RESUMEN
Emerging influenza A viruses (IAV) bear the potential to cause pandemics with unpredictable consequences for global human health. In particular, the WHO has declared avian H5 and H7 subtypes as high-risk candidates, and continuous surveillance of these viruses as well as the development of novel, broadly acting antivirals, are key for pandemic preparedness. In this study, we sought to design T-705 (Favipiravir) related inhibitors that target the RNA-dependent RNA polymerase and evaluate their antiviral efficacies against a broad range of IAVs. Therefore, we synthesized a library of derivatives of T-705 ribonucleoside analogues (called T-1106 pronucleotides) and tested their ability to inhibit both seasonal and highly pathogenic avian influenza viruses in vitro. We further showed that diphosphate (DP) prodrugs of T-1106 are potent inhibitors of H1N1, H3N2, H5N1, and H7N9 IAV replication. Importantly, in comparison to T-705, these DP derivatives achieved 5- to 10-fold higher antiviral activity and were non-cytotoxic at the therapeutically active concentrations. Moreover, our lead DP prodrug candidate showed drug synergy with the neuraminidase inhibitor oseltamivir, thus opening up another avenue for combinational antiviral therapy against IAV infections. Our findings may serve as a basis for further pre-clinical development of T-1106 prodrugs as an effective countermeasure against emerging IAVs with pandemic potential.
RESUMEN
The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influenza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favorable overall configuration.
Asunto(s)
Subtipo H7N9 del Virus de la Influenza A , Gripe Humana , Humanos , Nucleósidos , Nucleótidos/metabolismo , Antivirales/farmacología , Antivirales/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismoRESUMEN
Zoonotic spillover, i.e. pathogen transmission from animal to human, has repeatedly introduced RNA viruses into the human population. In some cases, where these viruses were then efficiently transmitted between humans, they caused large disease outbreaks such as the 1918 flu pandemic or, more recently, outbreaks of Ebola and Coronavirus disease. These examples demonstrate that RNA viruses pose an immense burden on individual and public health with outbreaks threatening the economy and social cohesion within and across borders. And while emerging RNA viruses are introduced more frequently as human activities increasingly disrupt wild-life eco-systems, therapeutic or preventative medicines satisfying the "one drug-multiple bugs"-aim are unavailable. As one central aspect of preparedness efforts, this review digs into the development of broadly acting antivirals via targeting viral genome synthesis with host- or virus-directed drugs centering around nucleotides, the genomes' universal building blocks. Following the first strategy, selected examples of host de novo nucleotide synthesis inhibitors are presented that ultimately interfere with viral nucleic acid synthesis, with ribavirin being the most prominent and widely used example. For directly targeting the viral polymerase, nucleoside and nucleotide analogues (NNAs) have long been at the core of antiviral drug development and this review illustrates different molecular strategies by which NNAs inhibit viral infection. Highlighting well-known as well as recent, clinically promising compounds, structural features and mechanistic details that may confer broad-spectrum activity are discussed. The final part addresses limitations of NNAs for clinical development such as low efficacy or mitochondrial toxicity and illustrates strategies to overcome these.
Asunto(s)
Antivirales/farmacología , Genoma Viral/efectos de los fármacos , Infecciones por Virus ARN/tratamiento farmacológico , Virus ARN/efectos de los fármacos , Virus ARN/genética , Animales , Antivirales/química , Antivirales/uso terapéutico , Humanos , Virus ARN/fisiologíaRESUMEN
The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.
Asunto(s)
Amidas/farmacología , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Betacoronavirus/genética , Infecciones por Coronavirus/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Pirazinas/farmacología , Amidas/farmacocinética , Animales , Antivirales/farmacocinética , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/virología , ARN Polimerasa Dependiente de ARN de Coronavirus , Modelos Moleculares , Mutagénesis/efectos de los fármacos , Pandemias , Neumonía Viral/virología , Pirazinas/farmacocinética , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2 , Análisis de Secuencia , Células Vero , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.
Asunto(s)
Amidas/farmacología , Antivirales/farmacología , Línea Celular , Pirazinas/farmacología , Virus ARN/efectos de los fármacos , Animales , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Línea Celular/virología , Chlorocebus aethiops , Perros , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Células HEK293/virología , Humanos , Células de Riñón Canino Madin Darby/efectos de los fármacos , Células de Riñón Canino Madin Darby/metabolismo , Células de Riñón Canino Madin Darby/virología , Ribonucleósidos/metabolismo , Células Vero/efectos de los fármacos , Células Vero/metabolismo , Células Vero/virologíaRESUMEN
We here disclose chemical synthesis of ribonucleoside 5'-monophosphate (RMP), -diphosphate (RDP), and -triphosphate (RTP) and cycloSal-, Di PPro-, and Tri PPPro nucleotide prodrugs of the antiviral pseudobase T-1105. Moreover, we include one nucleoside diphosphate prodrug of the chemically less stable T-705. We demonstrate efficient T-1105-RDP and -RTP release from the Di PPro and Tri PPPro compounds by esterase activation. Using crude enzyme extracts, we saw rapid phosphorylation of T-1105-RDP into T-1105-RTP. In sharp contrast, phosphorylation of T-1105-RMP was not seen, indicating a yet unrecognized bottleneck in T-1105's metabolic activation. Accordingly, Di PPro and Tri PPPro compounds displayed improved cell culture activity against influenza A and B virus, which they retained in a mutant cell line incapable of activating the nucleobase parent. T-1105-RTP had a strong inhibitory effect against isolated influenza polymerase, and Di PPro-T-1105-RDP showed 4-fold higher potency in suppressing one-cycle viral RNA synthesis versus T-1105. Hence, our T-1105-RDP and -RTP prodrugs improve antiviral potency and achieve efficient metabolic bypass.
Asunto(s)
Amidas/química , Amidas/farmacología , Orthomyxoviridae/efectos de los fármacos , Profármacos/metabolismo , Pirazinas/química , Pirazinas/farmacología , Ribonucleótidos/química , Amidas/metabolismo , Animales , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Perros , Células de Riñón Canino Madin Darby , Pirazinas/metabolismoRESUMEN
T-705 (favipiravir) is a fluorinated hydroxypyrazine carboxamide that exhibits antiviral activities against a variety of RNA viruses. Given the lack of potent agents to combat these infections caused by a large number of high-impact pathogens, significant emphasis has been put on studies of the antiviral properties of T-705 and its mechanism of action. T-705 acts as a nucleobase analogue; it is therefore metabolized to the corresponding ribonucleoside triphosphate intracellularly. Herein we report a reliable synthesis of T-705-ribonucleoside as well as its 5'-monophosphate. Moreover, we disclose detailed studies on the remarkable lability of the heterocycle when attached to ribose under very mild conditions, as typically applied in biochemical studies.