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1.
Molecules ; 27(1)2021 Dec 30.
Article in English | MEDLINE | ID: covidwho-1580564

ABSTRACT

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


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Computational Biology/methods , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Evaluation, Preclinical , Flavonoids/pharmacology , SARS-CoV-2/enzymology , Allosteric Site , COVID-19/virology , Catalytic Domain , Drug Design , Humans , Intestinal Absorption , Molecular Docking Simulation
2.
Elife ; 102021 10 07.
Article in English | MEDLINE | ID: covidwho-1456505

ABSTRACT

The absence of 'shovel-ready' anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.


To multiply and spread from cell to cell, the virus responsible for COVID-19 (also known as SARS-CoV-2) must first replicate its genetic information. This process involves a 'polymerase' protein complex making a faithful copy by assembling a precise sequence of building blocks, or nucleotides. The only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), remdesivir, consists of a nucleotide analog, a molecule whose structure is similar to the actual building blocks needed for replication. If the polymerase recognizes and integrates these analogs into the growing genetic sequence, the replication mechanism is disrupted, and the virus cannot multiply. Most approaches to study this process seem to indicate that remdesivir works by stopping the polymerase and terminating replication altogether. Yet, exactly how remdesivir and other analogs impair the synthesis of new copies of the virus remains uncertain. To explore this question, Seifert, Bera et al. employed an approach called magnetic tweezers which uses a magnetic field to manipulate micro-particles with great precision. Unlike other methods, this technique allows analogs to be integrated under conditions similar to those found in cells, and to be examined at the level of a single molecule. The results show that contrary to previous assumptions, remdesivir does not terminate replication; instead, it causes the polymerase to pause and backtrack (which may appear as termination in other techniques). The same approach was then applied to other nucleotide analogs, some of which were also found to target the SARS-CoV-2 polymerase. However, these analogs are incorporated differently to remdesivir and with less efficiency. They also obstruct the polymerase in distinct ways. Taken together, the results by Seifert, Bera et al. suggest that magnetic tweezers can be a powerful approach to reveal how analogs interfere with replication. This information could be used to improve currently available analogs as well as develop new antiviral drugs that are more effective against SARS-CoV-2. This knowledge will be key at a time when treatments against COVID-19 are still lacking, and may be needed to protect against new variants and future outbreaks.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Nucleotides/pharmacology , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Cell Line , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays/methods , Humans , Models, Theoretical , Nucleotides/metabolism , RNA, Viral , SARS-CoV-2/enzymology , Stochastic Processes , Virus Replication/drug effects
3.
Sci Rep ; 11(1): 17748, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1412634

ABSTRACT

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Fullerenes/pharmacology , Antiviral Agents/therapeutic use , COVID-19/epidemiology , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/ultrastructure , Coronavirus Protease Inhibitors/chemistry , Coronavirus Protease Inhibitors/pharmacology , Coronavirus Protease Inhibitors/therapeutic use , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/ultrastructure , Crystallography, X-Ray , Fullerenes/chemistry , Fullerenes/therapeutic use , Humans , Molecular Dynamics Simulation , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleic Acid Synthesis Inhibitors/therapeutic use , Pandemics/prevention & control , RNA, Viral/biosynthesis , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure
4.
PLoS Comput Biol ; 17(9): e1009384, 2021 09.
Article in English | MEDLINE | ID: covidwho-1405333

ABSTRACT

Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, a well-known kinase inhibitor- Sorafenib showed a significant inhibition and dampened viral load in SARS-CoV-2 infected cells. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Protein Domains , SARS-CoV-2/drug effects , Catalytic Domain , Computer Simulation , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans
5.
Drug Res (Stuttg) ; 71(8): 462-472, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1404894

ABSTRACT

BACKGROUND: Replication of SARS-CoV-2 depends on viral RNA-dependent RNA-polymerase (RdRp). Remdesivir, the broad-spectrum RdRp inhibitor acts as nucleoside-analogues (NAs). Remdesivir has initially been repurposed as a promising drug against SARS-CoV-2 infection with some health hazards like liver damage, allergic reaction, low blood-pressure, and breathing-shortness, throat-swelling. In comparison, theaflavin-3'-O-gallate (TFMG), the abundant black tea component has gained importance in controlling viral infection. TFMG is a non-toxic, non-invasive, antioxidant, anticancer and antiviral molecule. RESULTS: Here, we analyzed the inhibitory effect of theaflavin-3'-O-gallate on SARS CoV-2 RdRp in comparison with remdesivir by molecular-docking study. TFMG has been shown more potent in terms of lower Atomic-Contact-Energy (ACE) and higher occupancy of surface area; -393.97 Kcal/mol and 771.90 respectively, favoured with lower desolvation-energy; -9.2: Kcal/mol. TFMG forms more rigid electrostatic and H-bond than remdesivir. TFMG showed strong affinity to RNA primer and template and RNA passage-site of RdRp. CONCLUSIONS: TFMG can block the catalytic residue, NTP entry site, cation binding site, nsp7-nsp12 junction with binding energy of -6. 72 Kcal/mol with Ki value of 11.79, and interface domain with binding energy of -7.72 and -6.16 Kcal/mol with Ki value of 2.21 and 30.71 µM. And most importantly, TFMG shows antioxidant/anti-inflammatory/antiviral effect on human studies.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Biflavonoids/pharmacology , COVID-19/drug therapy , Catechin/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/pharmacology , Gallic Acid/analogs & derivatives , Molecular Docking Simulation , SARS-CoV-2/drug effects , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/pharmacology , Antiviral Agents/chemistry , Biflavonoids/chemistry , COVID-19/virology , Catalytic Domain , Catechin/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/chemistry , Gallic Acid/chemistry , Gallic Acid/pharmacology , Protein Conformation , SARS-CoV-2/enzymology , Structure-Activity Relationship
6.
Phys Chem Chem Phys ; 23(36): 20117-20128, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1404891

ABSTRACT

The ongoing pandemic caused by SARS-CoV-2 emphasizes the need for effective therapeutics. Inhibition of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) by nucleotide analogs provides a promising antiviral strategy. One common group of RdRp inhibitors, 2'-modified nucleotides, are reported to exhibit different behaviors in the SARS-CoV-2 RdRp transcription assay. Three of these analogs, 2'-O-methyl UTP, Sofosbuvir, and 2'-methyl CTP, act as effective inhibitors in previous biochemical experiments, while Gemcitabine and ara-UTP show no inhibitory activity. To understand the impact of the 2'-modification on their inhibitory effects, we conducted extensive molecular dynamics simulations and relative binding free energy calculations using the free energy perturbation method on SARS-CoV-2 replication-transcription complex (RTC) with these five nucleotide analogs. Our results reveal that the five nucleotide analogs display comparable binding affinities to SARS-CoV-2 RdRp and they can all be added to the nascent RNA chain. Moreover, we examine how the incorporation of these nucleotide triphosphate (NTP) analogs will impact the addition of the next nucleotide. Our results indicate that 2'-O-methyl UTP can weaken the binding of the subsequent NTP and consequently lead to partial chain termination. Additionally, Sofosbuvir and 2'-methyl CTP can cause immediate termination due to the strong steric hindrance introduced by their bulky 2'-methyl groups. In contrast, nucleotide analogs with smaller substitutions, such as the fluorine atoms and the ara-hydroxyl group in Gemcitabine and ara-UTP, have a marginal impact on the polymerization process. Our findings are consistent with experimental observations, and more importantly, shed light on the detailed molecular mechanism of SARS-CoV-2 RdRp inhibition by 2'-substituted nucleotide analogs, and may facilitate the rational design of antiviral agents to inhibit SARS-CoV-2 RdRp.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Nucleotides/pharmacology , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/chemistry , Humans , Microbial Sensitivity Tests , Models, Molecular , Nucleic Acid Conformation , Nucleotides/chemistry , SARS-CoV-2/enzymology
7.
Sci Rep ; 11(1): 17748, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1397901

ABSTRACT

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Fullerenes/pharmacology , Antiviral Agents/therapeutic use , COVID-19/epidemiology , COVID-19/virology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/ultrastructure , Coronavirus Protease Inhibitors/chemistry , Coronavirus Protease Inhibitors/pharmacology , Coronavirus Protease Inhibitors/therapeutic use , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/ultrastructure , Crystallography, X-Ray , Fullerenes/chemistry , Fullerenes/therapeutic use , Humans , Molecular Dynamics Simulation , Nucleic Acid Synthesis Inhibitors/chemistry , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleic Acid Synthesis Inhibitors/therapeutic use , Pandemics/prevention & control , RNA, Viral/biosynthesis , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/ultrastructure
8.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1387102

ABSTRACT

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Coronavirus RNA-Dependent RNA Polymerase/genetics , Ebolavirus/drug effects , SARS-CoV-2/drug effects , Virus Replication/drug effects , Adenosine Monophosphate/genetics , Adenosine Monophosphate/metabolism , Alanine/genetics , Alanine/metabolism , Antiviral Agents/metabolism , Base Pairing , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/metabolism , Models, Molecular , Protein Biosynthesis/drug effects , RNA/genetics , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism
9.
Viruses ; 13(8)2021 08 11.
Article in English | MEDLINE | ID: covidwho-1355046

ABSTRACT

SARS-CoV-2 has caused an extensive pandemic of COVID-19 all around the world. Key viral enzymes are suitable molecular targets for the development of new antivirals against SARS-CoV-2 which could represent potential treatments of the corresponding disease. With respect to its essential role in the replication of viral RNA, RNA-dependent RNA polymerase (RdRp) is one of the prime targets. HeE1-2Tyr and related derivatives were originally discovered as inhibitors of the RdRp of flaviviruses. Here, we present that these pyridobenzothiazole derivatives also significantly inhibit SARS-CoV-2 RdRp, as demonstrated using both polymerase- and cell-based antiviral assays.


Subject(s)
Antiviral Agents/pharmacology , Benzothiazoles/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , SARS-CoV-2/drug effects , Virus Replication/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , SARS-CoV-2/enzymology , SARS-CoV-2/physiology
10.
Biochem Biophys Res Commun ; 571: 26-31, 2021 09 24.
Article in English | MEDLINE | ID: covidwho-1312941

ABSTRACT

The pandemic of SARS-CoV-2 has necessitated expedited research efforts towards finding potential antiviral targets and drug development measures. While new drug discovery is time consuming, drug repurposing has been a promising area for elaborate virtual screening and identification of existing FDA approved drugs that could possibly be used for targeting against functions of various proteins of SARS-CoV-2 virus. RNA dependent RNA polymerase (RdRp) is an important enzyme for the virus that mediates replication of the viral RNA. Inhibition of RdRp could inhibit viral RNA replication and thus new virus particle production. Here, we screened non-nucleoside antivirals and found three out of them to be strongest in binding to RdRp out of which two retained binding even using molecular dynamic simulations. We propose these two drugs as potential RdRp inhibitors which need further in-depth testing.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Amides/pharmacology , Antiviral Agents/chemistry , Benzimidazoles/pharmacology , COVID-19/virology , Carbamates/pharmacology , Catalytic Domain , Computer Simulation , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Cyclopropanes/pharmacology , Drug Evaluation, Preclinical , Drug Repositioning , Fluorenes/pharmacology , Humans , Lactams, Macrocyclic/pharmacology , Molecular Docking Simulation , Molecular Dynamics Simulation , Pandemics , Proline/analogs & derivatives , Proline/pharmacology , Protein Conformation , Quinoxalines/pharmacology , Sulfonamides/pharmacology
11.
Comput Biol Med ; 135: 104613, 2021 08.
Article in English | MEDLINE | ID: covidwho-1293684

ABSTRACT

The newly emerged Coronavirus Disease 2019 (COVID-19) rapidly outspread worldwide and now is one of the biggest infectious pandemics in human society. In this study, the inhibitory potential of 99 secondary metabolites obtained from endophytic fungi was investigated against the new coronavirus RNA-dependent RNA polymerase (RdRp) using computational methods. A sequence of blind and targeted molecular dockings was performed to predict the more potent compounds on the viral enzyme. In the next step, the five selected compounds were further evaluated by molecular dynamics (MD) simulation. Moreover, the pharmacokinetics of the metabolites was assessed using SwissADME server. The results of molecular docking showed that compounds 18-methoxy cytochalasin J, (22E,24R)-stigmasta-5,7,22-trien-3-ß-ol, beauvericin, dankasterone B, and pyrrocidine A had higher binding energy than others. The findings of MD and SwissADME demonstrated that two fungal metabolites, 18-methoxy cytochalasin J and pyrrocidine A had better results than others in terms of protein instability, strong complex formation, and pharmacokinetic properties. In conclusion, it is recommended to further evaluate the compounds 18-methoxy cytochalasin J and pyrrocidine A in the laboratory as good candidates for inhibiting COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Fungi/chemistry , SARS-CoV-2/drug effects , Molecular Docking Simulation , Molecular Dynamics Simulation , RNA-Dependent RNA Polymerase
12.
Biochem J ; 478(13): 2425-2443, 2021 07 16.
Article in English | MEDLINE | ID: covidwho-1289982

ABSTRACT

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.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Evaluation, Preclinical , SARS-CoV-2/enzymology , Small Molecule Libraries/pharmacology , Animals , Benzoates/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Assays , Fluorescence Resonance Energy Transfer , High-Throughput Screening Assays , Holoenzymes/metabolism , Reproducibility of Results , SARS-CoV-2/drug effects , Small Molecule Libraries/chemistry , Suramin/pharmacology , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
13.
Pharmacol Rep ; 73(6): 1754-1764, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1283833

ABSTRACT

BACKGROUND: SARS-CoV-2 is a newly emerged human coronavirus that severely affected human health and the economy. The viral RNA-dependent RNA polymerase (RdRp) is a crucial protein target to stop virus replication. The adenosine derivative, remdesivir, was authorized for emergency use 10 months ago by the United States FDA against COVID-19 despite its doubtful efficacy against SARS-CoV-2. METHODS: A dozen modifications based on remdesivir are tested against SARS-CoV-2 RdRp using combined molecular docking and dynamics simulation in this work. RESULTS: The results reveal a better binding affinity of 11 modifications compared to remdesivir. Compounds 8, 9, 10, and 11 show the best binding affinities against SARS-CoV-2 RdRp conformations gathered during 100 ns of the Molecular Dynamics Simulation (MDS) run (- 8.13 ± 0.45 kcal/mol, - 8.09 ± 0.67 kcal/mol, - 8.09 ± 0.64 kcal/mol, and - 8.07 ± 0.73 kcal/mol, respectively). CONCLUSIONS: The present study suggests these four compounds as potential SARS-CoV-2 RdRp inhibitors, which need to be validated experimentally.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Adenosine/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/chemistry , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Design , Adenosine Monophosphate/chemistry , Alanine/chemistry , Binding Sites , COVID-19 , Computer Simulation , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , SARS-CoV-2/pathogenicity
14.
Science ; 373(6551): 236-241, 2021 07 09.
Article in English | MEDLINE | ID: covidwho-1266364

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.


Subject(s)
Coenzymes/metabolism , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Cyclic N-Oxides/pharmacology , Iron/metabolism , SARS-CoV-2/drug effects , Sulfur/metabolism , Amino Acid Motifs , Animals , Antiviral Agents/pharmacology , Binding Sites , Catalytic Domain , Chlorocebus aethiops , Coenzymes/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/pharmacology , Iron/chemistry , Protein Domains , RNA Helicases/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Spin Labels , Sulfur/chemistry , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Zinc/metabolism
15.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1263454

ABSTRACT

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Coronavirus RNA-Dependent RNA Polymerase/genetics , Ebolavirus/drug effects , SARS-CoV-2/drug effects , Virus Replication/drug effects , Adenosine Monophosphate/genetics , Adenosine Monophosphate/metabolism , Alanine/genetics , Alanine/metabolism , Antiviral Agents/metabolism , Base Pairing , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Enzyme Inhibitors/metabolism , Models, Molecular , Protein Biosynthesis/drug effects , RNA/genetics , RNA/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism
16.
Molecules ; 26(11)2021 Jun 07.
Article in English | MEDLINE | ID: covidwho-1259549

ABSTRACT

Despite the fact that COVID-19 vaccines are already available on the market, there have not been any effective FDA-approved drugs to treat this disease. There are several already known drugs that through drug repositioning have shown an inhibitory activity against SARS-CoV-2 RNA-dependent RNA polymerase. These drugs are included in the family of nucleoside analogues. In our efforts, we synthesized a group of new nucleoside analogues, which are modified at the sugar moiety that is replaced by a quinazoline entity. Different nucleobase derivatives are used in order to increase the inhibition. Five new nucleoside analogues were evaluated with in vitro assays for targeting polymerase of SARS-CoV-2.


Subject(s)
Antiviral Agents/chemical synthesis , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemical synthesis , Nucleosides/analogs & derivatives , Nucleosides/chemical synthesis , SARS-CoV-2/enzymology , Chemistry, Pharmaceutical/methods , In Vitro Techniques , SARS-CoV-2/drug effects
17.
Curr Opin Virol ; 49: 127-138, 2021 08.
Article in English | MEDLINE | ID: covidwho-1252630

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has had a catastrophic impact on human health and the world economy. The response of the scientific community was unparalleled, and a combined global effort has resulted in the creation of vaccines in a shorter time frame than previously unimaginable. Reflecting this concerted effort, the structural analysis of the etiological agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has progressed with an unprecedented pace. Since the onset of the pandemic, over 1000 high-resolution structures of a broad range of SARS-CoV-2 proteins have been solved and made publicly available. These structures have aided in the identification of numerous potential druggable targets and have contributed to the design of different vaccine candidates. This opinion article will discuss the impact of high-resolution structures in understanding SARS-CoV-2 biology and explore their role in the development of vaccines and antivirals.


Subject(s)
COVID-19/prevention & control , SARS-CoV-2/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/epidemiology , COVID-19 Vaccines/immunology , COVID-19 Vaccines/therapeutic use , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Humans , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
18.
Curr Opin Virol ; 49: 81-85, 2021 08.
Article in English | MEDLINE | ID: covidwho-1225185

ABSTRACT

The nucleotide analogue prodrug remdesivir remains the only FDA-approved antiviral small molecule for the treatment of infection with SARS-CoV-2. Biochemical studies revealed that the active form of the drug targets the viral RNA-dependent RNA polymerase and causes delayed chain-termination. Delayed chain-termination is incomplete, but the continuation of RNA synthesis enables a partial escape from viral proofreading. Remdesivir becomes embedded in the copy of the RNA genome that later serves as a template. Incorporation of an incoming nucleotide triphosphate is now inhibited by the modified template. Knowledge on the mechanism of action matters. Enzymatic inhibition links to antiviral effects in cell cultures, animal models and viral load reduction in patients, which provides the logical chain that is expected for a direct acting antiviral. Hence, remdesivir also serves as a benchmark in current drug development efforts that will hopefully lead to orally available treatments to the benefit of a broader population.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/pharmacology , Alanine/therapeutic use , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Prodrugs/pharmacology , Prodrugs/therapeutic use , RNA, Viral/biosynthesis , RNA, Viral/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Substrate Specificity , Virus Replication/drug effects
19.
J Med Virol ; 93(1): 389-400, 2021 01.
Article in English | MEDLINE | ID: covidwho-1206780

ABSTRACT

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the harm caused by coronaviruses to the world cannot be underestimated. Recently, a novel coronavirus (severe acute respiratory syndrome coronavirus-2 [SARS-CoV-2]) initially found to trigger human severe respiratory illness in Wuhan City of China in 2019, has infected more than six million people worldwide by 21 June 2020, and which has been recognized as a public health emergency of international concern as well. And the virus has spread to more than 200 countries around the world. However, the effective drug has not yet been officially licensed or approved to treat SARS-Cov-2 and SARS-Cov infection. NSP12-NSP7-NSP8 complex of SARS-CoV-2 or SARS-CoV, essential for viral replication and transcription, is generally regarded as a potential target to fight against the virus. According to the NSP12-NSP7-NSP8 complex (PDB ID: 7BW4) structure of SARS-CoV-2 and the NSP12-NSP7-NSP8 complex (PDB ID: 6NUR) structure of SARS-CoV, NSP12-NSP7 interface model, and NSP12-NSP8 interface model were established for virtual screening in the present study. Eight compounds (Nilotinib, Saquinavir, Tipranavir, Lonafarnib, Tegobuvir, Olysio, Filibuvir, and Cepharanthine) were selected for binding free energy calculations based on virtual screening and docking scores. All eight compounds can combine well with NSP12-NSP7-NSP8 in the crystal structure, providing drug candidates for the treatment and prevention of coronavirus disease 2019 and SARS.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Molecular Docking Simulation , SARS Virus/drug effects , SARS-CoV-2/drug effects , Drug Discovery/methods , Models, Molecular , Small Molecule Libraries
20.
Chem Biol Interact ; 343: 109480, 2021 Jul 01.
Article in English | MEDLINE | ID: covidwho-1193249

ABSTRACT

Polyphenolics and 1,3,4-oxadiazoles are two of the most potent bioactive classes of compounds in medicinal chemistry, since both are known for their diverse pharmacological activities in humans. One of their prominent activities is the antimicrobial/antiviral activities, which are much apparent when the key functional structural moieties of both of them meet into the same compounds. The current COVID-19 pandemic motivated us to computationally screen and evaluate our library of previously-synthesized 2-(3,4,5-trihydroxyphenyl)-1,3,4-oxadiazoles against the major SARS-CoV-2 protein targets. Interestingly, few ligands showed promising low binding free energies (potent inhibitory interactions/affinities) with the active sites of some coronaviral-2 enzymes, specially the RNA-dependent RNA polymerase (nCoV-RdRp). One of them was 5,5'-{5,5'-[(1R,2R)-1,2-dihydroxyethane-1,2-diyl]bis(1,3,4-oxadiazole-5,2-diyl)}dibenzene-1,2,3-triol (Taroxaz-104), which showed significantly low binding energies (-10.60 and -9.10 kcal/mol) with nCoV-RdRp-RNA and nCoV-RdRp alone, respectively. These binding energies are even considerably lower than those of remdesivir potent active metabolite GS-443902 (which showed -9.20 and -7.90 kcal/mol with the same targets, respectively). Further computational molecular investigation revealed that Taroxaz-104 molecule strongly inhibits one of the potential active sites of nCoV-RdRp (the one with which GS-443902 molecule mainly interacts), since it interacts with at least seven major active amino acid residues of its predicted pocket. The successful repurposing of Taroxaz-104 has been achieved after the promising results of the anti-COVID-19 biological assay were obtained, as the data showed that Taroxaz-104 exhibited very significant anti-COVID-19 activities (anti-SARS-CoV-2 EC50 = 0.42 µM) with interesting effectiveness against the new strains/variants of SARS-CoV-2. Further investigations for the development of Taroxaz-104 and its coming polyphenolic 2,5-disubstituted-1,3,4-oxadiazole derivatives as anti-COVID-19 drugs, through in vivo bioevaluations and clinical trials research, are urgently needed.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Oxadiazoles/pharmacology , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/metabolism , Catalytic Domain , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Drug Repositioning , Enzyme Inhibitors/metabolism , Microbial Sensitivity Tests , Molecular Docking Simulation , Oxadiazoles/metabolism , Protein Binding , SARS-CoV-2/enzymology , Vero Cells
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