AAC Accepted Manuscript Posted Online 26 January 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.04779-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Synergistic suppression of dengue virus replication using a combination of nucleoside
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analogs and nucleoside synthesis inhibitors
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Running Title: Synergy between nucleoside and its synthesis inhibitor
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Kim Long Yeo, Yen-Liang Chen, Hao Ying Xu, Hongping Dong, Qing-Yin Wang,
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Fumiaki Yokokawa, Pei-Yong Shi*
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Novartis Institute for Tropical Diseases
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10 Biopolis Road, #05-01 Chromos Building
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Singapore 138670
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Singapore
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*Corresponding author:
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Pei-Yong Shi:
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Email: [email protected]
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ABSTRACT Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans.
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Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is
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a common practice in antiviral treatment and a potential approach to search for new treatment for
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infectious pathogens. In this study, we performed a combination treatment in cell culture using
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three distinct classes of inhibitors, including ribavirin (a guanosine analog with several antiviral
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mechanisms), brequinar (a pyrimidine biosynthesis inhibitor), and INX-08189 (a guanosine
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analog). The compound pairs were evaluated for their antiviral activities using a DENV-2
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luciferase replicon assay. Our result indicated combination of Ribavirin + INX-08189 exhibited
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strong antiviral synergy. This result suggests that synergy can be achieved with compound pairs
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in which one compound suppresses the synthesis of the nucleoside for which the other compound
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is a corresponding nucleoside analog. In addition, we found that treatment of cells with brequinar
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alone could activate interferon-stimulated response element (ISRE); furthermore, brequinar and
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NITD-982 (another pyrimidine biosynthesis inhibitor) can potentiate the interferon-induced
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ISRE activation. Compared with brequinar, treatment of cells with ribavirin alone could also
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induce ISRE activation, but to a less extend; however, when cells were co-treated with ribavirin
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and interferon-β, ribavirin did not augment the interferon-induced ISRE activation.
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INTRODUCTION Over 2.5 billion people worldwide are at risk from dengue virus (DENV) infection, with
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390 million human infections and 96 million cases with disease manifestations each year (1).
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DENV is endemic throughout tropical and subtropical climates, and found mostly in urban and
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semi-urban areas. This positive sense single-stranded RNA virus is mainly transmitted by the
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Aedes Aegypti mosquito and classified under the genus Flavivirus in the family Flaviviridae.
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Other notable viruses in this group include yellow fever virus, Japanese encephalitis virus, West
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Nile virus, and tick-borne encephalitis virus. Currently, neither antiviral nor vaccine is approved
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for DENV. Care for hospitalized dengue patients is supportive, mainly through optimal
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replenishment of body fluids. Treatment is intensive for those who succumb to the severe forms
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of the disease, dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF).
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Multiples approaches have been taken to identify inhibitors of DENV (2, 3). Small
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molecule inhibitors have been reported to target various DENV proteins, including capsid (4, 5),
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envelope (6), protease (7, 8), non-structural protein (NS) 4B (9, 10), methyltransferase (11, 12),
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and RNA-dependent RNA polymerase (13-17). Inhibitions of host factors important for viral
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replication and compounds with immune modulation activities have also been pursued for
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potential treatment of DENV infections, including iminosugars (18), cholesterol inhibitors (19),
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chloroquine (20), and prednisolone (21).
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Ribavirin is a drug with broad-spectrum of antiviral activity. Ribavirin in combination
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with pegylated interferon (IFN)-α in the past has been the standard treatment regime for hepatitis
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C virus (HCV), a virus related to DENV from family Flaviviridae. Besides HCV, ribavirin had
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also shown some success in the treatment of respiratory syncytial virus (22) and Lassa fever
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virus (23). Ribavirin is a guanosine analog with several antiviral mechanisms, one of which is to
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inhibit de novo biosynthesis of guanine nucleotides through direct binding to cellular inosine
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monophosphate dehydrogenase (IMPDH) (24). Depletion of intracellular pool of nucleoside
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triphosphates was proposed to be a major antiviral mechanism for ribavirin to inhibit flavivirus
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(25). In addition, ribavirin could function as a mutagen to increase error catastrophe (26) and
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potentiated the antiviral activity of interferon (IFN)-α/β by augmenting the expression of ISGs
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(IFN-stimulated genes) (27). Similar to ribavirin, brequinar also has a broad antiviral spectrum
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against both positive and negative-stranded RNA viruses (28, 29). Brequinar inhibits de novo
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biosynthesis of uracil nucleotides by inhibiting cellular dihydroxyorotate dehydrogenase
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(DHODH) (30). Depletion of intracellular pyrimidine triphosphates is the main antiviral
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mechanism for brequinar (28). Brequinar was first identified and developed as an anti-metabolite
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in cancer and immunosuppression therapy; since tumor cells rely heavily on de novo nucleotide
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synthesis, lowering the pyrimidine synthesis (by brequinar) could interfere with the rapid
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proliferation of lymphocytes (31).
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Combination therapy is commonly practiced in anti-infective treatment to minimize drug
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resistance. Although there is no clinically approved antivirals for DENV, it is of interest to
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examine whether compounds that are in clinical use or in preclinical development for other
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viruses inhibit DENV; and, if so, whether these compounds could have synergistic effect on anti-
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DENV activity when used in combination. In this study, we selected three clinical and preclinical
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compounds (Brequinar , Ribavirin, and INX-08189) with known anti-DENV activities and
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examined their combinatory antiviral activities in a cell culture system. The results showed that
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combination of the guanosine analog INX-08189 with GTP pool-depleting ribavirin inhibited
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DENV in a synergistic manner. The observed synergy could potentially be used to reduce the
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doses and, therefore, increase the safety margins of inhibitors to achieve therapeutic window in
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vivo. In addition, we found that brequinar and another uridine biosynthesis inhibitor can
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potentiate interferon-induced interferon-stimulated response element (ISRE) activation.
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MATERIALS AND METHODS
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Cells and culture media
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Huh-7 cells stably expressing the luciferase-reporting DENV-2 [New Guinea C (NGC)
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strain] replicon was described previously (32). These cells were maintained at 37oC and 5% CO2
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in Dulbecco’s modified Eagle medium (DMEM; high glucose; Life Technologies) supplemented
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with 2 mM L-glutamine (Life Technologies), 0.1 mM non-essential amino acid (Life
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Technologies), 10 µg/mL puromycin (Clontech), 1% penicillin/streptomycin (Life
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Technologies), and 10% fetal bovine serum (FBS; Hyclone). The replicon cells seeded for
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antiviral assay were maintained in phenol red-free DMEM (Life Technologies) supplemented
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with 1 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine, 0.1 mM Non-essential
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Amino Acids (NEAA) (Life Technologies), 1% penicillin/streptomycin, and 2% FBS. HEK 293-
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T cells were maintained at 37oC and 5% CO2 in DMEM (low glucose) supplemented with 0.1
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mM NEAA, 1% penicillin/streptomycin, and 10% FBS. HEK 293-T cells seeded for assays were
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maintained with 2% FBS instead.
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Compounds
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Ribavirin (CAS number 36791-04-5), brequinar sodium salt hydrate, and recombinant
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human interferon-β (IFN-β) were purchased from Sigma-Aldrich. INX-08189 and NITD-982
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(29) were synthesized in-house. All compounds were dissolved in Dimethyl sulfoxide (DMSO).
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DENV replicon antiviral assay
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The replicon assay was performed as described previously (32). Briefly, replicon cells
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were treated with 2 or 3-fold serial dilutions of test compounds. After incubation at 37oC for 48
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h, luciferase substrate (ViviRen, Promega) was added according to manufacturer’s protocol.
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Luminescence was measured using the Clarity luminescence reader (BioTek) with an integration
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time of 0.1 second. The concentrations of compounds that decreased the luciferase expression by
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50% (EC50) and 90% (EC90) were calculated by nonlinear regression analysis (GraphPad Prism).
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Compound synergy analysis was performed using Chalice Analyzer (Zalicus).
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Cell viability assay
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Cell viability was measured using the CellTiter-Glo luminescent cell viability assay
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(CTG, Promega) according to manufacturer’s protocol. Approximately 1.5×104 replicon cells or
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2×104 HEK 293-T cells were seeded in a 96-well plate in a total volume of 100 µl. After 16 h of
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incubation, the cells were treated with test compound. After another 48 h, 25 µL of CTG was
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added into to each well and the cells incubated at room temperature for 20 min. Luminescence
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was read with an integration time of 0.1 second using the Clarity luminescence reader (BioTek).
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Plasmids and transfection of HEK 293-T cells for IFN-stimulated response element (ISRE)
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induction assay
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Construct containing a firefly luciferase reporter under the control of an ISRE gene
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promoter (pISRE-TA-Luc) and construct containing a renilla luciferase reporter under the
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control of the herpes simplex virus type 2 thymidine kinase gene (HSV-TK) promoter (pGL4.74-
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hRluc/TK) were purchased from Clontech and Promega, respectively. Batch transfection of HEK
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293-T cell was performed with jetPRIME (Polyplus). For one 96-well culture plate, 12 µg each
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of pISRE-TA-Luc and pGL4.74-hRluc/TK were diluted in 600 µl of jetPRIME transfection
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buffer. Forty-eight µl of jetPRIME was then added, mixed, and incubated for 10 min at room
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temperature. This mixture was added to 2.4 ×106 cells in a final volume of 12 ml DMEM
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containing 0.1 mM NEAA, 1% penicillin/streptomycin, and 2% FBS. Finally 100 µl of this cell
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suspension was added to each well of the microplate containing 1 µl of compound. Cells were
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incubated for 48 h at 37oC in the presence of 5% CO2. Luciferase expression was assayed using
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the Dual-Glo Stop & Glo assay system (Promega) according to manufacturer’s
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recommendations. Briefly, media was removed from the wells containing cells and the cells were
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washed twice with Phosphate buffered saline (PBS) (Life Technologies). Cells were then lysed
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for 20 min at room temperature on an orbital shaker. Subsequently, 20 µl aliquot of cell lysate
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from each well was pipetted into wells of a white wall, white bottom plate. Firefly luciferase
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expression was measured by injecting 100 µl firefly luciferase substrate into each well.
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Expression was measured 2 seconds later on a Clarity luminescence reader (BioTek) using a 10-
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second integration time. After the first 20 seconds reading, 100 µl of Stop & Glo reagent was
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injected into each well. Renilla luciferase expression was measured 2 seconds later with an
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integration time of 10 seconds.
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Statistical analysis
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All the EC50s were calculated using 4 point non-linear regression curve fitting with variable
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slope and no constrains on both the top and bottom values in the PRISM software. Chalice
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Analyzer (33) was used to evaluate synergy. The theories of Loewe additivity, Bliss independent
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and highest single agent (HSA) were described in details in the references (34-36).
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RESULTS
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Antiviral activity of individual compounds
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We selected three compounds (ribavirin, brequinar, and INX-08189) to test their combination antiviral activities (left panels, Fig. 1A-C). Each of the selected compounds has
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distinct mode-of-actions (see Introduction). A DENV-2 luciferase replicon assay was used to
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measure antiviral activity throughout the study. Prior to combination testing, we first examined
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the anti-DENV activity of individual compounds (middle panels) and cytotoxicity (right panels).
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Figure 1D summarizes the EC50, EC90, and CC50 values of each compound. The results
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demonstrate that all selected compounds have anti-DENV activities with good therapeutic
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window in cell culture.
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Antiviral activity of combination treatment We measured antiviral activities of different compound pairs such that the inhibitor of
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nucleoside pathway is paired with a nucleoside inhibitor, INX-08189. To examine whether
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combinatory treatments have synergistic or additive effects, we tested each compound at seven
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concentrations (excluding a control without compound) with dose matrix centered on the EC50
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value. The left panels in Figure 2 show the percentages of replicon inhibition under each dose
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matrix (left panel, Fig. 2A). Chalice Analyzer was used to calculate the Loewe excess (right
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panel, Fig. 2A), Bliss excess (left panels, Fig. 2B), and HSA excess values (right panels, Fig.
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2B). These parameters are commonly used to indicate the excess percentage inhibition; the
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excess percentage inhibition is calculated by deducting the expected percentage inhibition values
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of various combinations, assuming non-synergy pairing in various models, from the
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experimental percentage inhibition values. These data allowed us to calculate the isobolograms,
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synergy scores, and best combination indices (CI) for each pair (Fig. 2C). In general, synergy
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score of >1 and CI of 5 µM and > 800 µM, respectively (Fig. 3D). The results indicate that, in the absence of
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exogenous IFN, treatment of cells with brequinar or ribavirin alone could induce ISRE
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activation.
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DHODH inhibitors potentiates the IFN-induced ISRE activation Since brequinar or ribavirin alone could induce ISRE activation, we examined whether
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these compounds could potentiate/enhance the ISRE activation when cells are co-incubated with
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exogenous IFN. To this end, HEK 293T cells were co-incubated with IFN-β and ribavirin or
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brequinar. As shown in Figure 4, compared with DMSO control, brequinar significantly
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potentiated ISRE activation when the cells were co-treated with 5,000 and 10,000 IU/ml IFN-β
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(p-value < 0.001); no potentiation was observed when the cells were co-treated with lower doses
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of IFN-β. In contrast, treatment with ribavirin plus IFN-β did not augment ISRE activation (Fig.
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4). The results indicate that brequinar, a DHODH inhibitor, could enhance the exogenous IFN-
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induced ISRE activation.
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Next, we asked whether other pyrimidine biosynthesis inhibitors could also potentiate the
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IFN-induced ISRE activation. To address this question, we used NITD-982, a compound that
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was recently reported as a potent inhibitor of DHODH (29). Treatment with NITD-982 at a non-
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cytotoxic concentration significantly augmented the ISRE activation when the cells were co-
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incubated with 10,000 IU/ml IFN-β (p-value < 0.001; Fig. 4). Taken together, the results
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demonstrate that DHODH inhibitors (brequinar and NITD-982) can potentiate the IFN-induced
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ISRE activation.
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DISCUSSION In this study, we explored the possibility of a combinatorial therapy selected from three
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compounds that have anti-DENV activities. Since INX-08189 is a potent nucleoside inhibitor for
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DENV (Fig. 1C), we chose this compound to perform combination analysis and tested the
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hypothesis that co-treatment with INX-08189 plus another inhibitor that perturbs the synthesis of
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natural triphosphate nucleotide(s) could lead to antiviral synergy. Our results showed that
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compound pair ribavirin + INX-08189 exhibited clear antiviral synergy with strong CI of 0.325
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(Fig. 2C). The combination has the potential to lower the dose of either compound by 3 times to
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achieve a similar level of viral inhibition (34); this could translate into a bigger therapeutic
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window for the treatment of DENV with these two compounds. The synergy is not due to the
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cytotoxicity of the combination treatment (Fig. 2D). It should be noted that synergy was
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observed in compound pairs in which one compound suppressed the synthesis of the nucleoside
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for which the other compound is a corresponding nucleoside analog. This is expected because
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reduction of the natural nucleoside pool (by the nucleoside synthesis inhibitor) increases the ratio
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of the nucleoside analog triphosphate versus the natural nucleoside triphosphate, making the
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nucleoside analog triphosphate more likely to be incorporated into the viral RNA and thus
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termination of viral RNA synthesis. Specifically, the synergy was found between ribavirin (a
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guanosine synthesis inhibitor) and INX-08189 (a guanosine analog).
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A similar combination antiviral approach was previously employed for HCV and HIV.
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For HCV, INX-08189 was tested in combination with ribavirin in the presence or absence of
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pegylated interferon (PEG-IFN) in HCV patients; unfortunately, adverse effects (heart and renal
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toxicity) were observed (38). This is not too surprising since INX-08189 had similar
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toxicological finding when administered as monotherapy (39). For HIV, a CTP synthase
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inhibitor (3’-deazauridine) strongly potentiated the anti-HIV-1 activity of a 5’-triphoaphate of
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the cytidine analog (3TC) and a 2,3’-dideoxy cytidine (ddC) in cell culture (40). One explanation
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for drug synergy is the parallel pathway inhibition model, which suggests that two drugs will be
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synergistic if they inhibit two proteins on parallel pathways essential for an observed phenotype
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(41). Our synergy results seem to support the parallel pathway inhibition model. However, we
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could not exclude other possible mechanisms that could also contribute to the observed synergy.
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It would be interesting to test whether the antiviral synergy of ribavirin + INX-08189
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observed in cell culture could be reproduced in the dengue AG129 mouse model. Since
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nucleoside inhibitor requires host kinases to convert to its active triphosphate form, caution
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should be taken when considering such in vivo study. Due to the potential species difference in
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converting INX-08189 to the triphosphate INX-08189 between human and mouse, the relevance
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of the non-human animal model should be carefully validated experimentally when analyzing
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nucleoside inhibitors.
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The potential involvement of IFN pathway in the observed antiviral activity was
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investigated. For brequinar, besides inhibiting pyrimidine synthesis, our results showed that
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brequinar alone could activate ISRE in cell culture; furthermore, brequinar could enhance the
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exogenous IFN-induced ISRE activation. Such enhancement was further validated with another
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pyrimidine biosynthesis inhibitor (NITD-982). The mechanisms of brequinar/NITD-982-
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mediated ISG activation and IFN-signaling enhancement remain to be determined. Nevertheless,
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these observations may have clinical implications. When patients receive therapy, DENV has
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already established its replication, triggering immune response to produce IFN and other
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cytokines (42, 43). If the patients were treated with brequinar, its ability to augment the IFN-
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induced ISRE activation should enhance the overall antiviral status of patients.
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The synergy observed in the ribavirin + INX-08189 treatment is unlikely to go through
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the ribavirin-mediated ISRE activation. This is because brequinar alone activated ISRE (Fig. 3B)
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but failed to synergize INX-08189. In addition, the activation level of IRES was modest even at
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high concentration of ribavirin.
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There is an urgent unmet medical need to develop a safe and effective antiviral for
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DENV infection. Although no single compound has been approved for clinical use, the
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possibility of repurposing clinically tried or approved drugs for DENV is a tractable option.
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Towards this approach, balapiravir, a cytidine nucleoside analog that was stopped for HCV
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clinical development, was tested in dengue patients; unfortunately, the compound did not show
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any efficacy in the dengue clinical trial (14). Similarly, celgosivir, a host alpha-glucosidase
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inhibitor (initially developed for HCV) also failed to show significant difference compared with
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placebo (44). An alternative approach to repurpose clinically approved drugs for DENV is to
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search for combination therapy. Such approach has two conceptual advantages. Firstly, synergy
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between two drugs would allow achieving efficacy at lower doses, leading to increased
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therapeutic window of potentially toxic compounds (45). Theoretical and experimental studies
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have shown that drugs that exhibit synergy for a specific effect are usually not synergistic for
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side effects (33, 46). Indeed, toxicity experiments suggest that the observed antiviral synergy of
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ribavirin + INX-08189 combination was not due to cytotoxicity (Fig. 2). However, due to the
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unpredictable nature of toxicity associated with nucleoside analog, caution should be taken when
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extrapolating the in vitro toxicity results. Secondly, combination treatment would minimize the
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chance of resistance. In the absence of effective monotherapy for DENV, a combination of two
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moderately effective drugs may be needed. This was evident in the treatment of HIV where only
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combination of drugs effectively reduces the viremia to undetectable level. These considerations
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make synergistic drug pairs ideal candidates for treatment of infectious pathogens. The current
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study used DENV-2 as a model to achieve proof-of-concept. Future studies are needed to expand
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the current observation in cell culture to an appropriate in vivo study.
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ACKNOWLEDGEMENTS We thank Chek Shik Lim, Christophe Leroy, and Joseph Lehar on data analysis on
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synergy. We thank colleagues at Novartis Institute for Tropical Diseases (NITD) for helpful
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discussions and help during the course of this study. All authors are employees of Novartis.
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21
459
FIGURE LEGENDS
460
Figure 1. Host nucleoside inhibitors and pre-clinical nucleoside analogues exhibit potent anti-
461
DENV replication activity with minimal cyototoxicty in cell culture. (A) The chemical structure
462
of ribavirin, a well-characterized inhibitor of IMPDH, is shown. The EC50 of ribavirin in the
463
Huh-7 NGC replicon is about 8.29 µM and the EC90 is about 61.77 µM. CC50 of ribavirin in
464
Huh-7-NGC replicon is more than 850 µM. (B) The structure of brequinar, a potent DHODH
465
inhibitor, is shown. Its EC50 in Huh-7 NGC replicon is about 51.5 nM and the EC90 is about
466
248.8 nM. The CC50 of brequinar in the Huh-7 NGC replicon is more than 5 µM. (C) The
467
chemical structure of the guanosine analog, INX-08189, is shown. The EC50 of INX-08189 in
468
the Huh-7 NGC replicon is about 14.48 nM and the EC90 is about 131.5 nM. The CC50 of INX-
469
08189 in the Huh-7 NGC replicon is more than 1 µM. (D) The EC50, EC90 and CC50 of
470
respective compounds are summarized, demonstrating that all the selected compounds have anti-
471
DENV activities with good therapeutic window in cell culture. The data is derived the average of
472
three to five independent experiments.
473 474
Figure 2. Chalice analysis of ribavirin and INX-08189 combination treatment on NGC replicon.
475
Luciferase activity was measured 48 h post-treatment with dosing matrix centered on the EC50
476
of each compound in the Huh-7 NGC replicon. (A) Dose matrix response values of the
477
combination showing the inhibition of luciferase activity (1 – treated / untreated) for the serially
478
diluted compounds. Inhibition values were analyzed by the Chalice software to generate
479
combinations’ excess values (the Loewe excess values are shown here). (B) The computed Bliss
480
and highest single agent (HSA) excess of each compound combination. Values are derived from
481
an average of 6 experiments. (C) Isobolograms, synergy score and best combination index (CI) at
22
482
90% inhibition of ribavirin and INX-08189. Values shown are derived from average of eight
483
experiments. (D) Cell viability of drug combination treatment. The concentrations of ribavirin
484
and INX-08189 were identical to those used in the dose matrix for antiviral assay (A). The
485
synergy calculation was derived from average of 8 datasets.
486 487
Figure 3. Dose-dependent induction of ISRE by IFN-β and nucleoside synthesis inhibitors. HEK
488
293T cells batch-transfected with pISRE-TA-Luc and pGL4.74-hRluc/TK, were simultaneously
489
treated with increasing concentrations of IFN-β, brequinar or ribavirin in individual wells. (A)
490
Treatment with IFN-β caused a maximum of 4-fold increase in ISRE activation while treatment
491
with (B) brequinar caused a maximum of 3-fold increase in ISRE activation. (C) Activation of
492
ISRE by ribavirin was modest, showing a maximum of 1.68-fold. (D) The induction of ISRE
493
activity by either host nucleoside inhibitors was not due to cytotoxicity as their CC50 values in
494
the HEK293-T cells were > 5 µM. The average results from two experiments are shown.
495 496
Figure 4. Induction of ISRE by IFN-β is potentiated in the presence of DHODH inhibitors. HEK
497
293-T cells were batch transfected with pISRE-TA-Luc and pGL4.74-hRluc/TK. Transfected
498
cells were simultaneously treated with increasing concentrations of IFN-β and the indicated
499
concentrations of respective compounds. 48 h post-treatment, luciferase activities were assayed
500
using the Dual-Glo Stop & Glo reagent. Co-treatment of HEK293T cells with brequinar and
501
5000 and 10, 000 IU/ml of IFN-β, significantly potentiated ISRE activation when compared to
502
DMSO control. There was no potentiation observed at lower doses of IFN-β. In contrast, co-
503
treatment of cells with ribavirin and IFN-β did not augment ISRE activation. Treatment of cells
23
504
with another pyrimidine biosynthesis inhibitor, NITD-982, with 10, 00 IU/ml of IFN-β, also
505
significantly augmented the ISRE activation.
24
Luminescence (%)
A
Ribavirin (log µM)
Luminescence (%)
B
Brequinar (log µM)
Luminescence (%)
C
Brequinar (µM)
EC50 = 14.2 ± 5.6 nM
INX-08189 (log µM)
INX-08189
D
EC50 = 53.4 ± 2.7 nM
Cell viability (%)
Brequinar
Ribavirin (µM)
Cell viability (%)
Ribavirin
EC50 = 3.18 ± 2.86 µM
Cell viability (%)
Figure 1
INX-08189 (µM)
Compound
EC50 (nM)
CC50 (µM)
Ribavirin
3,178 ± 2860
> 800
Brequinar
53.4 ± 2.7
>5
INX-08189
14.2 ± 5.6
>1
Figure 2 Loewe excess
Dose matrix
11 .14
1.2
Ribavirin (µM)
11 1.2 0
0
.14
Ribavirin (µM)
100
100
A
0
0.014
0.12
0.11
0
1
0.014
B
1
100 11 0
0.14
1.2
Ribavirin (µM)
100 11 1.2
Ribavirin (µM)
0.14 0
0.012
0.11
0 0.0014
1
Cytotoxicity of synergy data
11
59
62
65
70
75
74
69
59
71
73
75
81
88
93
88
71
89
90
93
98 102 110 104 84
99 102 104 105 108 112 107 88 1.2
Ribavirin (µM)
100
D
103 105 107 110 113 114 110 93 100 101 105 104 106 109 107 94 100 101 101 103 106 112 108 97 100 102 101 103 108 110 108 92 0
INX-08189 (0.11 µM)
1
.14
Synergy score: 2.2 ± 0.0475 Best CI: 0.325 ± 0.0143
0.11
0
Isobologram
0.012
INX-08189 (µM)
INX-08189 (µM)
Ribavirin (100 µM)
0.11
Huh-7 NGC replicon HSA excess
Huh-7 NGC replicon Bliss excess
0 0.0014
C
0.12
INX-08189 (µM)
INX-08189 (µM)
0.014
0.12
0.11
INX-08189 (µM)
1
Cell viability (%)
Cell viability (%)
ISRE activity (fold increase) ISRE activity (fold increase)
ISRE activity (fold increase)
Figure 3
A
B IFN-β (IU/mL)
C Brequinar (µM)
D Ribavirin (µM)
Brequinar (µM)
Ribavirin (µM)
Figure 4 1 % DMSO 1 mM Ribavirin 5 µM Brequinar 10 µM NITD-982
15 10
*** ***
*** p value < 0.001 ***
5
IFN- (IU / mL)
0 10 00
50 00
25 00
12 50
0
0
Fold-induction of ISRE
20
1 2
Synergistic suppression of dengue virus replication using a combination of nucleoside
3
analogs and nucleoside synthesis inhibitors
4 5
Running Title: Synergy between nucleoside and its synthesis inhibitor
6 7
Kim Long Yeo, Yen-Liang Chen, Hao Ying Xu, Hongping Dong, Qing-Yin Wang,
8
Fumiaki Yokokawa, Pei-Yong Shi*
9 10
Novartis Institute for Tropical Diseases
11
10 Biopolis Road, #05-01 Chromos Building
12
Singapore 138670
13
Singapore
14 15
*Corresponding author:
16
Pei-Yong Shi:
17
Email: [email protected]
18
1
19 20
ABSTRACT Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans.
21
Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is
22
a common practice in antiviral treatment and a potential approach to search for new treatment for
23
infectious pathogens. In this study, we performed a combination treatment in cell culture using
24
three distinct classes of inhibitors, including ribavirin (a guanosine analog with several antiviral
25
mechanisms), brequinar (a pyrimidine biosynthesis inhibitor), and INX-08189 (a guanosine
26
analog). The compound pairs were evaluated for their antiviral activities using a DENV-2
27
luciferase replicon assay. Our result indicated combination of Ribavirin + INX-08189 exhibited
28
strong antiviral synergy. This result suggests that synergy can be achieved with compound pairs
29
in which one compound suppresses the synthesis of the nucleoside for which the other compound
30
is a corresponding nucleoside analog. In addition, we found that treatment of cells with brequinar
31
alone could activate interferon-stimulated response element (ISRE); furthermore, brequinar and
32
NITD-982 (another pyrimidine biosynthesis inhibitor) can potentiate the interferon-induced
33
ISRE activation. Compared with brequinar, treatment of cells with ribavirin alone could also
34
induce ISRE activation, but to a less extend; however, when cells were co-treated with ribavirin
35
and interferon-β, ribavirin did not augment the interferon-induced ISRE activation.
36 37
2
38 39
INTRODUCTION Over 2.5 billion people worldwide are at risk from dengue virus (DENV) infection, with
40
390 million human infections and 96 million cases with disease manifestations each year (1).
41
DENV is endemic throughout tropical and subtropical climates, and found mostly in urban and
42
semi-urban areas. This positive sense single-stranded RNA virus is mainly transmitted by the
43
Aedes Aegypti mosquito and classified under the genus Flavivirus in the family Flaviviridae.
44
Other notable viruses in this group include yellow fever virus, Japanese encephalitis virus, West
45
Nile virus, and tick-borne encephalitis virus. Currently, neither antiviral nor vaccine is approved
46
for DENV. Care for hospitalized dengue patients is supportive, mainly through optimal
47
replenishment of body fluids. Treatment is intensive for those who succumb to the severe forms
48
of the disease, dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF).
49
Multiples approaches have been taken to identify inhibitors of DENV (2, 3). Small
50
molecule inhibitors have been reported to target various DENV proteins, including capsid (4, 5),
51
envelope (6), protease (7, 8), non-structural protein (NS) 4B (9, 10), methyltransferase (11, 12),
52
and RNA-dependent RNA polymerase (13-17). Inhibitions of host factors important for viral
53
replication and compounds with immune modulation activities have also been pursued for
54
potential treatment of DENV infections, including iminosugars (18), cholesterol inhibitors (19),
55
chloroquine (20), and prednisolone (21).
56
Ribavirin is a drug with broad-spectrum of antiviral activity. Ribavirin in combination
57
with pegylated interferon (IFN)-α in the past has been the standard treatment regime for hepatitis
58
C virus (HCV), a virus related to DENV from family Flaviviridae. Besides HCV, ribavirin had
59
also shown some success in the treatment of respiratory syncytial virus (22) and Lassa fever
60
virus (23). Ribavirin is a guanosine analog with several antiviral mechanisms, one of which is to
3
61
inhibit de novo biosynthesis of guanine nucleotides through direct binding to cellular inosine
62
monophosphate dehydrogenase (IMPDH) (24). Depletion of intracellular pool of nucleoside
63
triphosphates was proposed to be a major antiviral mechanism for ribavirin to inhibit flavivirus
64
(25). In addition, ribavirin could function as a mutagen to increase error catastrophe (26) and
65
potentiated the antiviral activity of interferon (IFN)-α/β by augmenting the expression of ISGs
66
(IFN-stimulated genes) (27). Similar to ribavirin, brequinar also has a broad antiviral spectrum
67
against both positive and negative-stranded RNA viruses (28, 29). Brequinar inhibits de novo
68
biosynthesis of uracil nucleotides by inhibiting cellular dihydroxyorotate dehydrogenase
69
(DHODH) (30). Depletion of intracellular pyrimidine triphosphates is the main antiviral
70
mechanism for brequinar (28). Brequinar was first identified and developed as an anti-metabolite
71
in cancer and immunosuppression therapy; since tumor cells rely heavily on de novo nucleotide
72
synthesis, lowering the pyrimidine synthesis (by brequinar) could interfere with the rapid
73
proliferation of lymphocytes (31).
74
Combination therapy is commonly practiced in anti-infective treatment to minimize drug
75
resistance. Although there is no clinically approved antivirals for DENV, it is of interest to
76
examine whether compounds that are in clinical use or in preclinical development for other
77
viruses inhibit DENV; and, if so, whether these compounds could have synergistic effect on anti-
78
DENV activity when used in combination. In this study, we selected three clinical and preclinical
79
compounds (Brequinar , Ribavirin, and INX-08189) with known anti-DENV activities and
80
examined their combinatory antiviral activities in a cell culture system. The results showed that
81
combination of the guanosine analog INX-08189 with GTP pool-depleting ribavirin inhibited
82
DENV in a synergistic manner. The observed synergy could potentially be used to reduce the
83
doses and, therefore, increase the safety margins of inhibitors to achieve therapeutic window in
4
84
vivo. In addition, we found that brequinar and another uridine biosynthesis inhibitor can
85
potentiate interferon-induced interferon-stimulated response element (ISRE) activation.
86 87
MATERIALS AND METHODS
88
Cells and culture media
89
Huh-7 cells stably expressing the luciferase-reporting DENV-2 [New Guinea C (NGC)
90
strain] replicon was described previously (32). These cells were maintained at 37oC and 5% CO2
91
in Dulbecco’s modified Eagle medium (DMEM; high glucose; Life Technologies) supplemented
92
with 2 mM L-glutamine (Life Technologies), 0.1 mM non-essential amino acid (Life
93
Technologies), 10 µg/mL puromycin (Clontech), 1% penicillin/streptomycin (Life
94
Technologies), and 10% fetal bovine serum (FBS; Hyclone). The replicon cells seeded for
95
antiviral assay were maintained in phenol red-free DMEM (Life Technologies) supplemented
96
with 1 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine, 0.1 mM Non-essential
97
Amino Acids (NEAA) (Life Technologies), 1% penicillin/streptomycin, and 2% FBS. HEK 293-
98
T cells were maintained at 37oC and 5% CO2 in DMEM (low glucose) supplemented with 0.1
99
mM NEAA, 1% penicillin/streptomycin, and 10% FBS. HEK 293-T cells seeded for assays were
100
maintained with 2% FBS instead.
101
Compounds
102
Ribavirin (CAS number 36791-04-5), brequinar sodium salt hydrate, and recombinant
103
human interferon-β (IFN-β) were purchased from Sigma-Aldrich. INX-08189 and NITD-982
104
(29) were synthesized in-house. All compounds were dissolved in Dimethyl sulfoxide (DMSO).
105
DENV replicon antiviral assay
5
106
The replicon assay was performed as described previously (32). Briefly, replicon cells
107
were treated with 2 or 3-fold serial dilutions of test compounds. After incubation at 37oC for 48
108
h, luciferase substrate (ViviRen, Promega) was added according to manufacturer’s protocol.
109
Luminescence was measured using the Clarity luminescence reader (BioTek) with an integration
110
time of 0.1 second. The concentrations of compounds that decreased the luciferase expression by
111
50% (EC50) and 90% (EC90) were calculated by nonlinear regression analysis (GraphPad Prism).
112
Compound synergy analysis was performed using Chalice Analyzer (Zalicus).
113
Cell viability assay
114
Cell viability was measured using the CellTiter-Glo luminescent cell viability assay
115
(CTG, Promega) according to manufacturer’s protocol. Approximately 1.5×104 replicon cells or
116
2×104 HEK 293-T cells were seeded in a 96-well plate in a total volume of 100 µl. After 16 h of
117
incubation, the cells were treated with test compound. After another 48 h, 25 µL of CTG was
118
added into to each well and the cells incubated at room temperature for 20 min. Luminescence
119
was read with an integration time of 0.1 second using the Clarity luminescence reader (BioTek).
120
Plasmids and transfection of HEK 293-T cells for IFN-stimulated response element (ISRE)
121
induction assay
122
Construct containing a firefly luciferase reporter under the control of an ISRE gene
123
promoter (pISRE-TA-Luc) and construct containing a renilla luciferase reporter under the
124
control of the herpes simplex virus type 2 thymidine kinase gene (HSV-TK) promoter (pGL4.74-
125
hRluc/TK) were purchased from Clontech and Promega, respectively. Batch transfection of HEK
126
293-T cell was performed with jetPRIME (Polyplus). For one 96-well culture plate, 12 µg each
127
of pISRE-TA-Luc and pGL4.74-hRluc/TK were diluted in 600 µl of jetPRIME transfection
128
buffer. Forty-eight µl of jetPRIME was then added, mixed, and incubated for 10 min at room
6
129
temperature. This mixture was added to 2.4 ×106 cells in a final volume of 12 ml DMEM
130
containing 0.1 mM NEAA, 1% penicillin/streptomycin, and 2% FBS. Finally 100 µl of this cell
131
suspension was added to each well of the microplate containing 1 µl of compound. Cells were
132
incubated for 48 h at 37oC in the presence of 5% CO2. Luciferase expression was assayed using
133
the Dual-Glo Stop & Glo assay system (Promega) according to manufacturer’s
134
recommendations. Briefly, media was removed from the wells containing cells and the cells were
135
washed twice with Phosphate buffered saline (PBS) (Life Technologies). Cells were then lysed
136
for 20 min at room temperature on an orbital shaker. Subsequently, 20 µl aliquot of cell lysate
137
from each well was pipetted into wells of a white wall, white bottom plate. Firefly luciferase
138
expression was measured by injecting 100 µl firefly luciferase substrate into each well.
139
Expression was measured 2 seconds later on a Clarity luminescence reader (BioTek) using a 10-
140
second integration time. After the first 20 seconds reading, 100 µl of Stop & Glo reagent was
141
injected into each well. Renilla luciferase expression was measured 2 seconds later with an
142
integration time of 10 seconds.
143
Statistical analysis
144
All the EC50s were calculated using 4 point non-linear regression curve fitting with variable
145
slope and no constrains on both the top and bottom values in the PRISM software. Chalice
146
Analyzer (33) was used to evaluate synergy. The theories of Loewe additivity, Bliss independent
147
and highest single agent (HSA) were described in details in the references (34-36).
148
RESULTS
149
Antiviral activity of individual compounds
150 151
We selected three compounds (ribavirin, brequinar, and INX-08189) to test their combination antiviral activities (left panels, Fig. 1A-C). Each of the selected compounds has
7
152
distinct mode-of-actions (see Introduction). A DENV-2 luciferase replicon assay was used to
153
measure antiviral activity throughout the study. Prior to combination testing, we first examined
154
the anti-DENV activity of individual compounds (middle panels) and cytotoxicity (right panels).
155
Figure 1D summarizes the EC50, EC90, and CC50 values of each compound. The results
156
demonstrate that all selected compounds have anti-DENV activities with good therapeutic
157
window in cell culture.
158 159 160
Antiviral activity of combination treatment We measured antiviral activities of different compound pairs such that the inhibitor of
161
nucleoside pathway is paired with a nucleoside inhibitor, INX-08189. To examine whether
162
combinatory treatments have synergistic or additive effects, we tested each compound at seven
163
concentrations (excluding a control without compound) with dose matrix centered on the EC50
164
value. The left panels in Figure 2 show the percentages of replicon inhibition under each dose
165
matrix (left panel, Fig. 2A). Chalice Analyzer was used to calculate the Loewe excess (right
166
panel, Fig. 2A), Bliss excess (left panels, Fig. 2B), and HSA excess values (right panels, Fig.
167
2B). These parameters are commonly used to indicate the excess percentage inhibition; the
168
excess percentage inhibition is calculated by deducting the expected percentage inhibition values
169
of various combinations, assuming non-synergy pairing in various models, from the
170
experimental percentage inhibition values. These data allowed us to calculate the isobolograms,
171
synergy scores, and best combination indices (CI) for each pair (Fig. 2C). In general, synergy
172
score of >1 and CI of 5 µM and > 800 µM, respectively (Fig. 3D). The results indicate that, in the absence of
205
exogenous IFN, treatment of cells with brequinar or ribavirin alone could induce ISRE
206
activation.
207 208 209
DHODH inhibitors potentiates the IFN-induced ISRE activation Since brequinar or ribavirin alone could induce ISRE activation, we examined whether
210
these compounds could potentiate/enhance the ISRE activation when cells are co-incubated with
211
exogenous IFN. To this end, HEK 293T cells were co-incubated with IFN-β and ribavirin or
212
brequinar. As shown in Figure 4, compared with DMSO control, brequinar significantly
213
potentiated ISRE activation when the cells were co-treated with 5,000 and 10,000 IU/ml IFN-β
214
(p-value < 0.001); no potentiation was observed when the cells were co-treated with lower doses
215
of IFN-β. In contrast, treatment with ribavirin plus IFN-β did not augment ISRE activation (Fig.
216
4). The results indicate that brequinar, a DHODH inhibitor, could enhance the exogenous IFN-
217
induced ISRE activation.
218
Next, we asked whether other pyrimidine biosynthesis inhibitors could also potentiate the
219
IFN-induced ISRE activation. To address this question, we used NITD-982, a compound that
220
was recently reported as a potent inhibitor of DHODH (29). Treatment with NITD-982 at a non-
10
221
cytotoxic concentration significantly augmented the ISRE activation when the cells were co-
222
incubated with 10,000 IU/ml IFN-β (p-value < 0.001; Fig. 4). Taken together, the results
223
demonstrate that DHODH inhibitors (brequinar and NITD-982) can potentiate the IFN-induced
224
ISRE activation.
225 226 227
DISCUSSION In this study, we explored the possibility of a combinatorial therapy selected from three
228
compounds that have anti-DENV activities. Since INX-08189 is a potent nucleoside inhibitor for
229
DENV (Fig. 1C), we chose this compound to perform combination analysis and tested the
230
hypothesis that co-treatment with INX-08189 plus another inhibitor that perturbs the synthesis of
231
natural triphosphate nucleotide(s) could lead to antiviral synergy. Our results showed that
232
compound pair ribavirin + INX-08189 exhibited clear antiviral synergy with strong CI of 0.325
233
(Fig. 2C). The combination has the potential to lower the dose of either compound by 3 times to
234
achieve a similar level of viral inhibition (34); this could translate into a bigger therapeutic
235
window for the treatment of DENV with these two compounds. The synergy is not due to the
236
cytotoxicity of the combination treatment (Fig. 2D). It should be noted that synergy was
237
observed in compound pairs in which one compound suppressed the synthesis of the nucleoside
238
for which the other compound is a corresponding nucleoside analog. This is expected because
239
reduction of the natural nucleoside pool (by the nucleoside synthesis inhibitor) increases the ratio
240
of the nucleoside analog triphosphate versus the natural nucleoside triphosphate, making the
241
nucleoside analog triphosphate more likely to be incorporated into the viral RNA and thus
242
termination of viral RNA synthesis. Specifically, the synergy was found between ribavirin (a
243
guanosine synthesis inhibitor) and INX-08189 (a guanosine analog).
11
244
A similar combination antiviral approach was previously employed for HCV and HIV.
245
For HCV, INX-08189 was tested in combination with ribavirin in the presence or absence of
246
pegylated interferon (PEG-IFN) in HCV patients; unfortunately, adverse effects (heart and renal
247
toxicity) were observed (38). This is not too surprising since INX-08189 had similar
248
toxicological finding when administered as monotherapy (39). For HIV, a CTP synthase
249
inhibitor (3’-deazauridine) strongly potentiated the anti-HIV-1 activity of a 5’-triphoaphate of
250
the cytidine analog (3TC) and a 2,3’-dideoxy cytidine (ddC) in cell culture (40). One explanation
251
for drug synergy is the parallel pathway inhibition model, which suggests that two drugs will be
252
synergistic if they inhibit two proteins on parallel pathways essential for an observed phenotype
253
(41). Our synergy results seem to support the parallel pathway inhibition model. However, we
254
could not exclude other possible mechanisms that could also contribute to the observed synergy.
255
It would be interesting to test whether the antiviral synergy of ribavirin + INX-08189
256
observed in cell culture could be reproduced in the dengue AG129 mouse model. Since
257
nucleoside inhibitor requires host kinases to convert to its active triphosphate form, caution
258
should be taken when considering such in vivo study. Due to the potential species difference in
259
converting INX-08189 to the triphosphate INX-08189 between human and mouse, the relevance
260
of the non-human animal model should be carefully validated experimentally when analyzing
261
nucleoside inhibitors.
262
The potential involvement of IFN pathway in the observed antiviral activity was
263
investigated. For brequinar, besides inhibiting pyrimidine synthesis, our results showed that
264
brequinar alone could activate ISRE in cell culture; furthermore, brequinar could enhance the
265
exogenous IFN-induced ISRE activation. Such enhancement was further validated with another
266
pyrimidine biosynthesis inhibitor (NITD-982). The mechanisms of brequinar/NITD-982-
12
267
mediated ISG activation and IFN-signaling enhancement remain to be determined. Nevertheless,
268
these observations may have clinical implications. When patients receive therapy, DENV has
269
already established its replication, triggering immune response to produce IFN and other
270
cytokines (42, 43). If the patients were treated with brequinar, its ability to augment the IFN-
271
induced ISRE activation should enhance the overall antiviral status of patients.
272
The synergy observed in the ribavirin + INX-08189 treatment is unlikely to go through
273
the ribavirin-mediated ISRE activation. This is because brequinar alone activated ISRE (Fig. 3B)
274
but failed to synergize INX-08189. In addition, the activation level of IRES was modest even at
275
high concentration of ribavirin.
276
There is an urgent unmet medical need to develop a safe and effective antiviral for
277
DENV infection. Although no single compound has been approved for clinical use, the
278
possibility of repurposing clinically tried or approved drugs for DENV is a tractable option.
279
Towards this approach, balapiravir, a cytidine nucleoside analog that was stopped for HCV
280
clinical development, was tested in dengue patients; unfortunately, the compound did not show
281
any efficacy in the dengue clinical trial (14). Similarly, celgosivir, a host alpha-glucosidase
282
inhibitor (initially developed for HCV) also failed to show significant difference compared with
283
placebo (44). An alternative approach to repurpose clinically approved drugs for DENV is to
284
search for combination therapy. Such approach has two conceptual advantages. Firstly, synergy
285
between two drugs would allow achieving efficacy at lower doses, leading to increased
286
therapeutic window of potentially toxic compounds (45). Theoretical and experimental studies
287
have shown that drugs that exhibit synergy for a specific effect are usually not synergistic for
288
side effects (33, 46). Indeed, toxicity experiments suggest that the observed antiviral synergy of
289
ribavirin + INX-08189 combination was not due to cytotoxicity (Fig. 2). However, due to the
13
290
unpredictable nature of toxicity associated with nucleoside analog, caution should be taken when
291
extrapolating the in vitro toxicity results. Secondly, combination treatment would minimize the
292
chance of resistance. In the absence of effective monotherapy for DENV, a combination of two
293
moderately effective drugs may be needed. This was evident in the treatment of HIV where only
294
combination of drugs effectively reduces the viremia to undetectable level. These considerations
295
make synergistic drug pairs ideal candidates for treatment of infectious pathogens. The current
296
study used DENV-2 as a model to achieve proof-of-concept. Future studies are needed to expand
297
the current observation in cell culture to an appropriate in vivo study.
298 299 300
ACKNOWLEDGEMENTS We thank Chek Shik Lim, Christophe Leroy, and Joseph Lehar on data analysis on
301
synergy. We thank colleagues at Novartis Institute for Tropical Diseases (NITD) for helpful
302
discussions and help during the course of this study. All authors are employees of Novartis.
303 304
14
305
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FIGURE LEGENDS
460
Figure 1. Host nucleoside inhibitors and pre-clinical nucleoside analogues exhibit potent anti-
461
DENV replication activity with minimal cyototoxicty in cell culture. (A) The chemical structure
462
of ribavirin, a well-characterized inhibitor of IMPDH, is shown. The EC50 of ribavirin in the
463
Huh-7 NGC replicon is about 8.29 µM and the EC90 is about 61.77 µM. CC50 of ribavirin in
464
Huh-7-NGC replicon is more than 850 µM. (B) The structure of brequinar, a potent DHODH
465
inhibitor, is shown. Its EC50 in Huh-7 NGC replicon is about 51.5 nM and the EC90 is about
466
248.8 nM. The CC50 of brequinar in the Huh-7 NGC replicon is more than 5 µM. (C) The
467
chemical structure of the guanosine analog, INX-08189, is shown. The EC50 of INX-08189 in
468
the Huh-7 NGC replicon is about 14.48 nM and the EC90 is about 131.5 nM. The CC50 of INX-
469
08189 in the Huh-7 NGC replicon is more than 1 µM. (D) The EC50, EC90 and CC50 of
470
respective compounds are summarized, demonstrating that all the selected compounds have anti-
471
DENV activities with good therapeutic window in cell culture. The data is derived the average of
472
three to five independent experiments.
473 474
Figure 2. Chalice analysis of ribavirin and INX-08189 combination treatment on NGC replicon.
475
Luciferase activity was measured 48 h post-treatment with dosing matrix centered on the EC50
476
of each compound in the Huh-7 NGC replicon. (A) Dose matrix response values of the
477
combination showing the inhibition of luciferase activity (1 – treated / untreated) for the serially
478
diluted compounds. Inhibition values were analyzed by the Chalice software to generate
479
combinations’ excess values (the Loewe excess values are shown here). (B) The computed Bliss
480
and highest single agent (HSA) excess of each compound combination. Values are derived from
481
an average of 6 experiments. (C) Isobolograms, synergy score and best combination index (CI) at
22
482
90% inhibition of ribavirin and INX-08189. Values shown are derived from average of eight
483
experiments. (D) Cell viability of drug combination treatment. The concentrations of ribavirin
484
and INX-08189 were identical to those used in the dose matrix for antiviral assay (A). The
485
synergy calculation was derived from average of 8 datasets.
486 487
Figure 3. Dose-dependent induction of ISRE by IFN-β and nucleoside synthesis inhibitors. HEK
488
293T cells batch-transfected with pISRE-TA-Luc and pGL4.74-hRluc/TK, were simultaneously
489
treated with increasing concentrations of IFN-β, brequinar or ribavirin in individual wells. (A)
490
Treatment with IFN-β caused a maximum of 4-fold increase in ISRE activation while treatment
491
with (B) brequinar caused a maximum of 3-fold increase in ISRE activation. (C) Activation of
492
ISRE by ribavirin was modest, showing a maximum of 1.68-fold. (D) The induction of ISRE
493
activity by either host nucleoside inhibitors was not due to cytotoxicity as their CC50 values in
494
the HEK293-T cells were > 5 µM. The average results from two experiments are shown.
495 496
Figure 4. Induction of ISRE by IFN-β is potentiated in the presence of DHODH inhibitors. HEK
497
293-T cells were batch transfected with pISRE-TA-Luc and pGL4.74-hRluc/TK. Transfected
498
cells were simultaneously treated with increasing concentrations of IFN-β and the indicated
499
concentrations of respective compounds. 48 h post-treatment, luciferase activities were assayed
500
using the Dual-Glo Stop & Glo reagent. Co-treatment of HEK293T cells with brequinar and
501
5000 and 10, 000 IU/ml of IFN-β, significantly potentiated ISRE activation when compared to
502
DMSO control. There was no potentiation observed at lower doses of IFN-β. In contrast, co-
503
treatment of cells with ribavirin and IFN-β did not augment ISRE activation. Treatment of cells
23
504
with another pyrimidine biosynthesis inhibitor, NITD-982, with 10, 00 IU/ml of IFN-β, also
505
significantly augmented the ISRE activation.
24
Luminescence (%)
A
Ribavirin (log µM)
Luminescence (%)
B
Brequinar (log µM)
Luminescence (%)
C
Brequinar (µM)
EC50 = 14.2 ± 5.6 nM
INX-08189 (log µM)
INX-08189
D
EC50 = 53.4 ± 2.7 nM
Cell viability (%)
Brequinar
Ribavirin (µM)
Cell viability (%)
Ribavirin
EC50 = 3.18 ± 2.86 µM
Cell viability (%)
Figure 1
INX-08189 (µM)
Compound
EC50 (nM)
CC50 (µM)
Ribavirin
3,178 ± 2860
> 800
Brequinar
53.4 ± 2.7
>5
INX-08189
14.2 ± 5.6
>1
Figure 2 Loewe excess
Dose matrix
11 .14
1.2
Ribavirin (µM)
11 1.2 0
0
.14
Ribavirin (µM)
100
100
A
0
0.014
0.12
0.11
0
1
0.014
B
1
100 11 0
0.14
1.2
Ribavirin (µM)
100 11 1.2
Ribavirin (µM)
0.14 0
0.012
0.11
0 0.0014
1
Cytotoxicity of synergy data
11
59
62
65
70
75
74
69
59
71
73
75
81
88
93
88
71
89
90
93
98 102 110 104 84
99 102 104 105 108 112 107 88 1.2
Ribavirin (µM)
100
D
103 105 107 110 113 114 110 93 100 101 105 104 106 109 107 94 100 101 101 103 106 112 108 97 100 102 101 103 108 110 108 92 0
INX-08189 (0.11 µM)
1
.14
Synergy score: 2.2 ± 0.0475 Best CI: 0.325 ± 0.0143
0.11
0
Isobologram
0.012
INX-08189 (µM)
INX-08189 (µM)
Ribavirin (100 µM)
0.11
Huh-7 NGC replicon HSA excess
Huh-7 NGC replicon Bliss excess
0 0.0014
C
0.12
INX-08189 (µM)
INX-08189 (µM)
0.014
0.12
0.11
INX-08189 (µM)
1
Cell viability (%)
Cell viability (%)
ISRE activity (fold increase) ISRE activity (fold increase)
ISRE activity (fold increase)
Figure 3
A
B IFN-β (IU/mL)
C Brequinar (µM)
D Ribavirin (µM)
Brequinar (µM)
Ribavirin (µM)
Figure 4 1 % DMSO 1 mM Ribavirin 5 µM Brequinar 10 µM NITD-982
15 10
*** ***
*** p value < 0.001 ***
5
IFN- (IU / mL)
0 10 00
50 00
25 00
12 50
0
0
Fold-induction of ISRE
20
