Synthesis and Biological Evaluation of Purine 2′‐Fluoro‐2′‐deoxyriboside ProTides as Anti‐influenza Virus Agents

Meneghesso, Silvia; Vanderlinden, Evelien; Brancale, Andrea; Balzarini, Jan; Naesens, Lieve; McGuigan, Christopher

doi:10.1002/cmdc.201200562

Cited by 14 publications

(16 citation statements)

References 41 publications

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“…After 48 hours incubation at 37°C, the cells were trypsinized, resuspended in culture medium at 15 Â 10 6 cells per ml, and transfected with the four vRNP reconstituting plasmids and the firefly and Renilla luciferase reporter plasmids. The procedure (Meneghesso et al, 2013) was adapted from the reverse genetics protocol published by Martínez-Sobrido and Garcia-Sastre (2010). Specifically, 1 ml of the transfection mixture contained: 2.4 Â 10 6 siRNA-treated HEK293T cells; 5 ml Lipofectamine-2000 (Invitrogen); 0.22 mg of each of the four vRNP-reconstituting plasmids; 0.086 mg of the firefly luciferase plasmid, and 0.020 mg of the Renilla luciferase plasmid.…”

Section: Methodsmentioning

confidence: 99%

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Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

et al. 2013

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6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-59-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-59-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution.Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having K m app values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.

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Section: Methodsmentioning

confidence: 99%

“…The vRNP reconstitution assay represents a convenient cell-based method to estimate the activity of compounds toward the influenza virus polymerase (Meneghesso et al, 2013). In this plasmid-based assay, the four influenza virus proteins constituting the vRNP complexes are generated in human HEK293T cells.…”

Section: Mdck-tgmentioning

confidence: 99%

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

et al. 2013

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“…ProTide prodrugs were employed to overcome the first (rate‐limiting) phosphorylation step, and enable intracellular delivery of the nucleoside 5ʹ‐monophosphate species . This ProTide concept was successfully applied to 2ʹ‐FdG and its uridine analogue 2ʹ‐FdU …”

Section: Strategies To Interfere With the Influenza Virus Polymerasementioning

confidence: 99%

The Influenza Virus Polymerase Complex: An Update on Its Structure, Functions, and Significance for Antiviral Drug Design

Stevaert

Naesens

2016

Medicinal Research Reviews

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138

151

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Influenza viruses cause seasonal epidemics and pandemic outbreaks associated with significant morbidity and mortality, and a huge cost. Since resistance to the existing anti‐influenza drugs is rising, innovative inhibitors with a different mode of action are urgently needed. The influenza polymerase complex is widely recognized as a key drug target, given its critical role in virus replication and high degree of conservation among influenza A (of human or zoonotic origin) and B viruses. We here review the major progress that has been made in recent years in unravelling the structure and functions of this protein complex, enabling structure‐aided drug design toward the core regions of the PA endonuclease, PB1 polymerase, or cap‐binding PB2 subunit. Alternatively, inhibitors may target a protein–protein interaction site, a cellular factor involved in viral RNA synthesis, the viral RNA itself, or the nucleoprotein component of the viral ribonucleoprotein. The latest advances made for these diverse pharmacological targets have yielded agents in advanced (i.e., favipiravir and VX‐787) or early clinical testing, besides several experimental inhibitors in various stages of development, which are all covered here.

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“…Viral ribonucleoproteins (vRNPs) were isolated from virions and purified by gradient ultracentrifugation (42)(43)(44)(45). These vRNPs contain the native PA-PB1-PB2 polymerase enzyme and, as the template for transcription, viral RNA complexed to nucleoprotein.…”

Section: Virusmentioning

confidence: 99%

“…After addition of the isolated vRNPs, the samples were incubated for 1 h at 30°C. The amount of radioactivity incorporated into viral RNA was quantified by a filter-based method as described elsewhere (42,43).…”

Section: Virusmentioning

confidence: 99%

Distinct Effects of T-705 (Favipiravir) and Ribavirin on Influenza Virus Replication and Viral RNA Synthesis

Vanderlinden

Vrancken

Houdt

et al. 2016

Antimicrob Agents Chemother

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100

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c T-705 (favipiravir) is a new antiviral agent in advanced clinical development for influenza therapy. It is supposed to act as an alternative substrate for the viral polymerase, causing inhibition of viral RNA synthesis or virus mutagenesis. These mechanisms were also proposed for ribavirin, an established and broad antiviral drug that shares structural similarity with T-705. We here performed a comparative analysis of the effects of T-705 and ribavirin on influenza virus and host cell functions. Influenza virusinfected cell cultures were exposed to T-705 or ribavirin during single or serial virus passaging. The effects on viral RNA synthesis and infectious virus yield were determined and mutations appearing in the viral genome were detected by whole-genome virus sequencing. In addition, the cellular nucleotide pools as well as direct inhibition of the viral polymerase enzyme were quantified. We demonstrate that the anti-influenza virus effect of ribavirin is based on IMP dehydrogenase inhibition, which results in fast and profound GTP depletion and an imbalance in the nucleotide pools. In contrast, T-705 acts as a potent and GTP-competitive inhibitor of the viral polymerase. In infected cells, viral RNA synthesis is completely inhibited by T-705 or ribavirin at >50 M, whereas exposure to lower drug concentrations induces formation of noninfectious particles and accumulation of random point mutations in the viral genome. This mutagenic effect is 2-fold higher for T-705 than for ribavirin. Hence, T-705 and ribavirin both act as purine pseudobases but profoundly differ with regard to the mechanism behind their antiviral and mutagenic effects on influenza virus.

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Synthesis and Biological Evaluation of Purine 2′‐Fluoro‐2′‐deoxyriboside ProTides as Anti‐influenza Virus Agents

Cited by 14 publications

References 41 publications

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

The Influenza Virus Polymerase Complex: An Update on Its Structure, Functions, and Significance for Antiviral Drug Design

Distinct Effects of T-705 (Favipiravir) and Ribavirin on Influenza Virus Replication and Viral RNA Synthesis

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