVID-19 activities (anti-SARS-CoV-2 EC50 = 0.31 and 1.01 μM, respectively). Further in vivo/clinical studies for the development of CoViTris2020 and ChloViD2020 as anti-COVID-19 medications are required. In brief, the ascent of CoViTris2020 and ChloViD2020 as the two lead members of the novel family of anti-COVID-19 polyphenolic 2,5-disubstituted-1,3,4-oxadiazole derivatives represents a promising hope in COVID-19 therapy. CoViTris2020 and ChloViD2020 inhibit SARS-CoV-2 life cycle with surprising EC50 values of 0.31 and 1.01 μM, respectively. CoViTris2020 strongly inhibits coronaviral-2 RdRp with exceptionally lower inhibitory binding energy of − 12.00 kcal/mol. [ABSTRACT FROM AUTHOR]

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 drug-like 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.

In an effort to identify functional-energetic correlations leading to the development of efficient anti-SARS-CoV-2 therapeutic agents, we have designed synthetic analogs of aurintricarboxylic acid (ATA), a heterogeneous polymeric mixture of structurally related linear homologs known to exhibit a host of biological properties, including antiviral activity. These derivatives are evaluated for their ability to interact with a plasma transporter protein (human serum albumin), eukaryotic (yeast) ribosomes, and a SARS-CoV-2 target, the RNA-dependent RNA polymerase (RdRp). The resultant data are critical for characterizing drug distribution, bioavailability, and effective inhibition of host and viral targets. Promising lead compounds are selected on the basis of their binding energetics which have been characterized and correlated with functional activities as assessed by inhibition of RNA replication and protein synthesis. Our results reveal that the activity of heterogeneous ATA is mimicked by linear compounds of defined molecular weight, with a dichlorohexamer salicylic-acid derivative exhibiting the highest potency. These findings are instrumental for optimizing the design of structurally defined ATA analogs that fulfill the requirements of an antiviral drug with respect to bioavailability, homogeneity, and potency, thereby expanding the arsenal of therapeutic regimens that are currently available to address the urgent need for effective SARS-CoV-2 treatment strategies.

A novel coronavirus disease (COVID-19) caused by SARS-CoV2 has now spread globally. Replication/transcription machinery of this virus consists of RNA-dependent RNA polymerase (nsp12 or RdRp) and its two cofactors nsp7 and nsp8 proteins. Hence, RdRp has emerged as a promising target to control COVID-19. In the present study, we are reporting a novel inhibitor VTRM1.1 against the RdRp protein of SARS CoV2. A series of antivirals were tested for binding to the catalytic residues of the active site of RdRp protein. In-silico screening, molecular mechanics, molecular dynamics simulation (MDS) analysis suggest ribavirin, and remdesivir have good interaction with the binding site of the RdRp protein as compared to other antiviral investigated. Hence, ribavirin and remdesivir were used for the denovo fragments based antiviral design. This design, along with docking and MDS analysis, identified a novel inhibitor VTRM1 that has better interaction with RdRp as compared to their parent molecules. Further, to produce a lead-like compound, retrosynthetic analysis, and combinatorial synthesis were performed, which produces 1000 analogs of VTRM1. These analogs were analysed by docking and MDS analysis that identified VTRM1.1 as a possible lead to inhibit RdRp protein. This lead has a good docking score, favourable binding energy and bind at catalytic residues of the active site of RdRp. The VTRM1.1 also interacts with RdRp in the presence of RNA primer and other cofactors. It was also seen that, VTRM1.1 do not have off-target in human. Therefore, the present study suggests a hybrid inhibitor VTRM1.1 for the RNA-dependent RNA polymerase of SARS CoV2 that may be useful to control infection caused by COVID-19. [ABSTRACT FROM AUTHOR]

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.

Summary: Autophagy is an evolutionarily conserved pathway in eukaryotes that delivers unwanted cytoplasmic materials to the lysosome/vacuole for degradation/recycling. Stimulated autophagy emerges as an integral part of plant immunity against intracellular pathogens.In this study, we used turnip mosaic virus (TuMV) as a model to investigate the involvement of autophagy in plant RNA virus infection.The small integral membrane protein 6K2 of TuMV, known as a marker of the virus replication site and an elicitor of the unfolded protein response (UPR), upregulates the selective autophagy receptor gene NBR1 in a UPR‐dependent manner. NBR1 interacts with TuMV NIb, the RNA‐dependent RNA polymerase of the virus replication complex (VRC), and the autophagy cargo receptor/adaptor protein ATG8f. The NIb/NBR1/ATG8f interaction complexes colocalise with the 6K2‐stained VRC. Overexpression of NBR1 or ATG8f enhances TuMV replication, and deficiency of NBR1 or ATG8f inhibits virus infection. In addition, ATG8f interacts with the tonoplast‐specific protein TIP1 and the NBR1/ATG8f‐containing VRC is enclosed by the TIP1‐labelled tonoplast. In TuMV‐infected cells, numerous membrane‐bound viral particles are evident in the vacuole.Altogether these results suggest that TuMV activates and manipulates UPR‐dependent NBR1‐ATG8f autophagy to target the VRC to the tonoplast to promote viral replication and virion accumulation. [ABSTRACT FROM AUTHOR]

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants. Image 1 • A scFv library previously screened in a Y2H system against a human rotavirus was tested against CMV 2a protein derivatives. • Positive scFvs were analyzed for their interaction with CMV 2a protein derivatives in mammalian transient expression system. • Tobacco plants transformed with four selected scFvs had reduced levels of CMV and showed premature recovery from infection. [ABSTRACT FROM AUTHOR]

The emergence of the Zika virus, which belongs to the Flaviviridae family, became a significant worldwide health issue due to its link with severe neurological complications. The RNA-dependent RNA polymerase (RdRp) of the Zika virus plays a significant part in the replication of the virus and is considered a promising candidate for antiviral drug identification. In this study, we employed computer-based drug discovery approaches to identify potential natural compounds that could act as inhibitors against the RdRp protein of the Zika virus. A comprehensive virtual screening strategy was implemented using the MTiOpenScreen webserver to identify natural compounds from the NP-Lib database. Four natural compounds having the ZINC ID - ZINC000253499147, ZINC000299817665, ZINC000044404209, and ZINC000253388535 were selected based on the binding score revealed during virtual screening. Molecular docking simulations of these selected compounds and reference compounds were performed to assess the binding affinities and the molecular bonds formed during the docking. Additionally, molecular dynamics (MD) simulations, endpoint free binding energy calculation and principal component analysis (PCA) were performed to evaluate the stability and dynamics of the protein-ligand complexes. These compounds exhibited favourable binding energies and formed stable interactions within the active site of the RdRp protein. Moreover, the molecular dynamics simulations revealed the robustness of the protein-ligand complexes, suggesting the potential for sustained inhibition. These findings provide valuable insights for the design and development of novel therapeutic interventions against Zika virus infection. Further experimental validation and optimization of the identified compounds are warranted to advance their potential translation into effective antiviral drugs.Communicated by Ramaswamy H. Sarma.