Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Sáez-Álvarez, Yanira; Arias, Armando; Águila, C. del; Agudo, Rubén

doi:10.1038/s41598-019-41998-1

Cited by 43 publications

(63 citation statements)

References 70 publications

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“…The fluorescence emitted was recorded in real-time using a TECAN F200 with excitation and emission filters at 485 and 520 nm, respectively. The assay records the synthesis of dsRNA in a reaction using a poly-U molecule as a template and ATP as the nucleotide substrate, which has been adapted from methods previously documented for the detection of Zika virus polymerase activity 34 . Reactions were performed in individual wells of black 384-well low volume round bottom plates.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for inhibition of the SARS-CoV-2 RNA polymerase by suramin

Yin

Luan

et al. 2021

Nat Struct Mol Biol

125

157

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evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of coronavirus disease 2019 (COVID-19), with over 84.66 million infections and 1.83 million deaths as reported on 3 January 2021 (refs. 1,2). SARS-CoV-2 is a positive-sense, single-stranded RNA virus. SARS-CoV-2 and several related beta-coronaviruses, including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), are highly pathogenic. Infections can lead to severe acute respiratory syndrome, loss of lung function and, in severe cases, death. Compared to SARS-CoV and MERS-CoV, SARS-CoV-2 has a higher capacity of human-to-human infections, which resulted in the rapidly growing pandemic 3. Finding an effective treatment for COVID-19, potentially also through drug repurposing, is an urgent but unmet medical need. Suramin (Fig. 1a) is a century-old drug that has been used to treat African sleeping sickness and river blindness 4,5. It has also been shown to be effective in inhibiting the replication of a wide range of viruses, including enteroviruses 6 , Zika virus 7 , Chikungunya 8 and Ebola viruses 9. The viral inhibition mechanisms of suramin are diverse, including inhibition of viral attachment, viral entry and release from host cells in part through interactions with viral capsid proteins 7,8,10,11. Recently, suramin has been shown to inhibit SARS-CoV-2 infection in cell culture by preventing cellular entry of the virus 12. Here we report that suramin is also a potent inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), an essential enzyme for the viral life cycle. The potency of suramin in biochemical RdRp inhibition assays is at least 20-fold more potent than remdesivir, the current Food and Drug Administration-approved nucleotide drug for the treatment of COVID-19. The activity of suramin in cell-based viral inhibition is similar to remdesivir because the highly negative charge of suramin prevents efficient cellular uptake. A cryogenic electron microscopy (cryo-EM) structure reveals that suramin binds to the RdRp active site, blocking the binding of both RNA template and primer strands. These results provide a structural template for the design of next generation suramin derivatives as SARS-CoV-2 RdRp inhibitors. Structural basis for inhibition of the SARS-CoV-2 RNA polymerase by suramin Wanchao Yin 1,

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Section: Methodsmentioning

confidence: 99%

Structural basis for inhibition of the SARS-CoV-2 RNA polymerase by suramin

Yin

Luan

et al. 2021

Nat Struct Mol Biol

125

157

View full text Add to dashboard Cite

show abstract

“…In addition to PicoGreen, other fluorophores have also been used to distinguish dsRNA from ssRNA, and they are useful for this type of RdRP assay. 52,53 Scintillation Proximity Assay (SPA) SPA has also been used in RdRP enzyme assays for HTS. 54 This assay relies on the incorporation of radioactive nucleotides to the newly synthesized RNA chain catalyzed by RdRP using a biotinylated primer-template in the presence of 3 H-GTP (Fig.…”

Section: Fluorometric Rdrp Activity Assaymentioning

confidence: 99%

RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery

et al. 2020

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COVID-19 respiratory disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global health issue since it emerged in December 2019. While great global efforts are underway to develop vaccines and to discover or repurpose therapeutic agents for this disease, as of this writing only the nucleoside drug remdesivir has been approved under Emergency Use Authorization to treat COVID-19. The RNA-dependent RNA polymerase (RdRP), a viral enzyme for viral RNA replication in host cells, is one of the most intriguing and promising drug targets for SARS-CoV-2 drug development. Because RdRP is a viral enzyme with no host cell homologs, selective SARS-CoV-2 RdRP inhibitors can be developed that have improved potency and fewer off-target effects against human host proteins and thus are safer and more effective therapeutics for treating COVID-19. This review focuses on biochemical enzyme and cell-based assays for RdRPs that could be used in high-throughput screening to discover new and repurposed drugs against SARS-CoV-2.

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“…We followed the novel fluorescence-based method for the detection of polymerase activity of EV71 3D pol (52).…”

Section: Fluorescence-based Activity Assay For Ev71 3d Polmentioning

confidence: 99%

The Pyrimidine Analog FNC Potently Inhibits the Replication of Multiple Enteroviruses

Yang

Zheng

et al. 2020

J Virol

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Human enteroviruses (EVs), including coxsackieviruses, the numbered enteroviruses, and echoviruses, cause a wide range of diseases, such as hand, foot, and mouth disease (HFMD), encephalitis, myocarditis, acute flaccid myelitis (AFM), pneumonia, and bronchiolitis. Therefore, broad-spectrum anti-EV drugs are urgently needed to treat EV infection. Here, we demonstrate that FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine), a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs, including enterovirus 71 (EV71), coxsackievirus A16 (CA16), CA6, EVD68, and coxsackievirus B3 (CVB3), at the nanomolar level. The antiviral mechanism of FNC involves mainly positive- and negative-strand RNA synthesis inhibition by targeting and competitively inhibiting the activity of EV71 viral RNA-dependent RNA polymerase (3Dpol), as demonstrated through quantitative real-time reverse transcription-PCR (RT-qPCR), in vitro 3Dpol activity, and isothermal titration calorimetry (ITC) experiments. We further demonstrated that FNC treatment every 2 days with 1 mg/kg of body weight in EV71 and CA16 infection neonatal mouse models successfully protected mice from lethal challenge with EV71 and CA16 viruses and reduced the viral load in various tissues. These findings provide important information for the clinical development of FNC as a broad-spectrum inhibitor of human EV pathogens. IMPORTANCE Human enterovirus (EV) pathogens cause various contagious diseases such as hand, foot, and mouth disease, encephalitis, myocarditis, acute flaccid myelitis, pneumonia, and bronchiolitis, which have become serious health threats. However, except for the EV71 vaccine on the market, there are no effective strategies to prevent and treat other EV pathogen infections. Therefore, broad-spectrum anti-EV drugs are urgently needed. In this study, we demonstrated that FNC, a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs at the nanomolar level. Further investigation revealed that FNC inhibits positive- and negative-strand RNA synthesis of EVs by interacting and interfering with the activity of EV71 viral RNA-dependent RNA polymerase (3Dpol). Our findings demonstrate for the first time that FNC is an effective broad-spectrum inhibitor for human EV pathogens.

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Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Cited by 43 publications

References 70 publications

Structural basis for inhibition of the SARS-CoV-2 RNA polymerase by suramin

Structural basis for inhibition of the SARS-CoV-2 RNA polymerase by suramin

RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery

The Pyrimidine Analog FNC Potently Inhibits the Replication of Multiple Enteroviruses

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