RESUMO
The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus , Microscopia Crioeletrônica , SARS-CoV-2 , Humanos , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/ultraestrutura , COVID-19/virologia , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , RNA Viral/biossíntese , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Especificidade por Substrato , Guanosina Trifosfato/metabolismo , Capuzes de RNARESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.
Assuntos
COVID-19/transmissão , COVID-19/virologia , Aptidão Genética , Mutação , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Cricetinae , Modelos Animais de Doenças , Humanos , Pulmão/virologia , Masculino , Mesocricetus/virologia , Modelos Biológicos , Mucosa Nasal/virologia , Testes de Neutralização , Estabilidade Proteica , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Técnicas de Cultura de Tecidos , Traqueia/virologia , Carga Viral , Vírion/química , Vírion/patogenicidade , Vírion/fisiologia , Replicação Viral/genéticaRESUMO
The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRps) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus and human metapneumovirus incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.
Assuntos
Antivirais , RNA Polimerase Dependente de RNA , Humanos , Antivirais/farmacologia , Antivirais/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/química , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/enzimologia , Metapneumovirus/efeitos dos fármacos , Nucleotídeos/química , Nucleotídeos/farmacologia , Nucleotídeos/metabolismoRESUMO
Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Modelos Moleculares , Vírus de RNA/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/química , Alanina/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Vírus de RNA de Sentido Negativo/efeitos dos fármacos , Vírus de RNA de Sentido Negativo/enzimologia , Vírus Nipah/efeitos dos fármacos , Vírus Nipah/enzimologia , Vírus de RNA de Cadeia Positiva/efeitos dos fármacos , Vírus de RNA de Cadeia Positiva/enzimologia , Vírus de RNA/efeitos dos fármacos , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Replicação Viral/efeitos dos fármacosRESUMO
The SARS-CoV-2 replication-transcription complex is an assembly of nonstructural viral proteins that collectively act to reproduce the viral genome and generate mRNA transcripts. While the structures of the individual proteins involved are known, how they assemble into a functioning superstructure is not. Applying molecular modeling tools, including protein-protein docking, to the available structures of nsp7-nsp16 and the nucleocapsid, we have constructed an atomistic model of how these proteins associate. Our principal finding is that the complex is hexameric, centered on nsp15. The nsp15 hexamer is capped on two faces by trimers of nsp14/nsp16/(nsp10)2, which then recruit six nsp12/nsp7/(nsp8)2 polymerase subunits to the complex. To this, six subunits of nsp13 are arranged around the superstructure, but not evenly distributed. Polymerase subunits that coordinate dimers of nsp13 are capable of binding the nucleocapsid, which positions the 5'-UTR TRS-L RNA over the polymerase active site, a state distinguishing transcription from replication. Analysis of the viral RNA path through the complex indicates the dsRNA that exits the polymerase passes over the nsp14 exonuclease and nsp15 endonuclease sites before being unwound by a convergence of zinc fingers from nsp10 and nsp14. The template strand is then directed away from the complex, while the nascent strand is directed to the sites responsible for mRNA capping. The model presents a cohesive picture of the multiple functions of the coronavirus replication-transcription complex and addresses fundamental questions related to proofreading, template switching, mRNA capping, and the role of the endonuclease.
Assuntos
Endorribonucleases/metabolismo , Modelos Moleculares , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Dimerização , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , SARS-CoV-2/isolamento & purificação , Transcrição Gênica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC50) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.13- to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Adenosina/análogos & derivados , Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Humanos , SARS-CoV-2/genéticaRESUMO
The urgent response to the COVID-19 pandemic required accelerated evaluation of many approved drugs as potential antiviral agents against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using cell-based, biochemical, and modeling approaches, we studied the approved HIV-1 nucleoside/tide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) and emtricitabine (FTC), as well as prodrugs tenofovir alafenamide (TAF) and tenofovir disoproxilfumarate (TDF) for their antiviral effect against SARS-CoV-2. A comprehensive set of in vitro data indicates that TFV, TAF, TDF, and FTC are inactive against SARS-CoV-2. None of the NRTIs showed antiviral activity in SARS-CoV-2 infected A549-hACE2 cells or in primary normal human lung bronchial epithelial (NHBE) cells at concentrations up to 50 µM TAF, TDF, FTC, or 500 µM TFV. These results are corroborated by the low incorporation efficiency of respective NTP analogs by the SARS-CoV-2 RNA-dependent-RNA polymerase (RdRp), and lack of the RdRp inhibition. Structural modeling further demonstrated poor fitting of these NRTI active metabolites at the SARS-CoV-2 RdRp active site. Our data indicate that the HIV-1 NRTIs are unlikely direct-antivirals against SARS-CoV-2, and clinicians and researchers should exercise caution when exploring ideas of using these and other NRTIs to treat or prevent COVID-19.
Assuntos
Fármacos Anti-HIV , Tratamento Farmacológico da COVID-19 , Infecções por HIV , HIV-1 , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Emtricitabina/farmacologia , Emtricitabina/uso terapêutico , Infecções por HIV/tratamento farmacológico , Humanos , Nucleosídeos/farmacologia , Nucleosídeos/uso terapêutico , Nucleotídeos/farmacologia , Pandemias , RNA Viral , RNA Polimerase Dependente de RNA , SARS-CoV-2 , Tenofovir/farmacologia , Tenofovir/uso terapêuticoRESUMO
Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Humanos , Pulmão , Proteínas do Tecido NervosoRESUMO
Herein we describe the discovery of IDX21437 35b, a novel RPd-aminoacid-based phosphoramidate prodrug of 2'-α-chloro-2'-ß-C-methyluridine monophosphate. Its corresponding triphosphate 6 is a potent inhibitor of the HCV NS5B RNA-dependent RNA polymerase (RdRp). Despite showing very weak activity in the in vitro Huh-7 cell based HCV replicon assay, 35b demonstrated high levels of active triphosphate 6 in mouse liver and human hepatocytes. A biochemical study revealed that the metabolism of 35b was mainly attributed to carboxyesterase 1 (CES1), an enzyme which is underexpressed in HCV Huh-7-derived replicon cells. Furthermore, due to its metabolic activation, 35b was efficiently processed in liver cells compared to other cell types, including human cardiomyocytes. The selected RP diastereoisomeric configuration of 35b was assigned by X-ray structural determination. 35b is currently in Phase II clinical trials for the treatment of HCV infection.
Assuntos
Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Uridina Monofosfato/análogos & derivados , Uridina/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Fígado/efeitos dos fármacos , Fígado/virologia , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Uridina/síntese química , Uridina/química , Uridina Monofosfato/síntese química , Uridina Monofosfato/química , Uridina Monofosfato/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismoRESUMO
Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory tract infections. There are no convenient small-animal infection models. Here, we show viral replication in the upper and lower airways of AG129 mice (double IFNα/ß and IFNγ receptor knockout mice) upon intranasal inoculation. By multiplex fluorescence RNAscope and immunohistochemistry followed by confocal microscopy, we demonstrate viral tropism to ciliated cells and club cells of the bronchiolar epithelium. HPIV-3 causes a marked lung pathology. No virus transmission of the virus was observed by cohousing HPIV-3-infected AG129 mice with other mice. Oral treatment with GS-441524, the parent nucleoside of remdesivir, reduced infectious virus titers in the lung, with a relatively normal histology. Intranasal treatment also affords an antiviral effect. Thus, AG129 mice serve as a robust preclinical model for developing therapeutic and prophylactic strategies against HPIV-3. We suggest further investigation of GS-441524 and its prodrug forms to treat HPIV-3 infection in humans.
Assuntos
Antivirais , Modelos Animais de Doenças , Pulmão , Camundongos Knockout , Vírus da Parainfluenza 3 Humana , Infecções por Respirovirus , Animais , Pulmão/virologia , Pulmão/patologia , Pulmão/efeitos dos fármacos , Camundongos , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Vírus da Parainfluenza 3 Humana/fisiologia , Antivirais/farmacologia , Infecções por Respirovirus/tratamento farmacológico , Infecções por Respirovirus/virologia , Humanos , Replicação Viral/efeitos dos fármacos , Feminino , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/deficiência , Adenosina/análogos & derivados , Adenosina/farmacologia , Tropismo Viral , Benzamidas , FtalimidasRESUMO
Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In this work, we show that ODV and its circulating parent nucleoside metabolite, GS-441524, have similar in vitro antiviral activity against filoviruses, including Marburg virus, Ebola virus, and Sudan virus (SUDV). We also report that once-daily oral ODV treatment of cynomolgus monkeys for 10 days beginning 24 hours after SUDV exposure confers 100% protection against lethal infection. Transcriptomics data show that ODV treatment delayed the onset of inflammation and correlated with antigen presentation and lymphocyte activation. Our results offer promise for the further development of ODV to control outbreaks of filovirus disease more rapidly.
Assuntos
Alanina , Antivirais , Ebolavirus , Doença pelo Vírus Ebola , Nucleosídeos , Pró-Fármacos , Animais , Administração Oral , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/prevenção & controle , Macaca fascicularis , Nucleosídeos/administração & dosagem , Nucleosídeos/farmacologia , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/farmacologia , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/farmacologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacologia , Antivirais/administração & dosagem , Antivirais/farmacologiaRESUMO
Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (Mpro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.
Assuntos
Antivirais , Pró-Fármacos , SARS-CoV-2 , Animais , SARS-CoV-2/efeitos dos fármacos , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Humanos , Camundongos , Administração Oral , Chlorocebus aethiops , Células Vero , Tratamento Farmacológico da COVID-19 , COVID-19/virologia , Replicação Viral/efeitos dos fármacos , Nucleosídeos/farmacologia , Nucleosídeos/uso terapêutico , Nucleosídeos/química , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Feminino , Modelos Animais de DoençasRESUMO
Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (1), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C-nucleoside GS-646089 (2) with broad antiviral activity against RSV (EC50 = 3-46 nM), human metapneumovirus (EC50 = 210 nM), human rhinovirus (EC50 = 54-61 nM), and enterovirus (EC50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [(S)-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo. 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.
Assuntos
Antivirais , Picornaviridae , Pró-Fármacos , Infecções por Vírus Respiratório Sincicial , Animais , Antivirais/farmacologia , Antivirais/química , Pró-Fármacos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/síntese química , Chlorocebus aethiops , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Humanos , Picornaviridae/efeitos dos fármacos , Relação Estrutura-Atividade , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Descoberta de Drogas , Nucleosídeos/química , Nucleosídeos/farmacologia , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/virologiaRESUMO
Remdesivir (GS-5734; VEKLURY) is a single diastereomer monophosphoramidate prodrug of an adenosine analog (GS-441524). Remdesivir is taken up by target cells and metabolized in multiple steps to form the active nucleoside triphosphate (GS-443902), which acts as a potent inhibitor of viral RNA-dependent RNA polymerases. Remdesivir and GS-441524 have antiviral activity against multiple RNA viruses. Here, we expand the evaluation of remdesivir's antiviral activity to members of the families Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae. Using cell-based assays, we show that remdesivir can inhibit infection of flaviviruses (such as dengue 1-4, West Nile, yellow fever, Zika viruses), picornaviruses (such as enterovirus and rhinovirus), and filoviruses (such as various Ebola, Marburg, and Sudan virus isolates, including novel geographic isolates), but is ineffective or is significantly less effective against orthomyxoviruses (influenza A and B viruses), or hepadnaviruses B, D, and E. In addition, remdesivir shows no antagonistic effect when combined with favipiravir, another broadly acting antiviral nucleoside analog, and has minimal interaction with a panel of concomitant medications. Our data further support remdesivir as a broad-spectrum antiviral agent that has the potential to address multiple unmet medical needs, including those related to antiviral pandemic preparedness.
Assuntos
Filoviridae , Doença pelo Vírus Ebola , Infecção por Zika virus , Zika virus , Humanos , Antivirais/farmacologia , Antivirais/uso terapêutico , Monofosfato de Adenosina , Alanina , Doença pelo Vírus Ebola/tratamento farmacológico , Infecção por Zika virus/tratamento farmacológicoRESUMO
Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 (2) into lung cells, thereby forming the bioactive triphosphate 2-NTP. 2-NTP, an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP. Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP, leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2, which supports development of 3 as a promising COVID-19 treatment.
Assuntos
COVID-19 , Pró-Fármacos , Chlorocebus aethiops , Humanos , Animais , SARS-CoV-2 , Tratamento Farmacológico da COVID-19 , Nucleosídeos , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , RNA Viral , Antivirais/farmacologia , Antivirais/uso terapêutico , FuranosRESUMO
Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective treatment strategies. Here, we characterize the antiviral activity of GS-5245, Obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-related Bat-CoV RsSHC014, Middle East Respiratory Syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (Mpro) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions.
RESUMO
Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.
Assuntos
Gammaherpesvirinae/imunologia , Infecções por Herpesviridae/metabolismo , Macaca mulatta/imunologia , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Antivirais/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Gammaherpesvirinae/genética , Produtos do Gene env/administração & dosagem , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Produtos do Gene gag/administração & dosagem , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Vetores Genéticos , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Imunidade Celular , Técnicas Imunoenzimáticas , Rim/citologia , Rim/metabolismo , Rim/virologia , Macaca mulatta/genética , Macaca mulatta/virologia , Testes de Neutralização , Plasmídeos , Recombinação Genética , Vacinas contra a SAIDS/genética , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vacinação , Carga Viral , Replicação ViralRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo, African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of <10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , Adenosina/análogos & derivados , Animais , Antivirais/uso terapêutico , Chlorocebus aethiops , Humanos , Pandemias , SARS-CoV-2RESUMO
Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the US FDA approved intravenous (IV) RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for non-hospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an IV administration of RDV at 10 mg/kg, an approximately 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed following inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with IV RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning approximately 8 hours post-infection. Moreover, the efficacy of inhaled RDV was similar to that of IV RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV.