Antiviral activity of artesunate towards wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses

Efferth, Thomas; Marschall, Manfred; Wang, Xin; Huong, Shu-Mei; Hauber, Ilona; Olbrich, Armin; Kronschnabl, Martina; Stamminger, Thomas; Huang, Eng‐Shang

doi:10.1007/s00109-001-0300-8

Cited by 170 publications

(143 citation statements)

References 36 publications

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“…Additional clinical isolates, R3 and R4 (12,44), were analyzed for the presence of pUL97 isoforms using an anti-pUL97 polyclonal antibody (Ulm). Three polypeptides were detected, consistent with the presence of isoforms (Fig.…”

Section: Resultsmentioning

confidence: 99%

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

Webel

Hakki

Prichard

et al. 2014

J Virol

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The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCEThe HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo.T he product of the human cytomegalovirus (HCMV) UL97 gene (pUL97) is a serine/threonine protein kinase that phosphorylates itself and multiple viral and host proteins (1). It is expressed early in infection (2, 3), localizes predominantly to the nucleus using amino-terminal nuclear localization signals (4, 5), and is also observed in the cytoplasm later in the infection cycle (2, 5, 6). It is also present in the HCMV virion (7). Genetic deletion of UL97 results in a severe (10-to 1,000-fold) replication defect in vitro (8-10), as does mutation of the K355 lysine residue, which is critical for kinase activity (6). UL97 is therefore an attractive target for antiviral drug development (1,(11)(12)(13)(14) as an alternative to the UL54 DNA polymerase target of all currently licensed HCMV antivirals, including ganciclovir (GCV).Maribavir (MBV) is a benzimidazole riboside pUL97 inhibitor that inhibits HCMV replication potently (15) but variably depending on cell culture conditions (16). While MBV showed promise in early clinical trials, two recently published phase III trials demonstrated that MBV was no more...

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Section: Resultsmentioning

confidence: 99%

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

Webel

Hakki

Prichard

et al. 2014

J Virol

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“…Cytotoxicity Assay-Cell viability was measured 7 days after adenovirus transduction and/or HCMV infection by determining lactate dehydrogenase (LDH) activity in the cell layer by using a CytoTox 96 non-radioactive cytotoxicity assay (Promega), as described elsewhere (17,18).…”

Section: Methodsmentioning

confidence: 99%

RICK Activates a NF-κB-dependent Anti-human Cytomegalovirus Response

Eickhoff¹,

Hanke²,

Stein-Gerlach³

et al. 2004

Journal of Biological Chemistry

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“…Artesunate (AS) and the parent compound artemisinin were reported to inhibit human cytomegalovirus (CMV) replication in vitro and in vivo (3,4). These findings were followed by a case report of AS therapy in an immunocompromised patient who had ganciclovir (GCV)-resistant CMV disease as well as preliminary data on CMV kinetics following treatment with AS (5,6).…”

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confidence: 99%

Unique and Highly Selective Anticytomegalovirus Activities of Artemisinin-Derived Dimer Diphenyl Phosphate Stem from Combination of Dimer Unit and a Diphenyl Phosphate Moiety

Forman

Mott

et al. 2013

Antimicrob Agents Chemother

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We report that the artemisinin-derived dimer diphenyl phosphate (DPP; dimer 838) is the most selective inhibitor of human cytomegalovirus (CMV) replication among a series of artemisinin-derived monomers and dimers. Dimer 838 was also unique in being an irreversible CMV inhibitor. The peroxide unit within artemisinins' chemical structures is critical to their activities, and its absence results in loss of anti-CMV activities. Surprisingly, the deoxy dimer of 838 retained modest anti-CMV activity, suggesting that the DPP moiety of dimer 838 contributes to its anti-CMV activities. DPP alone did not inhibit CMV replication, but triphenyl phosphate (TPP) had modest CMV inhibition, although its selectivity index was low. Artemisinin DPP derivatives dimer 838 and monomer diphenyl phosphate (compound 558) showed stronger CMV inhibition and a higher selectivity index than their analogs lacking the DPP unit. An add-on and removal assay revealed that removing DPP derivatives (compounds 558 and 838) but not the non-DPP backbones (artesunate and compound 606) at 24 h postinfection (hpi) already resulted in dominant CMV inhibition. CMV inhibition was fully irreversible with 838 and partially irreversible with 558, while non-DPP artemisinins were reversible inhibitors. While all artemisinin derivatives and TPP reduced the expression of the CMV immediate early 2 (IE2), UL44, and pp65 proteins at or after 48 hpi, only TPP inhibited the expression of both IE1 and IE2. Combination of a non-DPP dimer (compound 606) with TPP was synergistic in CMV inhibition, while ganciclovir and TPP were additive. Although TPP shared structural similarity with monomer DPP (compound 558) and dimer DPP (compound 838), its pattern of CMV inhibition was significantly different from the patterns of the artemisinins. These findings demonstrate that the DPP group contributes to the unique activities of compound 838.A rtemisinins, compounds that became first-choice therapy for malaria, have recently received gained interest because of their antiviral activities (1, 2). Artesunate (AS) and the parent compound artemisinin were reported to inhibit human cytomegalovirus (CMV) replication in vitro and in vivo (3, 4). These findings were followed by a case report of AS therapy in an immunocompromised patient who had ganciclovir (GCV)-resistant CMV disease as well as preliminary data on CMV kinetics following treatment with AS (5, 6). Although AS inhibited GCV-resistant CMV strains in vitro, therapy was ineffective in controlling GCVresistant CMV in a renal transplant recipient (7,8). Since the serum concentrations achieved with the current dosing regimen of AS are unlikely to fully inhibit CMV replication and because higher doses may result in significant toxicities, improved and safe artemisinins with enhanced anti-CMV activity are desired (9). We have reported that artemisinin-derived dimers are highly selective inhibitors of CMV replication in vitro and are significantly more selective than their monomeric counterparts but do not have increased toxicity in h...

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Antiviral activity of artesunate towards wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses

Cited by 170 publications

References 36 publications

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

RICK Activates a NF-κB-dependent Anti-human Cytomegalovirus Response

Unique and Highly Selective Anticytomegalovirus Activities of Artemisinin-Derived Dimer Diphenyl Phosphate Stem from Combination of Dimer Unit and a Diphenyl Phosphate Moiety

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