Human Cytomegalovirus Inhibition by Cardiac Glycosides: Evidence for Involvement of the hERG Gene

Kapoor, Arun; Cai, Hongyi; Forman, Michael; He, Ran; Shamay, Meir; Arav‐Boger, Ravit

doi:10.1128/aac.00898-12

Cited by 51 publications

(59 citation statements)

References 53 publications

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“…Ouabain and digoxin have been described to inhibit virus infection through targeting immediate-early gene expression but are considerably more toxic than convallatoxin (Kapoor et al, 2012). Interestingly, cardiac glycosides ouabain and peruvoside were included in the compound library tested, but they were excluded as hit compounds because they did not reduce YFP expression >70% (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle

et al. 2015

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Human cytomegalovirus (CMV) is a latent and persistent virus whose proliferation increases morbidity and mortality of immune-compromised individuals. The current anti-CMV therapeutics targeting the viral DNA polymerase or the major immediate-early (MIE) gene locus are somewhat effective at limiting CMV-associated disease. However, due to low bioavailability, severe toxicity, and the development of drug resistant CMV strains following prolonged treatment, current anti-CMV therapeutics are insufficient. To help address this shortfall, we established a high-content assay to identify inhibitors targeting CMV entry and the early steps of infection. The infection of primary human fibroblasts with a variant of the CMV laboratory strain AD169 expressing a chimeric IE2-yellow fluorescence protein (YFP) (AD169IE2-YFP) provided the basis for the high-content assay. The localization of IE2-YFP to the nucleus shortly following an AD169IE2-YFP infection induced a robust fluorescent signal that was quantified using confocal microscopy. The assay was optimized to achieve outstanding assay fitness and high Z′ scores. We then screened a bioactive chemical library consisting of 2080 compounds and identified hit compounds based on the decrease of fluorescence signal from IE2-YFP nuclear expression. The hit compounds likely target various cellular processes involved in the early steps of infection including capsid transport, chromatin remodeling, and viral gene expression. Extensive secondary assays confirmed the ability of a hit compound, convallatoxin, to inhibit infection of both laboratory and clinical CMV strains and limit virus proliferation. Collectively, the data demonstrate that we have established a robust high-content screen to identify compounds that limit the early steps of the CMV life cycle, and that novel inhibitors of early infection events may serve as viable CMV therapeutics.

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Section: Discussionmentioning

confidence: 99%

Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle

et al. 2015

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“…The combination of GCV and all artemisinins (AS, dimer 838, dimer 606) demonstrated strong synergistic activity, and that of GCV and cardiac glycosides (digoxin, digitoxin, and ouabain) resulted in an additive effect. The two separate classes of CMV inhibitors, artemisinins and cardiac glycosides, were previously reported to have a higher slope than GCV, suggesting that these inhibitors belong to different classes of CMV inhibitors in which participation of multiple copies of the drug target may occur (24,37). For other chronic viral infections, such as those with HIV, the slope was found to be an important factor not only in distinguishing between drug classes with a known mechanism of action but also in contributing to reduced antiviral activity even when the EC 50 was unchanged in resistant virus mutants (39,40).…”

Section: Discussionmentioning

confidence: 99%

“…The compounds used in this study and their mechanisms of action, when known, are listed in Table 1. Artemisinins (monomers and dimers), cardiac glycosides, and U0126 were reported to inhibit CMV replication and were therefore selected for this study (24)(25)(26)(27)(28)(29). The multikinase inhibitor sunitinib has not previously been reported to inhibit CMV replication, but other kinase inhibitors have been shown to inhibit CMV in vitro (30).…”

Section: Methodsmentioning

confidence: 99%

“…We reported that the CMV inhibitors artemisinins and cardiac glycosides acted earlier than GCV in CMV-infected cells, and limited combination studies indicated that GCV plus artemisinins had synergistic activities, while GCV plus digoxin showed only additive effects on CMV inhibition (23,24). We now report a comprehensive analysis of multiple drug combinations.…”

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confidence: 99%

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In Vitro Combination of Anti-Cytomegalovirus Compounds Acting through Different Targets: Role of the Slope Parameter and Insights into Mechanisms of Action

Cai

Kapoor

et al. 2014

Antimicrob Agents Chemother

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cConventional therapy for human cytomegalovirus (CMV) relies on inhibition of the viral DNA polymerase. Ganciclovir (GCV) is the first-line therapy, but when GCV-resistant strains emerge, alternative therapies are extremely limited and are associated with significant toxicities. Combination of anti-CMV agents that act on different targets or stages of virus replication has not been well studied, mostly because of the limited number of anti-CMV agents. We report our investigation of combinations of agents that inhibit CMV by targeting the viral DNA polymerase, cellular kinases, or other cell/virus mechanisms yet to be discovered. The selected compounds differed by the slopes of their dose-response curve: compounds with a slope of 1 (GCV) representing one target or noncooperativity and compounds with high slopes indicating positive cooperativity. Analysis of anti-CMV drug combinations using the Bliss model (which accounts for the slope parameter) distinguished between combinations with synergistic, antagonistic, and additive activities. The combination of GCV and foscarnet was slightly synergistic; strong synergism was found when GCV was used with artemisinin-derived monomers or dimers or the MEK inhibitor U0126. The combination of GCV and cardiac glycosides (digoxin, digitoxin, and ouabain) was additive. The monomeric artemisinin artesunate was synergistic when combined with U0126 or the multikinase inhibitor sunitinib. However, the combination of artemisinin-derived dimers (molecular weights, 606 and 838) and U0126 or sunitinib was antagonistic. These results demonstrate that members of a specific drug class show similar patterns of combination with GCV and that the slope parameter plays an important role in the evaluation of drug combinations. Lastly, antagonism between different classes of CMV inhibitors may assist in target identification and improve the understanding of CMV inhibition by novel compounds. C ytomegalovirus (CMV) is the most common cause of congenitally acquired infection in the United States and is a major pathogen in solid organ transplant recipients and patients with AIDS (1-3). Anti-CMV compounds have been used with varied success in these patient populations, but the complexity of CMV disease and the need for prolonged courses of therapy for virus suppression result in serious side effects and the emergence of resistant viral mutants (4-8). The FDA-approved anti-CMV drugs ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) belong to a single class of inhibitors, all targeting the viral DNA polymerase. The development and clinical evaluation of compounds that act on new viral targets, for example, the UL97 kinase inhibitor maribavir (9-11) and the terminase inhibitor AIC246 (12, 13), are under way. Cellular targets that could abrogate virus replication are also being studied as potential anti-CMV compounds (14). The role of anticellular antiviral inhibitors in CMV therapy is not defined as of yet; however, the potential use of such agents as either monotherapy (salvage therapy) or combi...

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“…Mycophenolate mofetil is an inosine monophosphate dehydrogenase inhibitor and is known to inhibit replication of other RNA viruses through depletion of the guanosine triphosphate pool (34,35). Strophanthin is a cardiac glycoside that inhibits the replication of other RNA viruses (36)(37)(38). A subset of compounds was also evaluated for their ability to inhibit EBOV in a murine model of infection.…”

Section: Discussionmentioning

confidence: 99%

A screen of approved drugs and molecular probes identifies therapeutics with anti–Ebola virus activity

et al. 2015

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Currently, no approved therapeutics exist to treat or prevent infections induced by Ebola viruses, and recent events have demonstrated an urgent need for rapid discovery of new treatments. Repurposing approved drugs for emerging infections remains a critical resource for potential antiviral therapies. We tested ~2600 approved drugs and molecular probes in an in vitro infection assay using the type species, Zaire ebolavirus. Selective antiviral activity was found for 80 U.S. Food and Drug Administration–approved drugs spanning multiple mechanistic classes, including selective estrogen receptor modulators, antihistamines, calcium channel blockers, and antidepressants. Results using an in vivo murine Ebola virus infection model confirmed the protective ability of several drugs, such as bepridil and sertraline. Viral entry assays indicated that most of these antiviral drugs block a late stage of viral entry. By nature of their approved status, these drugs have the potential to be rapidly advanced to clinical settings and used as therapeutic countermeasures for Ebola virus infections.

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Human Cytomegalovirus Inhibition by Cardiac Glycosides: Evidence for Involvement of the hERG Gene

Cited by 51 publications

References 53 publications

Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle

Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle

In Vitro Combination of Anti-Cytomegalovirus Compounds Acting through Different Targets: Role of the Slope Parameter and Insights into Mechanisms of Action

A screen of approved drugs and molecular probes identifies therapeutics with anti–Ebola virus activity

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