Artemisinin-Derived Dimer Diphenyl Phosphate Is an Irreversible Inhibitor of Human Cytomegalovirus Replication

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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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confidence: 73%

Section: Resultsmentioning

confidence: 99%

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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

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“…AS cannot form a dimer, but chemical synthesis resulted in several artemisinin-derived dimers, including dimer 606. Studies describing the anti-CMV activity of AS and dimer 606 have shown that the latter was significantly more active than AS, much more than two units of monomers combined (9,16,17). The compounds were dissolved in dimethyl sulfoxide (DMSO), and stocks of 10 mM were stored at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

Roy

Kapoor

et al. 2015

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dArtemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G 0 /G 1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G 1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G 1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G 1 /S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition.A rtemisinins, drugs of choice for malaria therapy, inhibit human cytomegalovirus (CMV) replication (1-4). Artesunate (AS) and the parent compound artemisinin inhibit CMV replication in vitro and in vivo. Variable responses to AS in case reports of CMV-infected patients have been attributed to the dosing regimen, duration of therapy, or tissue penetration of AS (5-8). In our effort to improve and uncover the anti-CMV activities of artemisinins, we reported on in vitro highly selective inhibition of CMV replication with artemisinin-derived dimers, significantly more than with their monomeric counterparts, without increasing toxicity in human foreskin fibroblasts (HFFs) (3, 9). Although similar effects on CMV replication were observed between monomers and dimers (timing of CMV inhibition, effects on DNA replication, and virus yield), dimers inhibited CMV at nanomolar concentrations and had a high slope of the dose-response curve, a measure of cooperativity in binding of multiple ligands to linked bi...

“…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%

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

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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...