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Article The Inhibits Human Cytomegalovirus Replication Via Iron Chelation

Jens-Uwe Vogel 1 , Sophie Schmidt 1, Daniel Schmidt 1, Florian Rothweiler 1, Benjamin Koch 2, Patrick Baer 2 , Holger Rabenau 1, Detlef Michel 3, Thomas Stamminger 3 , Martin Michaelis 4,* and Jindrich Cinatl 1,*

1 Institut für Medizinische Virologie, Universitätsklinikum, Goethe-Universität, Paul Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany; [email protected] (J.-U.V.); [email protected] (S.S.); [email protected] (D.S.); [email protected] (F.R.); [email protected] (H.R.) 2 Medizinische Klinik III, Nephrologie, Klinikum der Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany; [email protected] (B.K.); [email protected] (P.B.) 3 Institut für Virologie, Universitätsklinikum Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany; [email protected] (D.M.); [email protected] (T.S.) 4 Industry Biotechnology Centre and School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK * Correspondence: [email protected] (M.M.); [email protected] (J.C.); Tel.: +44-1227-82-7804 (M.M.); +49-69-678665-72 (J.C.);  Received: 1 November 2019; Accepted: 18 December 2019; Published: 20 December 2019 

Abstract: The agonist eltrombopag was successfully used against human cytomegalovirus (HCMV)-associated refractory to immunomodulatory and antiviral drugs. These effects were ascribed to the effects of eltrombopag on . Here, we tested whether eltrombopag may also exert direct antiviral effects. Therapeutic eltrombopag concentrations inhibited HCMV replication in human fibroblasts and adult mesenchymal stem cells infected with six different virus strains and drug-resistant clinical isolates. Eltrombopag also synergistically increased the anti-HCMV activity of the mainstay drug ganciclovir. Time-of-addition experiments suggested that eltrombopag interfered with HCMV replication after virus entry. Eltrombopag was effective in thrombopoietin receptor-negative cells, and the addition of Fe3+ prevented the anti-HCMV effects, indicating that it inhibits HCMV replication via iron chelation. This may be of particular interest for the treatment of cytopenias after hematopoietic transplantation, as HCMV reactivation is a major reason for transplantation failure. Since therapeutic eltrombopag concentrations are effective against drug-resistant viruses, and synergistically increase the effects of ganciclovir, eltrombopag is also a drug-repurposing candidate for the treatment of therapy-refractory HCMV disease.

Keywords: human cytomegalovirus; antiviral therapy; eltrombopag; thrombopietin receptor agonist; drug resistance; iron chelation

1. Introduction Eltrombopag is a thrombopoietin receptor (also known as c-Mpl or MPL) agonist that is used for the treatment of thrombocytopenia, including virus-associated thrombocytopenia [1–3]. Its use has also been suggested for the treatment of cytopenias after transplantations and case reports support its safety and efficacy [4–9].

Cells 2020, 9, 31; doi:10.3390/cells9010031 www.mdpi.com/journal/cells Cells 2020, 9, 31 2 of 12

Human cytomegalovirus (HCMV) reactivation and HCMV-associated disease are leading reasons for the failure of hematopoietic stem cell transplantations [10–12]. Anti-HCMV drugs, including ganciclovir, cidofovir, and foscarnet, are available, but their use is associated with severe side effects [13]. In particular, the use of ganciclovir (and its prodrug valganciclovir), the mainstay treatment for cytomegalovirus disease, is associated with severe hematological side effects, including thrombocytopenia [14–16]. A case report described the use of eltrombopag in an immunocompetent patient who suffered from human cytomegalovirus (HCMV)-associated thrombocytopenia [17]. Immunosuppressive treatment for thrombocytopenia (prednisone, intravenous immunoglobulin, dapsone), in combination with antiviral therapy (ganciclovir/valganciclovir, HCMV hyperimmune globulin), only resulted in a temporary response with subsequent relapse. A change to eltrombopag, intended to increase platelet counts without immunosuppressive therapy, resulted in a durable increase in platelet levels, no evidence of HCMV viraemia, and the resolution of symptoms [17]. The observed effects were attributed to eltrombopag overcoming HCMV-induced suppression of platelet production [17]. However, we hypothesized that direct antiviral effects may also have contributed to the beneficial outcome in the case report of the patient with HCMV-associated thrombocytopenia [17]. Indeed, we found that eltrombopag exerts anti-HCMV effects via iron chelation.

2. Materials and Methods

2.1. Drugs Eltrombopag (the orally active ethanolamine salt of eltrombopag olamine) was purchased from Selleck Chemicals (via Absource Diagnostics GmbH, Munich, Germany), deferasirox and ganciclovir from MedChemExpress (via Hycultec, Beutelsbach, Germany), and cidofovir from Cayman Chemical (via Biomol GmbH, Hamburg, Germany).

2.2. Cells and Viruses Primary human foreskin fibroblasts (HFFs) and adipose-derived adult mesenchymal stem cells (ASCs) were cultivated as previously described [18,19]. The wild-type HCMV strain, Hi91, was isolated from the urine of an AIDS patient with HCMV retinitis, as described previously [20]. HCMV strains Davis and Towne were received from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Virus stocks were prepared in HFFs maintained in minimal essential medium (MEM), supplemented with 4% fetal calf serum (FCS). The U1, U59, and U75 are patient isolates, which were isolated as previously described [20,21]. Virus stocks were prepared in HFFs maintained in MEM supplemented with 4% FCS. Murine cytomegalovirus (Smith strain, catalogue number VR-1399) was obtained from ATCC, and virus stocks were prepared in NIH/3T3 mouse fibroblasts (ATCC) maintained in MEM supplemented with 4% FCS. DNA isolation, amplification, and sequencing were performed as previously described [21], using established primers [22].

2.3. Virus Infectivity Assay In 96-well microtiter plates, confluent cultures of HFF cells or ASCs were incubated with HCMV at the indicated multiplicities of infection (MOIs). After incubation for 1-h, cells were washed with phosphate-buffered saline (PBS) and incubated in MEM containing 4% FCS and serial dilutions of the indicated substances. As described previously [18,23], cells producing HCMV-specific antigens were detected 24 h post infection by immunoperoxidase staining, using monoclonal directed against the UL123-coded 72 kDa immediate early antigen 1 (IEA1) (Mouse Anti CMV IEA, MAB8131, Millipore, Temecula, CA, USA), and 120 h post infection by immunoperoxidase staining, using monoclonal Cells 2020, 9, 31 3 of 12 antibodies directed against UL55-encoded late antigen (LA) gB, (kindly provided by K. Radsak, Institut für Virologie, Marburg, Germany), as previously described. Drug concentrations that reduced HCMV antigen expression by 50%, inhibitory concentration (IC50), were calculated using Calcusyn (Biosoft, Cambridge, UK). Effects of eltrombopag on murine cytomegalovirus were determined by visual scoring of cytopathogenic effect (CPE) formation (detected 120 h post infection) in MOI 1-infected murine NIH/3T3 fibroblasts.

2.4. Drug Combination Studies Drugs were combined at equimolar concentrations or used as single agents. Combined effects were determined by staining for HCMV LA. Combination indices (CIs) were calculated at different levels of inhibition (50% inhibition, CI50; 75% inhibition, CI75; 90% inhibition, CI90; 95% inhibition, CI95) by the method of Chou and Talalay [24], using CalcuSyn software version 1.0 (Biosoft, Cambridge, United Kingdom). Weighted average CI values (CIwt) were calculated as (CI + 2 CI + 3 CI 50 × 75 × 90 + 4 CI )/10. CIwt values 0.7 indicate synergistic effects, CIwt values >0.7 and 0.9 moderately × 95 ≤ ≤ synergistic effects, CIwt values >0.9 and 1.2 additive effects, CIwt values >1.2 and 1.45 moderately ≤ ≤ antagonistic effects, and CIwt values >1.45 antagonistic effects [24].

2.5. Viability Assay Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay as described previously [23]. Five thousand cells were seeded per well in 96-well microtiter plates and incubated with culture medium containing serial dilutions of the indicated substances. After five days of incubation, MTT (1 mg/mL) was added, and after an additional 4 h, cells were lysed in a buffer containing 20% (w/v) sodium dodecyl sulfate (SDS) and 50% N,N-dimethylformamide adjusted to pH 4.5. Absorbance was determined at 570 nm for each well using a 96-well multiscanner. After subtracting background absorbance, cell viability was expressed in percent relative to untreated control cells. Drug concentrations that reduced cell viability by 50% (CC50) were calculated using CalcuSyn (Biosoft, Cambridge, UK). The MTT assay measures metabolic activity in the mitochondria. To confirm viability results by a second assay, the CellTiter-Glo assay (Promega, Walldorf, Germany), which measures cellular ATP production, was used according to the manufacturer’s instructions.

2.6. Virus Yield Assay The amount of infectious virus was determined by virus yield assay in a single-cycle assay format, as previously described [23]. Virus titres were expressed as 50% of tissue culture infectious dose (TCID50/mL), 120 h post infection.

2.7. Immunoblotting Immunoblotting was performed as previously described [23]. In brief, cells were lysed in a Triton X-100 sample buffer and separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were detected using specific antibodies against ß-actin (3598R-100-BV, BioVision via BioCat, Heidelberg, Germany) or HCMV 45 kDa LA (MBS320051, MyBioSource via Biozol, Echingen, Germany), and were visualized by enhanced chemiluminescence using a commercially available (Thermo Scientific, Schwerte, Germany).

2.8. Statistics Values presented are the mean S.D. of three independent biological repeats. Comparisons ± between two groups were performed using the Student’s t-test, three and more groups were compared Cells 2020, 9, 31 4 of 12 by ANOVA, followed by the Student–Newman–Keuls test. Data groups were considered significantly different at p < 0.05.

3. ResultsCells 2020, 9, 31 4 of 12

3.1. Eltrombopag3. Results Inhibits HCMV Replication in Human Foreskin Fibroblasts by Interference with Late Processes of the Replication Cycle 3.1. Eltrombopag Inhibits HCMV Replication in Human Foreskin Fibroblasts by Interference with Late EltrombopagProcesses of the did Replication not aff Cycleect HCMV Hi91-induced immediate early antigen (IEA) expression (Figure S1),Eltrombopag but inhibited did HCMVnot affect Hi91-inducedHCMV Hi91-induced LA expression immediate early with antigen an IC 50(IEA)of 415expression nM in HFFs (Figure1(FigureA,B). EltrombopagS1), but inhibited concentrations HCMV Hi91-induced of up LA to 25expressionµM did with not an reduce IC50 of 415 the nM viability in HFFs of (Figure proliferating HFFs by1A,B). 50%, Eltrombopag as determined concentrations by MTT assayof up to (Figure 25 µM 1didA). not Cell reduce viability the viability determination of proliferating by CellTiter-Glo HFFs resultedby in 50%, similar as determined results (HFF by viabilityMTT assay at 25(FigureµM: 1A). 53 Cell4 µ M).viability Hence, determination the selectivity by CellTiter-Glo index, cytotoxicity resulted in similar results (HFF viability at 25 µM:± 53 ± 4 µM). Hence, the selectivity index, concentration (CC50)/IC50, is higher than 60.2 (Figure1A). Higher MOIs were associated with higher cytotoxicity concentration (CC50)/IC50, is higher than 60.2 (Figure 1A). Higher MOIs were associated IC values (Figure1C). At MOI 1, the highest MOI investigated in HFFs, the eltrombopag IC 50 with higher IC50 values (Figure 1C). At MOI 1, the highest MOI investigated in HFFs, the eltrombopag 50 was 3844IC50 nM. was The 3844 observed nM. The observed eltrombopag eltrombopag concentrations concentrations are withinare within the the range range of of therapeutic therapeutic plasma concentrations,plasma concentrations, which have which been describedhave been described to exceed to 45exceedµM[ 4525 µM,26 [25,26].].

ABVirus control Eltrombopag 10µM Eltrombopag 2.5µM 120

100

80

60

40 (% virus control) virus (% 20 IC50 (nM): 415 ± 197 HCMV LA expression LA HCMV IC90 (nM): 3379 ± 1088 0 0.1 1 10 100 1,000 10,000 Eltrombopag (nM) Eltrombopag Eltrombopag Eltrombopag 0.625µM 156nM 39nM

120

100 Eltrombopag (µM)

80 0 5 0.5 0.05 0.005 60 k s s k c u ck us ck iru irus r irus ir Mo V V Mock Vi Mo V Moc V Mo

(% control) (% 40 HFF viability HFF 20 HCMV LA CC50 (nM): > 25,000 0 1 10 100 1,000 10,000 100,000 ß-actin Eltrombopag (nM) C 120

100 ● MOI 1: IC50 (nM): 3844 ± 705, IC90 (nM): 8531 ± 793

80 ◼ MOI 0.1: IC50 (nM): 855 ± 429, IC90 (nM): 7830 ± 3758 60 ◆ MOI 0.01: IC50 (nM): 251 ± 135, IC90 (nM): 3476 ± 1330

40 (% virus control) 20 HCMVLA expression 0 0.1 1 10 100 1,000 10,000 Eltrombopag (nM)

Figure 1.FigureEffects 1. Effects of eltrombopag of eltrombopag on on human human cytomegalovirus cytomegalovirus (HCMV) (HCMV) late antigen late antigen (LA) expression (LA) expression in in primaryprimary human human foreskin foreskin fibroblastsfibroblasts (HFFs). (HFFs). (A) (RepresentativeA) Representative dose–res dose–responseponse curves showing curves the showing the effectseffects of of eltrombopag eltrombopag onon HCMV LA LA expression expression and andHFF HFFviability viability (as determined (as determined after 120 h after of 120 h incubation). Eltrombopag concentrations that reduce HCMV LA expression by 50% (inhibitory of incubation). Eltrombopag concentrations that reduce HCMV LA expression by 50% (inhibitory concentration (IC)50) or 90% (IC90) and cell viability by 50% (cytotoxicity concentration (CC)50) relative concentrationto untreated (IC)50 controls) or 90% are (ICalso90 provided.) and cell Eltrombopa viabilityg by was 50% continuously (cytotoxicity present concentration from the time (CC) of virus50) relative to untreatedinfection. controls (B) Representative are also provided. photogra Eltrombopagphs and Western was continuously blots demonstrating present fromthe theeffects time of of virus infection.eltrombopag (B) Representative on HCMV photographsLA expression. and In Western(A,B), HFFs blots were demonstrating infected with theHCMV effects strain of eltrombopagHi91 on HCMV(multiplicity LA expression. of infectionIn (MOI) (A,B 0.02).), HFFs HCMV were LA infectedexpression with was detected HCMV 120 strain h post Hi91 infection. (multiplicity (C) of infectionRepresentative (MOI) 0.02). dose–response HCMV LA curves expression and IC50 was values detected indicating 120 effects h post of eltrombopag infection. on (C HCMV) Representative LA expression in HFFs infected with different MOIs of HCMV strain Hi91 as detected 120 h post infection. dose–response curves and IC50 values indicating effects of eltrombopag on HCMV LA expression in

HFFs infected with different MOIs of HCMV strain Hi91 as detected 120 h post infection. Cells 2020, 9, 31 5 of 12 Cells 2020, 9, 31 5 of 12

Eltrombopag-inducedEltrombopag-induced inhibition inhibition of of HCMV HCMV LA LA translated translated into into reduced reduced virus virus replication replication as as indicatedindicated by by virus virus yield yield assay assay (Figure (Figure 2A).2A). At a concentration of 10 µM,µM, eltrombopag eltrombopag reduced reduced virus virus titrestitres by by 1.8 1.8 × 10104-fold4-fold and and at at 500 500 nM nM still still by by 15-fold. 15-fold. ×

FigureFigure 2. 2. EffectsEffects of of eltrombopag eltrombopag on on HCMV HCMV replication replication and and at at different different stages stages of of the the viral viral replication replication cycle.cycle. HFFs HFFs were were infected infected with with HCMV HCMV strain strain Hi91 Hi91 (MOI (MOI 0.02). 0.02). HCMV HCMV LA expression LA expression and virus and titres virus weretitres detected were detected 120 h post 120 hinfection. post infection. (A) Virus (A) Virustitres titresin the in absence the absence or presence or presence of eltrombopag. of eltrombopag. (B)

Representative(B) Representative dose–response dose–response curves curves and IC and50 values IC50 valuesindicating indicating the effects the of eff eltrombopagects of eltrombopag on HCMV on LAHCMV expression LA expression after 24 h afterof pre-treatment, 24 h of pre-treatment, after treatment after during treatment the during1-h adsorp thetion 1-h adsorptionperiod, after period, drug additionafter drug post addition infection post following infection followingthe 1-h virus the 1-hadsorption virus adsorption period, period,after drug after addition drug addition 24 h post 24 h infection,post infection, and after and drug after addition drug addition 48 h post 48 h infection. post infection. * p < 0.05 * p < 0.05

TheThe HCMV HCMV replication replication cycle cycle is is divided divided into into three three phases phases characterized characterized by by the the expression expression of of immediateimmediate early, early, early, early, and and late late viral viral . genes. Immediate Immediate early early genes genes are are transcribed transcribed immediately immediately after after infectioninfection and and do do not not depend depend on on synthesis synthesis of of vira virall DNA DNA or or transcription transcription of of proteins. proteins. Delayed Delayed early early proteinsproteins are are represented represented by by the the viral viral DNA DNA polymerase, polymerase, and and other other viral viral functions functions required required for for viral viral DNADNA synthesis, synthesis, and and some some viral viral structural structural proteins. proteins. La Latete genes genes encode encode mostly mostly structural structural proteins proteins used used inin viral viral assembly assembly and and packaging, packaging, and and are are generally generally expressed expressed subsequent subsequent to to delayed delayed early early genes genes [27]. [27]. ToTo better better define define which which phases phases of of the the viral viral replicat replicationion cycle cycle are are affected affected by by eltrombopag, eltrombopag, the the drug drug waswas added added at at different different time time points points (Figure (Figure 22B,B, TableTable S1).S1). Pre-incubation and drug addition during thethe 1-h 1-h virus virus adsorption adsorption period period did did not, not, or or only only mo modestly,destly, affect affect virus virus replication. replication. This This shows shows that that eltrombopageltrombopag does does not not primarily primarily interfere interfere with with virus virus binding binding to to host host cells cells and and virus virus internalization, internalization, butbut needs needs to to be be present present during during virus virus replication replication to to exert exert its its anti-HCMV anti-HCMV effects. effects. Drug Drug addition addition 1-h 1-h or or 2424 h h post post infection was susufficientfficient toto achieveachieve maximum maximum inhibition inhibition of of HCMV HCMV LA LA expression expression (Figure (Figure2B, 2B,Table Table S1). S1). This, This, together together with with the the observed observed lack lack ofof inhibitioninhibition ofof HCMV immediate early early antigen antigen (IEA)(IEA) expression, expression, indicates indicates that that eltrombopag eltrombopag inhibits inhibits the the late late stages stages of of the the HCMV HCMV replication replication cycle cycle characterizedcharacterized byby LALA expression. expression. Drug Drug addition addition 48 h48 post h infectionpost infection resulted resulted in reduced in reduced effects compared effects comparedto drug addition to drug 1-h addition or 24 h1-h post or infection24 h post (Figure infection2B, (Figure Table S1). 2B, Table S1). 3.2. Eltrombopag Inhibits HCMV Expression via Iron Chelation 3.2. Eltrombopag Inhibits HCMV Expression via Iron Chelation Eltrombopag was developed as a thrombopoietin receptor agonist [1–3]. However, it is unlikely Eltrombopag was developed as a thrombopoietin receptor agonist [1–3]. However, it is unlikely that eltrombopag inhibits HCMV replication via thrombopoietin receptor activation, because fibroblasts that eltrombopag inhibits HCMV replication via thrombopoietin receptor activation, because do not express the thrombopoietin receptor [28]. In agreement, eltrombopag also inhibited murine fibroblasts do not express the thrombopoietin receptor [28]. In agreement, eltrombopag also inhibited cytomegalovirus replication in murine NIH/3T3 fibroblasts (Figure3A), although eltrombopag does murine cytomegalovirus replication in murine NIH/3T3 fibroblasts (Figure 3A), although not target the murine thrombopoietin receptor [29]. eltrombopag does not target the murine thrombopoietin receptor [29].

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FigureFigure 3. 3. EltrombopagEltrombopag inhibits inhibits HCMV HCMV infection infection by by iron iron depletion. depletion. ( (AA)) Representative Representative dose–response dose–response curvecurve indicating indicating the the effects effects of ofeltrombopag eltrombopag on cyto on cytopathogenicpathogenic effect e ff(CPE)ect (CPE) formation formation (detected (detected 120 h post120 hinfection), post infection), in murine in murine cytomegalovirus cytomegalovirus (MOI (MOI1)-infected 1)-infected murine, murine, NIH/3T3 NIH fibroblasts,/3T3 fibroblasts, and

eltrombopagand eltrombopag concentration concentration that reduces that reduces CPE formation CPE formation by 50% by (IC 50%50), relative (IC50), relativeto untreated to untreated control. Thecontrol. findings The findingsindicate indicatethat eltrombopag that eltrombopag interferes interferes with with cytomegalovirus cytomegalovirus replication replication by by thrombopoietin-receptor-independentthrombopoietin-receptor-independent effects, effects, si sincence eltrombopag eltrombopag does does not not activate activate the the murine thrombopoietinthrombopoietin receptor. receptor. The The investigated investigated eltrombo eltrombopagpag concentrations concentrations did did no nott affect affect NIH/3T3 NIH/3T3 cell viability. ((BB)) Representative Representative growth growth curve curv indicatinge indicating the e fftheects effects of equimolar of equimolar concentrations concentrations of Fe(III)Cl of3

Fe(III)Clon the anti-HCMV3 on the anti-HCMV effects of effects eltrombopag, of eltrombopag, as indicated as indicated by HCMV by HCMV LA expression LA expression in HCMV in HCMV Hi91 (MOI 0.02)-infected human foreskin fibroblasts (HFFs) 120 h post infection. Equimolar Fe(III)Cl Hi91 (MOI 0.02)-infected human foreskin fibroblasts (HFFs) 120 h post infection. Equimolar Fe(III)Cl3 concentrationsconcentrations circumvent circumvent the anti-H anti-HCMVCMV effects effects exerted by eltrombopag.

Eltrombopag is also an iron chelator [2,30,31], and iron chelators have been shown to inhibit Eltrombopag is also an iron chelator [2,30,31], and iron chelators have been shown to inhibit HCMV replication [32–38]. The addition of equimolar Fe3+ concentrations was shown to inhibit HCMV replication [32–38]. The addition of equimolar Fe3+ concentrations was shown to inhibit pharmacological action of eltrombopag that are caused via iron chelation [31]. Hence, we investigated pharmacological action of eltrombopag that are caused via iron chelation [31]. Hence, we investigated eltrombopag in combination with equimolar Fe(III)Cl3 concentrations to investigate whether iron eltrombopag in combination with equimolar Fe(III)Cl3 concentrations to investigate whether iron chelation is the mechanism by which eltrombopag exerts its anti-HCMV effects (Figure3B). Equimolar chelation is the mechanism by which eltrombopag exerts its anti-HCMV effects (Figure 3B). Fe(III)Cl3 concentrations prevented the anti-HCMV effects of eltrombopag (Figure3B). This suggests Equimolar Fe(III)Cl3 concentrations prevented the anti-HCMV effects of eltrombopag (Figure 3B). that iron chelation is the main mechanism of eltrombopag’s anti-HCMV activity. In agreement, This suggests that iron chelation is the main mechanism of eltrombopag’s anti-HCMV activity. In eltrombopag exerted antagonistic effects in combination with the iron chelator deferasirox (Table S2), agreement, eltrombopag exerted antagonistic effects in combination with the iron chelator which may indicate that both compounds share the same antiviral mechanism. deferasirox (Table S2), which may indicate that both compounds share the same antiviral mechanism. 3.3. Eltrombopag Exerts Synergistic Effects with Ganciclovir 3.3. Eltrombopag Exerts Synergistic Effects with Ganciclovir Next, we tested eltrombopag in combination with ganciclovir, the mainstay of anti-HCMV Next, we tested eltrombopag in combination with ganciclovir, the mainstay of anti-HCMV therapies [13]. The combination of equimolar eltrombopag and ganciclovir concentrations resulted therapies [13]. The combination of equimolar eltrombopag and ganciclovir concentrations resulted in in synergistic anti-HCMV effects (Figure4), which is illustrated by a weighted average combination synergistic anti-HCMV effects (Figure 4), which is illustrated by a weighted average combination index (CIWT) of 0.17 0.03, as determined by the method of Chou and Talalay [24]. According to this index (CIWT) of 0.17 ±± 0.03, as determined by the method of Chou and Talalay [24]. According to this method, combined effects are considered to be synergistic at a CIWT of 0.7 [24]. The combination of method, combined effects are considered to be synergistic at a CIWT of ≤0.7 [24]. The combination of eltrombopag and foscarnet also displayed synergistic effects (Table S2). eltrombopag and foscarnet also displayed synergistic effects (Table S2).

Cells 2020, 9, 31 7 of 12 Cells 2020, 9, 31 7 of 12 A ganciclovir 100 eltrombopag 80 combination *

60 * * * 40 *

20 *

HCMV LA expression (% virus control) (% virus expression LA HCMV * 0 detectable not 10,000 2,500 625 156.25 39.06 9.77 2.44 0.61 Drug concentration (nM) B 1.00

0.75

0.50 CI values 0.25

0 CI50 CI75 CI90 CI95 CIWT

FigureFigure 4. Antiviral 4. Antiviral eff effectsects of of eltrombopag eltrombopag in in combination combination with with ganciclovir. ganciclovir. (A) Effects (A)Eff ofects equimolar of equimolar drugdrug concentrations concentrations on on HCMV HCMV LALA expression in in HCMV HCMV Hi91 Hi91 (MOI (MOI 0.02) 0.02) HFFs HFFs 120 h 120 post h infection. post infection. * p <* p0.05 < 0.05 compared compared to to either either singlesingle treatment; (B (B) )Combination Combination indices indices (CIs) (CIs) at different at diff erentlevels levelsof of inhibition and weighted average CI values (CIwt) calculated as (CI50 + 2 × CI75 + 3 × CI90 + 4 × CI95)/10 inhibition and weighted average CI values (CIwt) calculated as (CI50 + 2 CI75 + 3 CI90 + 4 [24]. CIwt values ≤0.7 indicate synergistic effects [24]. × × × CI )/10 [24]. CIwt values 0.7 indicate synergistic effects [24]. 95 ≤ 3.4.3.4. Eltrombopag Eltrombopag Is EIsff Effectiveective in in Di Differentfferent CellCell TypesTypes and against against Different Different Viru Viruss Strains Strains and and Isolates Isolates Including Drug-ResistantIncluding Drug-Resistant Ones Ones Finally,Finally, we we investigated investigated the the eeffectsffects of eltrombo eltrombopagpag against against a broader a broader range range of laboratory of laboratory virus virus strains and clinical isolates in HFFs, and primary-adipose-derived ASCs, another cell type that strains and clinical isolates in HFFs, and primary-adipose-derived ASCs, another cell type that supports supports HCMV replication [39]. The laboratory HCMV strains included Davis [40] and Towne [41] HCMV replication [39]. The laboratory HCMV strains included Davis [40] and Towne [41] in addition in addition to Hi91. The clinical isolates U1, U59, and U75 were isolated from the urine of patients, as to Hi91.previously The clinical described isolates [20,21]. U1, U1 U59,and U59 and harbor U75 were an A987G isolated mutation from in the the urine HCMV of patients,DNA polymerase as previously describedUL54 (Table [20,21 1),]. which U1 and is known U59 harbor to confer an combined A987G mutationganciclovir in and the cidofovir HCMV resistance DNA polymerase [42,43]. U1 UL54 (Tablealso1), displays which isa knownC607Y mutation to confer in combined the HCMV ganciclovir kinase UL97 and (Table cidofovir 1), which resistance is associated [ 42,43 with]. U1 also displaysganciclovir a C607Y resistance mutation [44,45]. in the In agreement, HCMV kinase U1 and UL97 U59 (Tablewere characterized1), which is associatedby high ganciclovir with ganciclovir and resistancecidofovir [44 ,IC4550].s In(Table agreement, 1), which U1 are and typically U59 were considered characterized to indicate by high resistance ganciclovir [46–48]. and cidofovirU75 also IC50s (Tabledisplayed1), which resistance are typically to ganciclovir considered and to cidofovir, indicate resistancealthough it [ 46does–48 ].not U75 harbor also displayedknown resistance resistance to ganciclovirmutations and (data cidofovir, not shown). although it does not harbor known resistance mutations (data not shown).

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Table 1. Sensitivity of laboratory HCMV strains and patient isolates to ganciclovir and cidofovir as indicated by HCMV LA expression in human foreskin fibroblasts infected at MOI 0.02 120 h post infection. Concentrations that reduce LA expression by 50% (IC50) are provided.

IC50 (µM) IC50 (µM) Cells 2020, 9, 31 8 of 12 Virus Strain/Isolate Resistance Mutations Ganciclovir Cidofovir Hi91 n/a 0.71 ± 0.57 0.31 ± 0.14 Table 1. SensitivityDavis of laboratory HCMV strains n/a and patient isolates 0.72 ± 0.31 to ganciclovir 0.17 ± and0.02 cidofovir as indicated by HCMVTowne LA expression in human n/a foreskin fibroblasts 0.61 ± infected 0.12 at0.14 MOI ± 0.01 0.02 120 h post infection. ConcentrationsU1 that reduce A987G LA expression *, C607Y by ** 50% (IC50 24) are± 10 provided. 5.2 ± 1.9 U59 A987G 23 ± 14 1.6 ± 0.5 IC50 (µM) IC50 (µM) * mutation in the HCMV DNA polymerase UL54, which confers resistance to ganciclovir and Virus Strain/Isolate Resistance Mutations Ganciclovir Cidofovir cidofovir [42,43]. ** mutation in the HCMV kinase UL97, which is associated with ganciclovir Hi91 n/a 0.71 0.57 0.31 0.14 resistance [44,45]. ± ± Davis n/a 0.72 0.31 0.17 0.02 ± ± Towne n/a 0.61 0.12 0.14 0.01 The eltrombopag IC50s ranged from 99 nM (U1 in HFFs) to 4331± nM (Hi91 in ASCs)± (Figure 5A, U1 A987G *, C607Y ** 24 10 5.2 1.9 ± ± Table S3). When U59compared across the two A987G cell types, the different 23 HCMV14 strains and 1.6 clinical0.5 isolates ± ± displayed* mutation similar in theeltrombopag HCMV DNA sensitivity, polymerase UL54,apart whichfrom confersU1, which resistance appeared to ganciclovir to be particularly and cidofovir sensitive [42,43]. to eltrombopag** mutation inin the HFFs HCMV and kinase ASCs UL97, (Figure which is5A). associated The average with ganciclovir HCMV resistance sensitivity [44,45 ].to eltrombopag was very similar in both cell types.

ToThe confirm eltrombopag the relevance IC50s ranged of iron from chelation 99 nM (U1 as in mechanism HFFs) to 4331 of nM the (Hi91 anti-HCMV in ASCs) action (Figure 5ofA, eltrombopagTable S3). When using compared a clinical across virus the twoisolate, cell types,U1-infected the di fferentHFFs HCMVwere strainstreated andwith clinical equimolar isolates concentrationsdisplayed similar of eltrombopag sensitivity,and Fe(III)Cl apart3. fromThe U1,presence which of appeared equimolar to be Fe particularly3+ concentrations sensitive preventedto eltrombopag the eltrombopag-induced in HFFs and ASCs (Figureinhibition5A). of The HCMV average LA HCMV expression sensitivity in U1-infected to eltrombopag cells in was a comparablevery similar fashion in both (Figure cell types. 5B), as in Hi91-infected cells (Figure 3B).

FigureFigure 5. 5.AntiviralAntiviral effects effects of ofeltrombopag eltrombopag determined determined in indifferent different cell cell types types infected infected by by different different HCMVHCMV strains strains and and clinical clinical isolates. isolates. HFFs HFFs were were infe infectedcted at atan an MOI MOI ofof 0.02 0.02 and and adipose-derived adipose-derived adult adult mesenchymalmesenchymal stem stem cells cells (ASCs) (ASCs) at atan an MOI MOI of of 5. 5.HCMV HCMV LA LA expression expression was was determined determined 120 120 h hpost post infection.infection. (A (A) ) Eltrombopag concentrationsconcentrations that that reduce reduce HCMV HCMV LA expression LA expression by 50% (ICby 5050%). Numerical (IC50). Numericalvalues are values provided are inprovided Table S3. in The Table eltrombopag S3. The eltrombopag concentration concentration that reduced ASCthat viabilityreduced byASC 50% viability(CC50) by was 50% 17,872 (CC50) 1302was 17,872 nM. ( B±) 1302 Representative nM. (B) Representative growth curve growth indicating curve the indicating effects of the equimolar effects ± ofconcentrations equimolar concentrations of Fe(III)Cl 3ofon Fe(III)Cl the anti-HCMV3 on the anti-HCMV effects ofeltrombopag effects of eltrombopag as indicated as byindicated HCMV by LA HCMVexpression LA expression in U1 (MOI in 0.02)-infectedU1 (MOI 0.02)-infected HFFs 120 hHFFs post 120 infection. h post infection.

4. DiscussionTo confirm the relevance of iron chelation as mechanism of the anti-HCMV action of eltrombopag using a clinical virus isolate, U1-infected HFFs were treated with equimolar concentrations Here, we show that the approved thrombopoietin receptor agonist3+ eltrombopag exerts anti- of eltrombopag and Fe(III)Cl3. The presence of equimolar Fe concentrations prevented the HCMVeltrombopag-induced effects in various inhibition cell types of infected HCMV with LA expressiona range of different in U1-infected virus strains cells in and a comparable clinical isolates, fashion including(Figure5 B),drug-resistant as in Hi91-infected ones. The cells observed (Figure 3ICB).50 values ranged from 99 nM to 4331 nM, which is in the range of therapeutic plasma concentrations that have been reported to exceed 45 µM [25,26]. Eltrombopag4. Discussion also synergistically increased the activity of the approved anti-HCMV drug ganciclovir. Our findings are in agreement with a case report on an immunocompetent patient, who suffered Here, we show that the approved thrombopoietin receptor agonist eltrombopag exerts anti-HCMV from HCMV-associated thrombocytopenia and recovered after eltrombopag therapy [17]. This effects in various cell types infected with a range of different virus strains and clinical isolates, including drug-resistant ones. The observed IC50 values ranged from 99 nM to 4331 nM, which is in the range of therapeutic plasma concentrations that have been reported to exceed 45 µM[25,26]. Eltrombopag also synergistically increased the activity of the approved anti-HCMV drug ganciclovir. Cells 2020, 9, 31 9 of 12

Our findings are in agreement with a case report on an immunocompetent patient, who suffered from HCMV-associated thrombocytopenia and recovered after eltrombopag therapy [17]. This response had originally been attributed to effects of eltrombopag on platelet production [17]. The possibility that eltrombopag may exert antiviral affects was not considered. Our current data show that therapeutic eltrombopag levels interfere with HCMV replication, which may have contributed to the beneficial clinical outcome. Notably, eltrombopag has also been shown to inhibit the replication of severe fever with thrombocytopenia syndrome virus, a member of the genus Banyangvirus (Phenuiviridae) [49]. The anti-HCMV effects of eltrombopag are unlikely to be caused by action on the thrombopoietin receptor, since eltrombopag was effective in cell types that do not express the thrombopoietin receptor, which is expressed in hematopoietic cells [28,29]. In agreement, eltrombopag also exerted antiviral effects in mouse fibroblasts infected with murine CMV, although the hematological effects of eltrombopag are known to be species-specific and to not affect mice [28,29]. Eltrombopag is also known to be an iron chelator [30,31]. The addition of Fe3+ prevented the eltrombopag-mediated anti-HCMV effects in strain Hi91 and clinical isolate U1-infected cells. Hence, our data suggest that eltrombopag inhibits HCMV replication via Fe3+ chelation. A number of different iron chelators, including desferrioxamine, diethylenetriaminepentaacetic acid (DTPA), and ethylenediaminedisuccinic acid (EDDS), were shown to inhibit HCMV replication [32–38]. However, the iron chelators tiron and ciclopirox olamine were not found to inhibit HCMV strain AD169 replication in MRC5 cells [50]. The experimental setup differed, as MRC5 cells were infected at a high MOI of 3, and no dose–response relationships were determined. Hence, a direct comparison is not possible. Notably, specific antiviral activity can easily be missed if the therapeutic window between antiviral and cytotoxic effects is relatively small. For example, desferrioxamine was found to inhibit HCMV replication at concentrations that did not decrease the viability of confluent fibroblasts but affected dividing cells [32]. In contrast, eltrombopag inhibits HCMV replication in concentrations that do not affect cell proliferation. Hence, the size of the therapeutic window that discriminates between anti-HCMV activity and antiproliferative and cytotoxic effects substantially differs among iron chelators. Eltrombopag seems to be an iron chelator that possesses a particularly preferential therapeutic window in terms of its anti-HCMV activity. Due to its effects on platelet counts and hematopoietic stem cells [2], however, the anti-HCMV effects of eltrombopag are primarily of relevance for anemia patients at risk of HCMV disease, for whom eltrombopag is indicated. Eltrombopag has been suggested for the treatment of cytopenias after hematopoietic stem cell transplantations and case reports support its safety and efficacy [4–9]. Since HCMV reactivation and HCMV-associated diseases are leading reasons for the failure of hematopoietic stem cell transplantations [10–12], antiviral effects exerted by eltrombopag may also contribute to improved therapy outcome. Notably, eltrombopag was effective against resistant clinical HCMV isolates, and resistance formation to the approved drugs is a major challenge after stem cell transplantation [11,12].

5. Conclusions Therapeutic eltrombopag concentrations inhibit HCMV replication via chelation of Fe3+ ions. Eltrombopag is effective against drug-resistant viruses and synergistically increases the effects of the mainstay anti-HCMV drug ganciclovir. The anti-HCMV activity of eltrombopag may be of particular interest for its use for the treatment of cytopenias after haematopoietic stem cell transplantation, as HCMV reactivation and disease is a major reason for transplantation failure.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/1/31/s1, Figure S1: Effects of eltrombopag on HCMV immediate early antigen (IEA) expression in primary human foreskin fibroblasts (HFFs), Table S1: Effects of eltrombopag addition at different time points during the HCMV replication, Table S2: Effects of eltrombopag in combination with deferasirox and foscarnet, Table S3: Effects of eltrombopag on HCMV late antigen (LA) expression in different cell types infected with different virus strains and isolates. Cells 2020, 9, 31 10 of 12

Author Contributions: J.-U.V., S.S., D.S., F.R., B.K., P.B., H.R., D.M., T.S. and J.C. performed experiments. All authors analysed data. M.M. and J.C. designed and conducted the study and wrote the first manuscript draft. All authors read and approved the final version. All authors have read and agreed to the published version of the manuscript. Funding: The work was funded by the Hilfe für krebskranke Kinder Frankfurt e.V. and the Frankfurter Stiftung für krebskranke Kinder. Conflicts of Interest: The authors declare no conflicts of interest.

References

1. Gill, H.; Wong, R.S.M.; Kwong, Y.L. From chronic immune thrombocytopenia to severe : Recent insights into the evolution of eltrombopag. Ther. Adv. Hematol. 2017, 8, 159–174. [CrossRef] 2. Scheinberg, P. Activity of eltrombopag in severe aplastic anemia. Blood Adv. 2018, 2, 3054–3062. [CrossRef] 3. Ghanima, W.; Cooper, N.; Rodeghiero, F.; Godeau, B.; Bussel, J.B. Thrombopoietin receptor : ten years later. Haematologica 2019, 104, 1112–1123. [CrossRef] 4. Master, S.; Dwary, A.; Mansour, R.; Mills, G.M.; Koshy, N. Use of Eltrombopag in Improving Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation. Case Rep. Oncol. 2018, 11, 191–195. [CrossRef] 5. Tang, C.; Chen, F.; Kong, D.; Ma, Q.; Dai, H.; Yin, J.; Li, Z.; Chen, J.; Zhu, X.; Mao, X.; Wu, D.; Tang, X. Successful treatment of secondary poor graft function post allogeneic hematopoietic stem cell transplantation with eltrombopag. J. Hematol. Oncol. 2018, 11, 103. [CrossRef] 6. Fu, H.; Zhang, X.; Han, T.; Mo, X.; Wang, Y.; Chen, H.; Han, W.; Wang, J.; Wang, F.; Yan, C.; Zhang, Y.; Sun, Y.; Liu, K.; Huang, X.; Xu, L. Eltrombopag is an effective and safe therapy for refractory thrombocytopenia after haploidentical hematopoietic stem cell transplantation. . Transplant. 2019, 54, 1310–1318. [CrossRef] 7. Guenther, K.L.; Cheruku, P.S.; Cash, A.; Smith, R.H.; Alvarado, L.J.; Burkett, S.; Townsley, D.M.; Winkler, T.; Larochelle, A. Eltrombopag promotes DNA repair in human hematopoietic stem and progenitor cells. Exp. Hematol. 2019, 73, 1–6. [CrossRef] 8. Marotta, S.; Marano, L.; Ricci, P.; Cacace, F.; Frieri, C.; Simeone, L.; Trastulli, F.; Vitiello, S.; Cardano, F.; Pane, F.; Risitano, A.M. Eltrombopag for post-transplant cytopenias due to poor graft function. Bone Marrow. Transplant. 2019, 54, 1346–1353. [CrossRef] 9. Rivera, D.; Bastida, J.M.; Lopez-Corral, L.; Sanchez-Guijo, F.; Cabrero, M.; Martin, A.; Perez, E.; Lopez-Parra, M.; Avendaño, A.; Veiga, A.; et al. Usefulness of eltrombopag for treating thrombocytopenia after allogeneic stem cell transplantation. Bone Marrow. Transplant. 2019, 54, 757–761. [CrossRef] 10. Cho, S.Y.; Lee, D.G.; Kim, H.J. Cytomegalovirus Infections after Hematopoietic Stem Cell Transplantation: Current Status and Future Immunotherapy. Int. J. Mol. Sci. 2019, 20, 2666. [CrossRef] 11. Ljungman, P.; de la Camara, R.; Robin, C.; Crocchiolo, R.; Einsele, H.; Hill, J.A.; Hubacek, P.; Navarro, D.; Cordonnier, C.; Ward, K.N.; et al. Guidelines for the management of cytomegalovirus infection in patients with haematological malignancies and after stem cell transplantation from the 2017 European Conference on Infections in Leukaemia (ECIL 7). Lancet Infect. Dis. 2019, 19, e260–e272. [CrossRef] 12. Pande, A.; Dubberke, E.R. Cytomegalovirus Infections of the Stem Cell Transplant Recipient and Hematologic Malignancy Patient. Infect. Dis. Clin. North Am. 2019, 33, 485–500. [CrossRef] 13. Britt, W.J.; Prichard, M.N. New therapies for human cytomegalovirus infections. Antiviral. Res. 2018, 159, 153–174. [CrossRef] 14. McGavin, J.K.; Goa, K.L. Ganciclovir: an update of its use in the prevention of cytomegalovirus infection and disease in transplant recipients. Drugs 2001, 61, 1153–1183. [CrossRef] 15. Busca, A.; de Fabritiis, P.; Ghisetti, V.; Allice, T.; Mirabile, M.; Gentile, G.; Locatelli, F.; Falda, M. Oral valganciclovir as preemptive therapy for cytomegalovirus infection post allogeneic stem cell transplantation. Transpl. Infect. Dis. 2007, 9, 102–107. [CrossRef] 16. Matsumoto, K.; Shigemi, A.; Ikawa, K.; Kanazawa, N.; Fujisaki, Y.; Morikawa, N.; Takeda, Y. Risk factors for ganciclovir-induced thrombocytopenia and leukopenia. Biol. Pharm. Bull. 2015, 38, 235–238. [CrossRef] 17. Simpson, J.D.; Matthews, G.V.; Brighton, T.A.; Joseph, J.E. Cytomegalovirus-associated thrombocytopenia treated with thrombopoietin receptor agonist. Intern. Med. J. 2016, 46, 1096–1099. [CrossRef] Cells 2020, 9, 31 11 of 12

18. Cinatl, J.; Cinatl, J.; Weber, B.; Rabenau, H.; Gümbel, H.O.; Chenot, J.F.; Scholz, M.; Encke, A.; Doerr, H.W. In vitro inhibition of human cytomegalovirus replication in human foreskin fibroblasts and endothelial cells by ascorbic acid 2-phosphate. Antiviral. Res. 1995, 27, 405–418. [CrossRef] 19. Baer, P.C.; Brzoska, M.; Geiger, H. Epithelial differentiation of human adipose-derived stem cells. Methods Mol. Biol. 2011, 702, 289–298. 20. Cinatl, J.Jr.; Kotchetkov, R.; Scholz, M.; Cinatl, J.; Vogel, J.U.; Driever, P.H.; Doerr, H.W. Human cytomegalovirus infection decreases expression of thrombospondin-1 independent of the tumor suppressor . Am. J. Pathol. 1999, 155, 285–292. [CrossRef] 21. Vogel, J.U.; Otte, J.; Koch, F.; Gümbel, H.; Doerr, H.W.; Cinatl, J., Jr. Role of human cytomegalovirus genotype polymorphisms in AIDS patients with cytomegalovirus retinitis. Med. Microbiol. Immunol. 2013, 202, 37–47. [CrossRef] 22. Michel, D.; Höhn, S.; Haller, T.; Jun, D.; Mertens, T. Aciclovir selects for ganciclovir-cross-resistance of human cytomegalovirus in vitro that is only in part explained by known mutations in the UL97 protein. J. Med. Virol. 2001, 65, 70–76. [CrossRef] 23. Michaelis, M.; Paulus, C.; Löschmann, N.; Dauth, S.; Stange, E.; Doerr, H.W.; Nevels, M.; Cinatl, J. Jr. The multi-targeted kinase inhibitor inhibits human cytomegalovirus replication. Cell Mol. Life Sci. 2011, 68, 1079–1090. [CrossRef] 24. Chou, T.C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 2006, 58, 621–681. [CrossRef] 25. Matthys, G.; Park, J.W.; McGuire, S.; Wire, M.B.; Bowen, C.; Williams, D.; Jenkins, J.; Peng, B. Clinical , platelet response, and safety of eltrombopag at supratherapeutic doses of up to 200 mg once daily in healthy volunteers. J. Clin. Pharmacol. 2011, 51, 301–308. [CrossRef] 26. Wire, M.B.; Fang, L.; Hussaini, A.; Kleha, J.F.; Theodore, D. Lack of clinically significant pharmacokinetic interaction between the thrombopoietin receptor agonist eltrombopag and hepatitis C virus protease inhibitors boceprevir and telaprevir. Antimicrob. Agents Chemother. 2014, 58, 6704–6709. [CrossRef] 27. Scholz, M.; Doerr, H.W.; Cinatl, J. Inhibition of cytomegalovirus immediate early expression: A therapeutic option? Antiviral Res. 2001, 49, 129–145. [CrossRef] 28. Kaushansky, K. Molecular mechanisms of thrombopoietin signaling. J. Thromb. Haemost. 2009, 7 (Suppl. S1), 235–238. [CrossRef] 29. Erickson-Miller, C.L.; Delorme, E.; Tian, S.S.; Hopson, C.B.; Landis, A.J.; Valoret, E.I.; Sellers, T.S.; Rosen, J.; Miller, S.G.; Luengo, J.I.; et al. Preclinical activity of eltrombopag (SB-497115), an oral, nonpeptide thrombopoietin receptor agonist. Stem. Cells 2009, 27, 424–430. [CrossRef] 30. Roth, M.; Will, B.; Simkin, G.; Narayanagari, S.; Barreyro, L.; Bartholdy, B.; Tamari, R.; Mitsiades, C.S.; Verma, A.; Steidl. Eltrombopag inhibits the proliferation of cells via reduction of intracellular iron and induction of differentiation. Blood 2012, 120, 386–394. [CrossRef] 31. Vlachodimitropoulou, E.; Chen, Y.L.; Garbowski, M.; Koonyosying, P.; Psaila, B.; Sola-Visner, M.; Cooper, N.; Hider, R.; Porter, J. Eltrombopag: A powerful chelator of cellular or extracellular iron(III) alone or combined with a second chelator. Blood 2017, 130, 1923–1933. [CrossRef] 32. Cinatl, J., Jr.; Cinatl, J.; Rabenau, H.; Gümbel, H.O.; Kornhuber, B.; Doerr, H.W. In vitro inhibition of human cytomegalovirus replication by desferrioxamine. Antiviral. Res. 1994, 25, 73–77. [CrossRef] 33. Cinatl, J. Jr.; Hoffmann, F.; Cinatl, J.; Weber, B.; Scholz, M.; Rabenau, H.; Stieneker, F.; Kabickova, H.; Blasko, M.; Doerr, H.W. In vitro inhibition of human cytomegalovirus replication by calcium trinatrium diethylenetriaminepentaacetic acid. Antiviral. Res. 1996, 31, 23–34. [CrossRef] 34. Kloover, J.S.; Scholz, M.; Cinatl, J., Jr.; Lautenschlager, I.; Grauls, G.E.; Bruggeman, C.A. Effect of desferrioxamine (DFO) and calcium trinatrium diethylenetriaminepentaacetic acid (DTPA) on rat cytomegalovirus replication in vitro and in vivo. Antiviral. Res. 1999, 44, 55–65. [CrossRef] 35. Martelius, T.; Scholz, M.; Krogerus, L.; Höckerstedt, K.; Loginov, R.; Bruggeman, C.; Cinatl, J. Jr.; Doerr, H.W.; Lautenschlager, I. Antiviral and immunomodulatory effects of desferrioxamine in cytomegalovirus-infected rat allografts with rejection. Transplantation 1999, 68, 1753–1761. [CrossRef] 36. Vogel, J.U.; Michaelis, M.; Neyts, J.; Blaheta, R.A.; Snoeck, R.; Andrei, G.; De Clercq, E.; Rabenau, H.F.; Kreuter, J.; Cinatl, J., Jr.; et al. Antiviral and immunomodulatory activity of the metal chelator ethylenediaminedisuccinic acid against cytomegalovirus in vitro and in vivo. Antiviral Res. 2002, 55, 179–188. [CrossRef] Cells 2020, 9, 31 12 of 12

37. Crowe, W.E.; Maglova, L.M.; Ponka, P.; Russell, J.M. Human cytomegalovirus-induced host cell enlargement is iron dependent. Am. J. Physiol. Cell Physiol. 2004, 287, C1023–C1030. [CrossRef] 38. Michaelis, M.; Langer, K.; Arnold, S.; Doerr, H.W.; Kreuter, J.; Cinatl, J., Jr. Pharmacological activity of DTPA linked to protein-based drug carrier systems. Biochem. Biophys. Res. Commun. 2004, 323, 1236–1240. [CrossRef] 39. Zwezdaryk, K.J.; Ferris, M.B.; Strong, A.L.; Morris, C.A.; Bunnell, B.A.; Dhurandhar, N.V.; Gimble, J.M.; Sullivan, D.E. Human cytomegalovirus infection of human adipose-derived stromal/stem cells restricts differentiation along the adipogenic lineage. Adipocyte 2015, 5, 53–64. [CrossRef] 40. Craig, J.M.; Macaulay, J.C.; Weller, T.H.; Wirth, P. Isolation of intranuclear inclusion producing agents from infants with illnesses resembling cytomegalic inclusion disease. Proc. Soc. Exp. Biol. Med. 1957, 94, 4–12. 41. Plotkin, S.A.; Furukawa, T.; Zygraich, N.; Huygelen, C. Candidate cytomegalovirus strain for human vaccination. Infect Immun. 1975, 12, 521–527. [PubMed] 42. Cihlar, T.; Fuller, M.D.; Cherrington, J.M. Characterization of drug resistance-associated mutations in the human cytomegalovirus DNA polymerase gene by using recombinant mutant viruses generated from overlapping DNA fragments. J. Virol. 1998, 72, 5927–5936. [PubMed] 43. Chou, S.; Marousek, G.; Bowlin, T.L. Cyclopropavir susceptibility of cytomegalovirus DNA polymerase mutants selected after antiviral drug exposure. Antimicrob. Agents Chemother. 2012, 56, 197–201. [CrossRef] [PubMed] 44. Baldanti, F.; Underwood, M.R.; Talarico, C.L.; Simoncini, L.; Sarasini, A.; Biron, K.K.; Gerna, G. The Cys607–>Tyr change in the UL97 phosphotransferase confers ganciclovir resistance to two human cytomegalovirus strains recovered from two immunocompromised patients. Antimicrob. Agents Chemother. 1998, 42, 444–446. 45. Chou, S.; Ercolani, R.J.; Sahoo, M.K.; Lefterova, M.I.; Strasfeld, L.M.; Pinsky, B.A. Improved detection of emerging drug-resistant mutant cytomegalovirus subpopulations by deep sequencing. Antimicrob. Agents Chemother. 2014, 58, 4697–4702. [CrossRef] 46. Lurain, N.S.; Chou, S. Antiviral drug resistance of human cytomegalovirus. Clin. Microbiol. Rev. 2010, 23, 689–712. [CrossRef] 47. Chou, S.; Ercolani, R.J.; Vanarsdall, A.L. Differentiated Levels of Ganciclovir Resistance Conferred by Mutations at Codons 591 to 603 of the Cytomegalovirus UL97 Kinase Gene. J. Clin. Microbiol. 2017, 55, 2098–2104. [CrossRef] 48. Chemaly, R.F.; Hill, J.A.; Voigt, S.; Peggs, K.S. In vitro comparison of currently available and investigational antiviral agents against pathogenic human double-stranded DNA viruses: A systematic literature review. Antiviral. Res. 2019, 163, 50–58. [CrossRef] 49. Yuan, S.; Chan, J.F.; Ye, Z.W.; Wen, L.; Tsang, T.G.; Cao, J.; Huang, J.; Chan, C.C.; Chik, K.K.; Choi, G.K.; et al. Screening of an FDA-Approved Drug Library with a Two-Tier System Identifies an Entry Inhibitor of Severe Fever with Thrombocytopenia Syndrome Virus. Viruses 2019, 11, 385. [CrossRef] 50. Sun, Y.; Bao, Q.; Xuan, B.; Xu, W.; Pan, D.; Li, Q.; Qian, Z. Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism. J Virol. 2018, 92, e00191-18. [CrossRef]

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