Synergistic Suppression of Dengue Virus Replication Using aCombination of Nucleoside Analogs and Nucleoside SynthesisInhibitors

Kim Long Yeo, Yen-Liang Chen, Hao Ying Xu, Hongping Dong, Qing-Yin Wang, Fumiaki Yokokawa, Pei-Yong Shi

Novartis Institute for Tropical Diseases, Singapore, Singapore

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Currently, there is no clinically ap-proved vaccine or antiviral for DENV. Combination therapy is a common practice in antiviral treatment and a potential ap-proach to search for new treatments for infectious pathogens. In this study, we performed a combination treatment in cell cul-ture by using three distinct classes of inhibitors, including ribavirin (a guanosine analog with several antiviral mechanisms),brequinar (a pyrimidine biosynthesis inhibitor), and INX-08189 (a guanosine analog). The compound pairs were evaluated forantiviral activity by use of a DENV-2 luciferase replicon assay. Our result indicated that the combination of ribavirin and INX-08189 exhibited strong antiviral synergy. This result suggests that synergy can be achieved with compound pairs in which onecompound suppresses the synthesis of the nucleoside for which the other compound is a corresponding nucleoside analog. Inaddition, we found that treatment of cells with brequinar alone could activate interferon-stimulated response elements (ISREs);furthermore, brequinar and NITD-982 (another pyrimidine biosynthesis inhibitor) potentiated interferon-induced ISRE activa-tion. Compared to treatment with brequinar, treatment of cells with ribavirin alone could also induce ISRE activation, but to alesser extent; however, when cells were cotreated with ribavirin and beta interferon, ribavirin did not augment the interferon-induced ISRE activation.

Over 2.5 billion people worldwide are at risk of dengue virus(DENV) infection, with 390 million human infections and 96

million cases with disease manifestations each year (1). DENV isendemic throughout tropical and subtropical climates and isfound mostly in urban and semiurban areas. This positive-sensesingle-stranded RNA virus is transmitted mainly by the Aedes ae-gypti mosquito and is classified under the genus Flavivirus in thefamily Flaviviridae. Other notable viruses in this group includeyellow fever virus, Japanese encephalitis virus, West Nile virus,and tick-borne encephalitis virus. Currently, neither an antiviralnor a vaccine is approved for DENV. Care for hospitalized denguepatients is supportive, mainly through optimal replenishment ofbody fluids. Treatment is intensive for those who succumb to thesevere forms of the disease, i.e., dengue shock syndrome (DSS)and dengue hemorrhagic fever (DHF).

Multiples approaches have been taken to identify inhibitors ofDENV (2, 3). Small-molecule inhibitors have been reported totarget various DENV proteins, including capsid (4, 5), envelope(6), protease (7, 8), nonstructural protein (NS) 4B (9, 10), meth-yltransferase (2, 11), and RNA-dependent RNA polymerase (12–16). Inhibition of host factors important for viral replication andof compounds with immunomodulation activities, including im-ino sugars (17), cholesterol inhibitors (18), chloroquine (19), andprednisolone (20), has also been pursued for potential treatmentof DENV infections.

Ribavirin is a drug with a broad spectrum of antiviral activity.Ribavirin in combination with pegylated alpha interferon (PEG-IFN-�) in the past has been the standard treatment regimen forhepatitis C virus (HCV), a virus from the family Flaviviridae that isrelated to DENV. Besides HCV, ribavirin had also shown somesuccess in the treatment of respiratory syncytial virus (21) andLassa fever virus (22). Ribavirin is a guanosine analog with severalantiviral mechanisms, one of which is to inhibit de novo biosyn-

thesis of guanine nucleotides through direct binding to cellularIMP dehydrogenase (IMPDH) (23). Depletion of the intracellularpool of nucleoside triphosphates was proposed to be a major an-tiviral mechanism for ribavirin to inhibit flaviviruses (24). In ad-dition, ribavirin could function as a mutagen to increase errorcatastrophe (25) and potentiated the antiviral activity of IFN-�/�by augmenting the expression of IFN-stimulated genes (ISGs)(26). Similar to ribavirin, brequinar also has a broad antiviralspectrum against both positive- and negative-strand RNA viruses(27, 28). Brequinar inhibits de novo biosynthesis of uracil nucleo-tides by inhibiting cellular dihydroxyorotate dehydrogenase(DHODH) (29). Depletion of intracellular pyrimidine triphos-phates is the main antiviral mechanism for brequinar (27). Bre-quinar was first identified and developed as an antimetabolite incancer and immunosuppression therapy; since tumor cells relyheavily on de novo nucleotide synthesis, lowering pyrimidine syn-thesis (by use of brequinar) may interfere with the rapid prolifer-ation of lymphocytes (30).

Combination therapy is commonly practiced in anti-infectivetreatment to minimize drug resistance. Although there are no

Received 12 November 2014 Returned for modification 12 December 2014Accepted 19 January 2015

Accepted manuscript posted online 26 January 2015

Citation Yeo KL, Chen Y-L, Xu HY, Dong H, Wang Q-Y, Yokokawa F, Shi P-Y. 2015.Synergistic suppression of dengue virus replication using a combination ofnucleoside analogs and nucleoside synthesis inhibitors. Antimicrob AgentsChemother 59:2086 –2093. doi:10.1128/AAC.04779-14.

Address correspondence to Pei-Yong Shi, pei_yong.shi@novartis.com, orYen-Liang Chen, yen_liang.chen@novartis.com.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.04779-14

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clinically approved antivirals for DENV, it is of interest to examinewhether compounds that are in clinical use or in preclinical devel-opment for other viruses inhibit DENV and, if so, whether thesecompounds have synergistic effects against DENV when used incombination. In this study, we selected three clinical and preclin-ical compounds (brequinar, ribavirin, and INX-08189) withknown anti-DENV activities and examined their combinatoryantiviral activities in a cell culture system. The results showed thatcombination of the guanosine analog INX-08189 with the GTPpool-depleting drug ribavirin inhibited DENV in a synergisticmanner. The observed synergy may potentially be used to reducethe doses and therefore to increase the safety margins of inhibitorsto achieve a therapeutic window in vivo. In addition, we found thatbrequinar and another uridine biosynthesis inhibitor can poten-tiate interferon-stimulated response element (ISRE) activation in-duced by interferon.

MATERIALS AND METHODSCells and culture media. Huh-7 cells stably expressing a luciferase-re-porting DENV-2 (New Guinea C [NGC] strain) replicon were describedpreviously (31). These cells were maintained at 37°C and 5% CO2 inDulbecco’s modified Eagle medium (DMEM) (high glucose; Life Tech-nologies) supplemented with 2 mM L-glutamine (Life Technologies), 0.1mM nonessential amino acids (NEAA; Life Technologies), 10 �g/ml pu-romycin (Clontech), 1% penicillin-streptomycin (Life Technologies),and 10% fetal bovine serum (FBS; HyClone). The replicon cells seeded forantiviral assay were maintained in phenol red-free DMEM (Life Technol-ogies) supplemented with 1 mM sodium pyruvate (Life Technologies), 2mM L-glutamine, 0.1 mM NEAA (Life Technologies), 1% penicillin-streptomycin, and 2% FBS. HEK 293T cells were maintained at 37°C and5% CO2 in DMEM (low glucose) supplemented with 0.1 mM NEAA, 1%penicillin-streptomycin, and 10% FBS. HEK 293T cells seeded for assayswere maintained with 2% FBS instead.

Compounds. Ribavirin (CAS number 36791-04-5), brequinar so-dium salt hydrate, and recombinant human IFN-� were purchased fromSigma-Aldrich. INX-08189 and NITD-982 (28) were synthesized in-house. All compounds were dissolved in dimethyl sulfoxide (DMSO).

DENV replicon antiviral assay. The replicon assay was performed asdescribed previously (31). Briefly, replicon cells were treated with 2- or3-fold serial dilutions of test compounds. After incubation at 37°C for 48h, a luciferase substrate (ViviRen; Promega) was added according to themanufacturer’s protocol. Luminescence was measured using a Clarity lu-minescence reader (BioTek), with an integration time of 0.1 s. The con-centrations of compounds that decreased luciferase expression by 50%(EC50) and 90% (EC90) were calculated by nonlinear regression analysis(GraphPad Prism). Compound synergy analysis was performed usingChalice Analyzer (Zalicus).

Cell viability assay. Cell viability was measured using the CellTiter-Glo (CTG) luminescence cell viability assay (Promega) according to themanufacturer’s protocol. Approximately 1.5 � 104 replicon cells or 2 �104 HEK 293T cells were seeded in a 96-well plate in a total volume of 100�l. After 16 h of incubation, the cells were treated with a test compound.After another 48 h, 25 �l of CTG was added to each well, and the cells wereincubated at room temperature for 20 min. Luminescence was read withan integration time of 0.1 s, using a Clarity luminescence reader (BioTek).

Plasmids and transfection of HEK 293T cells for ISRE inductionassay. A construct containing a firefly luciferase reporter gene under thecontrol of an ISRE gene promoter (pISRE-TA-Luc) and a construct con-taining a Renilla luciferase reporter gene under the control of the herpessimplex virus type 2 thymidine kinase gene (HSV-TK) promoter(pGL4.74-hRluc/TK) were purchased from Clontech and Promega, re-spectively. Batch transfection of HEK 293T cells was performed withjetPRIME (Polyplus). For one 96-well culture plate, 12 �g each of pISRE-TA-Luc and pGL4.74-hRluc/TK was diluted in 600 �l of jetPRIME trans-

fection buffer. Forty-eight microliters of jetPRIME was then added,mixed, and incubated for 10 min at room temperature. This mixture wasadded to 2.4 � 106 cells in a final volume of 12 ml DMEM containing 0.1mM NEAA, 1% penicillin-streptomycin, and 2% FBS. Finally, 100 �l ofthis cell suspension was added to each well of the microplate, containing 1�l of compound. Cells were incubated for 48 h at 37°C in the presence of5% CO2. Luciferase expression was assayed using the Dual-Glo Stop &Glo assay system (Promega) according to the manufacturer’s recommen-dations. Briefly, medium was removed from the wells containing cells,and the cells were washed twice with phosphate-buffered saline (PBS)(Life Technologies). Cells were then lysed for 20 min at room temperatureon an orbital shaker. Subsequently, a 20-�l aliquot of cell lysate from eachwell was pipetted into a well of a white-wall, white-bottom plate. Fireflyluciferase expression was measured by injecting 100 �l firefly luciferasesubstrate into each well. Expression was measured 2 s later on a Clarityluminescence reader (BioTek), using a 10-s integration time. After thefirst 20-s reading, 100 �l of Stop & Glo reagent was injected into each well.Renilla luciferase expression was measured 2 s later, with an integrationtime of 10 s.

Statistical analysis. All the EC50s were calculated using 4-point non-linear regression curve fitting with a variable slope and no constraints onboth the top and bottom values, using Prism software. Chalice Analyzer(32) was used to evaluate synergy. The theories of Loewe additivity, Blissindependence, and the highest single agent (HSA) were previously de-scribed in detail (33–35).

RESULTSAntiviral activities of individual compounds. We selected threecompounds (ribavirin, brequinar, and INX-08189) to test theircombined antiviral activities (Fig. 1A to C, left panels). Each of theselected compounds has a distinct mode of action (see the intro-duction). A DENV-2 luciferase replicon assay was used to measureantiviral activity throughout the study. Prior to combination test-ing, we first examined the anti-DENV activities of the individualcompounds (Fig. 1A to C, middle panels), as well as their cytotox-icities (right panels). Figure 1D summarizes the EC50, EC90, andCC50 (concentration of the compound leading to 50% cell deathas measured by CellTiter-Glo) values of each compound. The re-sults demonstrate that all selected compounds have anti-DENVactivities with a good therapeutic window in cell culture.

Antiviral activities of combination treatments. We measuredantiviral activities of different compound pairs in which an inhib-itor of the nucleoside pathway was paired with a nucleoside inhib-itor, INX-08189. To examine whether combinatory treatmentshave synergistic or additive effects, we tested each compound atseven concentrations (excluding a control without compound),with a dose matrix centered on the EC50. Figure 2A, left panel,shows the percentages of replicon inhibition for each dose in thedose matrix. Chalice Analyzer was used to calculate the Loeweexcess (Fig. 2A, right panel), Bliss excess (Fig. 2B, left panel), andHSA excess (Fig. 2B, right panel) values. These parameters arecommonly used to indicate the excess percent inhibition; the ex-cess percent inhibition is calculated by deducting the expectedpercent inhibition values of various combinations, assuming non-synergy pairing in various models, from the experimental percentinhibition values. These data allowed us to calculate the isobolo-gram, synergy score, and best combination index (CI) for eachpair (Fig. 2C). In general, synergy scores of �1 and CI of �1indicate that a combination treatment has a synergistic effect; asynergy score of 1 and a CI of 1 indicate that a combination treat-ment has only an additive effect (33). To assess whether synergycould be achieved at high inhibition levels, we set the isobologram

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level at 0.9 to capture meaningful synergy with a 90% viral reduc-tion (equivalent to a 1-log10 reduction). Among the compoundpairs, only ribavirin plus INX-08189 generated synergy, with asynergy score of 2.2 � 0.0475 and a CI of 0.325 � 0.0143 (Fig. 2C).The observed synergy was not due to cytotoxicity, as there was nosignificant cytotoxicity for all the combinations tested (Fig. 2D).The compound pair of a guanosine nucleoside analog and brequi-

nar did not show any antiviral synergy (data not shown). Collec-tively, the results demonstrate that various compound pairs havedifferent synergistic or additive antiviral effects.

Treatment of cells with brequinar or ribavirin alone activatesISRE. Recent studies suggested that inhibitors of IMPDH orDHODH induce a state of cell stress that potentiates (i.e., furtherincreases the potency of) the type I IFN response (26, 36). The

FIG 1 Host nucleoside inhibitors and preclinical nucleoside analogs exhibit potent anti-DENV-replication activities, with minimal cytotoxicities in cell culture.(A) The chemical structure of ribavirin, a well-characterized inhibitor of IMPDH, is shown. The EC50 of ribavirin for the Huh-7 NGC replicon is about 8.29 �M,and the EC90 is about 61.77 �M. The CC50 of ribavirin for the Huh-7 NGC replicon is �850 �M. (B) The structure of brequinar, a potent DHODH inhibitor,is shown. Its EC50 for the Huh-7 NGC replicon is about 51.5 nM, and the EC90 is about 248.8 nM. The CC50 of brequinar for the Huh-7 NGC replicon is �5 �M.(C) The chemical structure of the guanosine analog INX-08189 is shown. The EC50 of INX-08189 for the Huh-7 NGC replicon is about 14.48 nM, and the EC90

is about 131.5 nM. The CC50 of INX-08189 for the Huh-7 NGC replicon is �1 �M. (D) The EC50, EC90, and CC50 values of the respective compounds aresummarized, demonstrating that all the selected compounds have anti-DENV activity, with a good therapeutic window, in cell culture. The data are averages forthree to five independent experiments.

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antiviral effect of these inhibitors may be contributed by bothpotentiation of the IFN response and depletion of intracellularnucleotides. We asked whether ribavirin- or brequinar-mediatedpotentiation of the IFN response could lead to the synergistic ef-

fect observed with the combination of ribavirin plus INX-08189.We cotransfected HEK 293T cells with the following two plasmids:(i) plasmid pISRE-TA-Luc contained the firefly luciferase geneunder the control of an ISRE, and the expression of firefly lucifer-

FIG 2 Chalice analysis of ribavirin and INX-08189 combination treatment of the NGC replicon. Luciferase activity was measured at 48 h posttreatment, with adosing matrix centered on the EC50 of each compound for the Huh-7 NGC replicon. (A) Dose matrix-response values for the combination, showing inhibitionof luciferase activity (1 treated value/untreated value) for the serially diluted compounds. Inhibition values were analyzed by Chalice software to generate thecombinations’ excess values (the Loewe excess values are shown here). (B) Computed Bliss and HSA excesses for the compound combination. Values are averagesfor six experiments. (C) Isobologram, synergy score, and best combination index (CI) at 90% inhibition for ribavirin and INX-08189. Values shown are averagesfor eight experiments. (D) Cell viability with drug combination treatment. The concentrations of ribavirin and INX-08189 were identical to those used in the dosematrix for antiviral activity assay (see panel A). The synergy calculation was derived from averages for eight data sets.

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ase was used to measure ISRE activation upon compound treat-ment; and (ii) plasmid pGL4.74-hRluc/TK contained the Renillaluciferase gene under the control of the HSV-TK promoter, andthe expression of Renilla luciferase was used to normalize trans-fection efficiency. The transfected cells were treated with increas-ing doses of IFN-� (Fig. 3A), brequinar (Fig. 3B), or ribavirin (Fig.3C) for 48 h before being assayed for luciferase expression. Foldinduction of the ISRE activity was measured by dividing the indi-vidual treatment signal by the mock treatment (1% DMSO) sig-nal. Although ISRE induction followed a dose-dependent re-sponse curve with all three inhibitors (Fig. 3A to C), the effect withribavirin treatment was modest compared to that with IFN-� orbrequinar treatment. Specifically, treatments with IFN-� and bre-quinar resulted in maximal 4-fold and 3-fold increases in ISREactivity, respectively. The effect of brequinar was quite pro-nounced even at low concentrations and started to plateau ataround 300 nM (Fig. 3B). Ribavirin treatment induced ISRE acti-vation by a maximum of 1.68-fold. The induction of ISRE activityby brequinar and ribavirin was not due to cytotoxicity, as theirCC50 values in HEK 293T cells were �5 �M and �800 �M, re-spectively (Fig. 3D). The results indicate that in the absence ofexogenous IFN, treatment of cells with brequinar or ribavirinalone can induce ISRE activation.

DHODH inhibitors potentiate IFN-induced ISRE activation.Since brequinar or ribavirin alone could induce ISRE activation,we examined whether these compounds could potentiate/en-hance the ISRE activation when cells were coincubated with exog-enous IFN. To this end, HEK 293T cells were coincubated withIFN-� and ribavirin or brequinar. As shown in Fig. 4, comparedwith the DMSO control, brequinar significantly potentiated ISREactivation when the cells were cotreated with 5,000 and 10,000IU/ml IFN-� (P � 0.001); no potentiation was observed when thecells were cotreated with lower doses of IFN-�. In contrast, treat-ment with ribavirin plus IFN-� did not augment ISRE activation(Fig. 4). The results indicate that brequinar, a DHODH inhibitor,can enhance exogenous IFN-induced ISRE activation.

Next, we asked whether other pyrimidine biosynthesis inhibi-tors can also potentiate IFN-induced ISRE activation. To addressthis question, we used NITD-982, a compound that was recentlyreported to be a potent inhibitor of DHODH (28). Treatment withNITD-982 at a noncytotoxic concentration significantly aug-mented ISRE activation when cells were coincubated with 10,000IU/ml IFN-� (P � 0.001) (Fig. 4). Taken together, the resultsdemonstrate that DHODH inhibitors (brequinar and NITD-982)can potentiate IFN-induced ISRE activation.

DISCUSSION

In this study, we explored the possibility of a combinatorial ther-apy selected from three compounds that have anti-DENV activi-

FIG 3 Dose-dependent induction of ISRE by IFN-� and nucleoside synthesisinhibitors. HEK 293T cells batch transfected with pISRE-TA-Luc andpGL4.74-hRluc/TK were simultaneously treated with increasing concentra-tions of IFN-�, brequinar, or ribavirin in individual wells. (A and B) Treat-ment with IFN-� (A) caused a maximum 4-fold increase in ISRE activation,while treatment with brequinar (B) caused a maximum 3-fold increase in ISREactivation. (C) Activation of ISRE by ribavirin was modest, showing a maxi-mum increase of 1.68-fold. (D) The induction of ISRE activity by either hostnucleoside inhibitor was not due to cytotoxicity, as the CC50 values for HEK293T cells were �5 �M. The average results for two experiments are shown.

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ties. Since INX-08189 is a potent nucleoside inhibitor for DENV(Fig. 1C), we chose this compound to perform combination anal-ysis and tested the hypothesis that cotreatment with INX-08189plus another inhibitor that perturbs the synthesis of a naturaltriphosphate nucleotide(s) will lead to antiviral synergy. Our re-sults showed that the compound pair ribavirin plus INX-08189exhibited clear antiviral synergy, with a strong CI of 0.325 (Fig.2C). The combination has the potential to lower the dose of eithercompound 3 times to achieve a level of viral inhibition similar tothat with either compound alone (33); this may translate into alarger therapeutic window for the treatment of DENV with thesetwo compounds. The synergy is not due to the cytotoxicity of thecombination treatment (Fig. 2D). It should be noted that synergywas observed for compound pairs in which one compound sup-pressed the synthesis of the nucleoside for which the other com-pound is a corresponding nucleoside analog. This was expected,because reduction of the natural nucleoside pool (by the nucleo-side synthesis inhibitor) increases the ratio of the nucleosideanalog triphosphate versus the natural nucleoside triphosphate,making the nucleoside analog triphosphate more likely to be in-corporated into the viral RNA, thus terminating viral RNA syn-thesis. Specifically, synergy was found between ribavirin (aguanosine synthesis inhibitor) and INX-08189 (a guanosine ana-log).

A similar combination antiviral approach was previously em-ployed for HCV and HIV. For HCV, INX-08189 was tested incombination with ribavirin in the presence or absence of pegy-lated interferon (PEG-IFN) in HCV patients; unfortunately, ad-verse effects (heart and renal toxicity) were observed (37). Thiswas not too surprising, as INX-08189 showed similar toxicologicalfindings when administered as monotherapy (38). For HIV, a CTPsynthase inhibitor (3=-deazauridine) strongly potentiated theanti-HIV-1 activity of a 5=-triphosphate of the cytidine analoglamivudine (3TC) and a 2,3=-dideoxycytidine (ddC) in cell cul-ture (39). One explanation for drug synergy is the parallel pathway

inhibition model, which suggests that two drugs will be synergisticif they inhibit two proteins in parallel pathways essential for anobserved phenotype (40). Our synergy results seem to support theparallel pathway inhibition model. However, we could not ex-clude other possible mechanisms that may also contribute to theobserved synergy.

It would be interesting to test whether the antiviral synergy ofribavirin plus INX-08189 observed in cell culture can be repro-duced in the AG129 mouse model of dengue. Since a nucleosideinhibitor requires host kinases to convert to its active triphosphateform, caution should be taken in considering such an in vivostudy. Due to the potential species difference in converting INX-08189 to the INX-08189 triphosphate between humans and mice,the relevance of the nonhuman animal model should be carefullyvalidated experimentally in analyzing nucleoside inhibitors.

The potential involvement of the IFN pathway in the observedantiviral activity was investigated. For brequinar, besides inhibit-ing pyrimidine synthesis, our results showed that brequinar alonecould activate ISRE in cell culture; furthermore, brequinar couldenhance the exogenous IFN-induced ISRE activation. Such anenhancement was further validated with another pyrimidine bio-synthesis inhibitor (NITD-982). The mechanisms of brequinar/NITD-982-mediated ISG activation and IFN signaling enhance-ment remain to be determined. Nevertheless, these observationsmay have clinical implications. When patients receive therapy,DENV has already established its replication, triggering an im-mune response to produce IFN and other cytokines (41, 42). Ifpatients are treated with brequinar, its ability to augment IFN-induced ISRE activation should enhance the overall antiviral sta-tus of patients.

The synergy observed with the ribavirin-plus-INX-08189treatment is unlikely to occur through ribavirin-mediated ISREactivation. This is because brequinar alone activated ISRE (Fig.3B) but failed to synergize with INX-08189. In addition, the acti-vation level of IRES was modest even at high concentrations ofribavirin.

There is an urgent unmet medical need to develop a safe andeffective antiviral for DENV infection. Although no single com-pound has been approved for clinical use, the possibility of repur-posing clinically tried or approved drugs for DENV is a tractableoption. To this end, balapiravir, a cytidine nucleoside analog thatwas stopped for HCV clinical development, was tested in denguepatients; unfortunately, the compound did not show any efficacyin the dengue clinical trial (13). Similarly, celgosivir, a host alpha-glucosidase inhibitor (initially developed for HCV), also failed toshow a significant difference compared to placebo (43). An alter-native approach for repurposing clinically approved drugs forDENV is to search for a combination therapy. Such an approachhas two conceptual advantages. First, synergy between two drugswould allow one to achieve efficacy at lower doses, leading to anincreased therapeutic window for potentially toxic compounds(44). Theoretical and experimental studies have shown that drugsthat exhibit synergy for a specific effect are usually not synergisticfor side effects (32, 45). Indeed, toxicity experiments suggestedthat the observed antiviral synergy of the ribavirin-plus-INX-08189 combination was not due to cytotoxicity (Fig. 2). However,due to the unpredictable nature of toxicity associated with nucle-oside analogs, caution should be taken in extrapolating in vitrotoxicity results. Second, combination treatment would minimizethe chance of resistance. In the absence of effective monotherapy

FIG 4 Induction of ISRE by IFN-� is potentiated in the presence of DHODHinhibitors. HEK 293T cells were batch transfected with pISRE-TA-Luc andpGL4.74-hRluc/TK. Transfected cells were simultaneously treated with in-creasing concentrations of IFN-� and the indicated concentrations of the re-spective compounds. At 48 h posttreatment, luciferase activities were assayedusing the Dual-Glo Stop & Glo reagent. Cotreatment of HEK 293T cells withbrequinar and 5,000 or 10,000 IU/ml of IFN-� significantly potentiated ISREactivation compared to that with the DMSO control. There was no potentia-tion observed at lower doses of IFN-�. In contrast, cotreatment of cells withribavirin and IFN-� did not augment ISRE activation. Treatment of cells withanother pyrimidine biosynthesis inhibitor, NITD-982, with 10,000 IU/ml ofIFN-� also significantly augmented the ISRE activation.

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for DENV, a combination of two moderately effective drugs maybe needed. This is evident in the treatment of HIV, where only acombination of drugs effectively reduces viremia to an undetect-able level. These considerations make synergistic drug pairs idealcandidates for treatment of infectious pathogens. The currentstudy used DENV-2 as a model to achieve proof of concept. Futurestudies are needed to expand the current observation in cell cul-ture to an appropriate in vivo study.

ACKNOWLEDGMENTS

We thank Chek Shik Lim, Christophe Leroy, and Joseph Lehar for dataanalysis of synergy. We thank colleagues at Novartis Institute for TropicalDiseases (NITD) for helpful discussions and help during the course of thisstudy.

We are all employees of Novartis.

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