Unique and highly selective anticytomegalovirus activities of artemisinin-derived dimer diphenyl phosphate stem from combination of dimer unit and a diphenyl phosphate moiety

Abstract

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.

Fig 1

Chemical structures of phosphate derivatives of artemisinin-derived monomers and dimers: the parent artemisinin-derived monomer and dimer (AS and compound 606, respectively), their MPP and DPP derivatives, and the nonartemisinins MPP, DPP, and TPP.

Fig 2

(A) Chemical structures of deoxy dimers 574 (deoxy dimer of compound 606) and 806 (deoxy dimer of compound 838). (B) Dose-response curves of deoxy dimers 574 and 806. The dose-dependent anti-CMV activity was measured for compounds 806 and 574 by determination of luciferase activity in CMV-infected HFFs at 72 hpi. The EC₅₀s and CC₅₀s of the four compounds appear in the inset. Mean values ± SDs of triplicate determinations from three independent experiments are shown.

Fig 3

The anti-CMV activity of MPP and DPP derivatives of dimer primary alcohol, compound 606 (A), and artemisinin monomer, AS (B). HFFs were infected and treated with monomeric or dimeric artemisinins at the indicated drug concentrations. Luciferase activity was measured at 72 hpi. Mean values ± SDs of triplicate determinations from at least three independent experiments are shown.

Fig 4

Anti-CMV activity of MPP, DPP, and TPP. CMV-infected HFFs were treated with MPP, DPP, and TPP, and luciferase activity was measured at 72 hpi. Mean values ± SDs of triplicate determinations from three independent experiments are shown.

Fig 5

Add-on and removal assays of artemisinin monomer (AS), monomer DPP (compound 558), dimer primary alcohol (compound 606), dimer DPP (compound 838), TPP, and GCV. Compounds were added (A) or removed (B) at 0, 6, 12, 24, 36, and 48 hpi. CMV inhibition was measured by determination of luciferase expression at 72 hpi. Mean values ± SDs of triplicate determinations from two independent experiments are shown.

Fig 6

Reversibility of CMV replication after treatment with artemisinin monomer (AS), monomer DPP (compound 558), artemisinin dimer primary alcohol (compound 606), dimer DPP (compound 838), TPP, and GCV. Compounds were present in CMV-infected cells over the indicated intervals (in days). Virus DNA in supernatants at 6 days postinfection was quantified by real-time PCR. Mean values ± SDs of triplicate determinations from two independent experiments are shown.

Fig 7

(A) Effect of pretreatment with artemisinin monomer (AS), monomer DPP (compound 558), dimer primary alcohol (compound 606), dimer DPP (compound 838), TPP, and GCV on CMV replication. (B) Inhibition of DNA replication, pp28 expression, and virus DNA yield by artemisinin monomer (AS), monomer DPP (compound 558), dimer primary alcohol (compound 606), dimer DPP (compound 838), TPP, and GCV. HFFs were treated with the indicated compounds for 24 h prior to infection. Compounds were removed just before infection. After infection, cells were grown in medium free of compounds. DNA replication in cell lysates collected at 48 hpi was measured by real-time PCR. pp28-luciferase activity was measured in cell lysates collected at 72 hpi. Virus DNA yield in supernatants collected at 96 hpi was measured by real-time PCR. Mean values ± SDs of triplicate determinations from three independent experiments are shown.

Fig 8

Effects of AS, 558, 606, 838, TPP, and GCV on CMV gene expression. The expression of CMV proteins was measured in infected HFFs. Compounds were added after virus infection, and cell lysates were collected for Western blotting at 24, 48, and 72 hpi.