Computational Chemistry to Repurposing Drugs for the Control of COVID-19

Thus far, in 2021, 219 countries with over 175 million people have been infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a positive sense, single-stranded RNA virus, and is the causal agent for coronavirus disease (COVID-19). Due to the urgency of the situation, virtual screening as a computational modeling method offers a fast and effective modality of identifying drugs that may be effective against SARS-CoV-2. There has been an overwhelming abundance of molecular docking against SARS-CoV-2 in the last year. Due to the massive volume of computational studies, this systematic review has been created to evaluate and summarize the findings of existing studies. Herein, we report on computational articles of drugs which target, (1) viral protease, (2) Spike protein-ACE 2 interaction, (3) RNA-dependent RNA polymerase, and (4) other proteins and nonstructural proteins of SARS-CoV-2. Based on the studies presented, there are 55 identified natural or drug compounds with potential anti-viral activity. The next step is to show anti-viral activity in vitro and translation to determine effectiveness into human clinical trials.

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RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽

10.3390/v13091738 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1738

Author(s):

Alesia A. Levanova ◽

Eeva J. Vainio ◽

Jarkko Hantula ◽

Minna M. Poranen

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Rna Virus ◽

Polymerization Reaction ◽

Rna Dependent Rna Polymerase ◽

Independent Manner ◽

Nucleotidyl Transferase ◽

Rna Polymerization ◽

The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

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Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

10.1101/2021.04.07.438807 ◽

2021 ◽

Author(s):

Agustina P. Bertolin ◽

Florian Weissmann ◽

Jingkun Zeng ◽

Viktor Posse ◽

Jennifer C. Milligan ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Virus ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Etiologic Agent ◽

Host Cells ◽

Rdrp Activity ◽

Chemical Library ◽

Rna Dependent Rna Polymerase

SummaryThe coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

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Molecular Characterization of a Novel Virga-like Virus Isolated From Wheat

10.21203/rs.3.rs-1177163/v1 ◽

2021 ◽

Author(s):

Hua Li ◽

Jun Guo ◽

ZhongHua Zhao ◽

Zhuangxin Ye ◽

Jianping Chen ◽

...

Keyword(s):

Rna Virus ◽

Open Reading Frames ◽

Polya Tail ◽

Rna Dependent Rna Polymerase ◽

Future Studies ◽

Putative Protein ◽

Host Growth ◽

Positive Sense ◽

Reading Frames

Abstract In this work, we report the isolation of a novel positive-sense single strand RNA virus from wheat, tentatively named Triticum aestivum-associated virga-like virus 1 (TaAVLV1). Further characterization revealed that the complete genome of TaAVLV1 was divided into two segments, RNA1 and RNA2, which were 3530 and 3466 nt long, excluding the polyA tail. These segments contained two open reading frames (ORFs). The ORF in RNA1 encoded an RNA-dependent RNA polymerase (RdRp), while the ORF in RNA2 encoded a putative protein carrying MET and HEL domains. Phylogenetic analysis based on the RdRp protein of each representative genus of Virgaviridae placed TaAVLV1 in the unclassified Virgaviridae clade of the Virgaviridae family. To our knowledge, this is the first report of virga-like virus isolated from wheat. Future studies will be conducted to examine its effect on host growth and development.

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Functional Characterization of the Cleavage Specificity of the Sapovirus Chymotrypsin-Like Protease

Journal of Virology ◽

10.1128/jvi.00693-08 ◽

2008 ◽

Vol 82 (16) ◽

pp. 8085-8093 ◽

Cited By ~ 7

Author(s):

Ivonne Robel ◽

Julia Gebhardt ◽

Jeroen R. Mesters ◽

Alexander Gorbalenya ◽

Bruno Coutard ◽

...

Keyword(s):

High Performance ◽

Molecular Mechanisms ◽

Rna Virus ◽

Functional Characterization ◽

Mass Spectrometry Analysis ◽

Viral Protease ◽

Spectrometry Analysis ◽

Cleavage Specificity ◽

Scissile Bond

ABSTRACT Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2′ positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2′ positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.

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Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

Biochemical Journal ◽

10.1042/bcj20210200 ◽

2021 ◽

Vol 478 (13) ◽

pp. 2425-2443 ◽

Cited By ~ 1

Author(s):

Agustina P. Bertolin ◽

Florian Weissmann ◽

Jingkun Zeng ◽

Viktor Posse ◽

Jennifer C. Milligan ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Virus ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Etiologic Agent ◽

Host Cells ◽

Rdrp Activity ◽

Chemical Library ◽

Rna Dependent Rna Polymerase

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication–transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

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Pharmacological Therapeutics Targeting RNA-Dependent RNA Polymerase, Proteinase and Spike Protein: From Mechanistic Studies to Clinical Trials for COVID-19

Journal of Clinical Medicine ◽

10.3390/jcm9041131 ◽

2020 ◽

Vol 9 (4) ◽

pp. 1131 ◽

Cited By ~ 35

Author(s):

Jiansheng Huang ◽

Wenliang Song ◽

Hui Huang ◽

Quancai Sun

Keyword(s):

Rna Polymerase ◽

The United States ◽

Spike Protein ◽

Therapeutic Approaches ◽

Rna Dependent Rna Polymerase ◽

S Protein ◽

Functional Relationships ◽

Human Lung Cells ◽

Novel Coronavirus

An outbreak of novel coronavirus-related pneumonia COVID-19, that was identified in December 2019, has expanded rapidly, with cases now confirmed in more than 211 countries or areas. This constant transmission of a novel coronavirus and its ability to spread from human to human have prompted scientists to develop new approaches for treatment of COVID-19. A recent study has shown that remdesivir and chloroquine effectively inhibit the replication and infection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, 2019-nCov) in vitro. In the United States, one case of COVID-19 was successfully treated with compassionate use of remdesivir in January of 2020. In addition, a clinically proven protease inhibitor, camostat mesylate, has been demonstrated to inhibit Calu-3 infection with SARS-CoV-2 and prevent SARS-2-spike protein (S protein)-mediated entry into primary human lung cells. Here, we systemically discuss the pharmacological therapeutics targeting RNA-dependent RNA polymerase (RdRp), proteinase and S protein for treatment of SARS-CoV-2 infection. This review should shed light on the fundamental rationale behind inhibition of SARS-CoV-2 enzymes RdRp as new therapeutic approaches for management of patients with COVID-19. In addition, we will discuss the viability and challenges in targeting RdRp and proteinase, and application of natural product quinoline and its analog chloroquine for treatment of coronavirus infection. Finally, determining the structural-functional relationships of the S protein of SARS-CoV-2 will provide new insights into inhibition of interactions between S protein and angiotensin-converting enzyme 2 (ACE2) and enable us to develop novel therapeutic approaches for novel coronavirus SARS-CoV-2.

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Comparison of Turnip Crinkle Virus RNA-Dependent RNA Polymerase Preparations Expressed in Escherichia coli or Derived from Infected Plants

Journal of Virology ◽

10.1128/jvi.76.4.1707-1717.2002 ◽

2002 ◽

Vol 76 (4) ◽

pp. 1707-1717 ◽

Cited By ~ 57

Author(s):

K. S. Rajendran ◽

J. Pogany ◽

P. D Nagy

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

De Novo ◽

Rna Virus ◽

In Vitro Assays ◽

Turnip Crinkle Virus ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

ABSTRACT Turnip crinkle virus (TCV) is a small, plus-sense, single-stranded RNA virus of plants. A virus-coded protein, p88, which is required for replication has been expressed and purified from Escherichia coli. In vitro assays revealed that the recombinant p88 has an RNA-dependent RNA polymerase (RdRp) activity and can also bind to RNA. Deletion of the N-terminal region in p88 resulted in a more active RdRp, while further deletions abolished RdRp activity. Comparison of the E. coli-expressed p88, the N-terminal deletion mutant of p88, and a TCV RdRp preparation obtained from infected plants revealed that these preparations show remarkable similarities in RNA template recognition and usage. Both the recombinant and the plant TCV RdRp preparations are capable of de novo initiation on both plus- and minus-strand satC and satD templates, which are small parasitic RNAs associated with TCV infections. In addition, these RdRp preparations can efficiently recognize the related Tomato bushy stunt virus promoter sequences, including the minus- and plus-strand initiation promoters. Heterologous viral and artificial promoters are recognized poorly by the recombinant and the plant TCV RdRps. Further comparison of the single-component recombinant TCV RdRp and the multicomponent plant TCV RdRp will help dissect the functions of various components of the TCV replicase.

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CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands

International Journal of Molecular Sciences ◽

10.3390/ijms22052614 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2614

Author(s):

Georgina Bezzi ◽

Ernesto J. Piga ◽

Andrés Binolfi ◽

Pablo Armas

Keyword(s):

Rna Virus ◽

Cellular Protein ◽

Molecular Approaches ◽

Genes Expression ◽

Positive Sense ◽

Replicative Intermediates ◽

Rna Genome ◽

First Time ◽

Negative Sense

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (−gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the −gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.

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Identification of a Conserved RNA-dependent RNA Polymerase (RdRp)-RNA Interface Required for Flaviviral Replication

Journal of Biological Chemistry ◽

10.1074/jbc.m116.724013 ◽

2016 ◽

Vol 291 (33) ◽

pp. 17437-17449 ◽

Cited By ~ 19

Author(s):

Kenneth Hodge ◽

Chairat Tunghirun ◽

Maliwan Kamkaew ◽

Thawornchai Limjindaporn ◽

Pa-thai Yenchitsomanus ◽

...

Keyword(s):

Dengue Virus ◽

Rna Polymerase ◽

Viral Protein ◽

Rna Virus ◽

Coding Region ◽

Rna Dependent Rna Polymerase ◽

Protein Coding ◽

The World ◽

Positive Sense ◽

Rna Motif

Dengue virus, an ∼10.7-kb positive-sense RNA virus, is the most common arthropod-communicated pathogen in the world. Despite dengue's clear epidemiological importance, mechanisms for its replication remain elusive. Here, we probed the entire dengue genome for interactions with viral RNA-dependent RNA polymerase (RdRp), and we identified the dominant interaction as a loop-forming ACAG motif in the 3′ positive-stranded terminus, complicating the prevailing model of replication. A subset of interactions coincides with known flaviviral recombination sites inside the viral protein-coding region. Specific recognition of the RNA element occurs via an arginine patch in the C-terminal thumb domain of RdRp. We also show that the highly conserved nature of the consensus RNA motif may relate to its tolerance to various mutations in the interacting region of RdRp. Disruption of the interaction resulted in loss of viral replication ability in cells. This unique RdRp-RNA interface is found throughout flaviviruses, implying possibilities for broad disease interventions.

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RNA ligands activate the Machupo virus polymerase and guide promoter usage

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1900790116 ◽

2019 ◽

Vol 116 (21) ◽

pp. 10518-10524 ◽

Cited By ~ 8

Author(s):

Jesse D. Pyle ◽

Sean P. J. Whelan

Keyword(s):

Rna Polymerase ◽

Rna Binding ◽

Rna Virus ◽

Binding Pocket ◽

Genomic Rna ◽

Rna Dependent Rna Polymerase ◽

Vitro Assay ◽

Guanine Residue ◽

Rna Ligands

Segmented negative-sense (SNS) RNA viruses initiate infection by delivering into cells a suite of genomic RNA segments, each sheathed by the viral nucleocapsid protein and bound by the RNA-dependent RNA-polymerase (RdRP). For the orthomyxovirus influenza and the bunyavirus La Crosse, the 5′ end of the genomic RNA binds as a hook-like structure proximal to the active site of the RdRP. Using an in vitro assay for the RNA-dependent RNA-polymerase (RdRP) of the arenavirus Machupo (MACV), we demonstrate that the 5′ genomic and antigenomic RNAs of both small and large genome segments stimulate activity in a promoter-specific manner. Functional probing of the activating RNAs identifies intramolecular base-pairing between positions +1 and +7 and a pseudotemplated 5′ terminal guanine residue as key for activation. Binding of structured 5′ RNAs is a conserved feature of all SNS RNA virus polymerases, implying that promoter-specific RdRP activation extends beyond the arenaviruses. The 5′ RNAs and the RNA binding pocket itself represent targets for therapeutic intervention.

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