Synergistic Suppression of Dengue Virus Replication Using a Combination of Nucleoside Analogs and Nucleoside Synthesis Inhibitors

Abstract

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Currently, there is no clinically approved vaccine or antiviral for DENV. Combination therapy is a common practice in antiviral treatment and a potential approach to search for new treatments for infectious pathogens. In this study, we performed a combination treatment in cell culture 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 for antiviral 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 one compound suppresses the synthesis of the nucleoside for which the other compound is a corresponding nucleoside analog. In addition, 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 activation. Compared to treatment with brequinar, treatment of cells with ribavirin alone could also induce ISRE activation, but to a lesser extent; however, when cells were cotreated with ribavirin and beta interferon, ribavirin did not augment the interferoninduced ISRE activation. O ver 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 is endemic throughout tropical and subtropical climates and is found mostly in urban and semiurban areas. This positive-sense single-stranded RNA virus is transmitted mainly by the Aedes aegypti mosquito and is classified under the genus Flavivirus in the family Flaviviridae. Other notable viruses in this group include yellow fever virus, Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus. Currently, neither an antiviral nor a vaccine is approved for DENV. Care for hospitalized dengue patients is supportive, mainly through optimal replenishment of body fluids. Treatment is intensive for those who succumb to the severe forms of the disease, i.e., dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF).Multiples approaches have been taken to identify inhibitors of DENV (2, 3). Small-molecule inhibitors have been reported to target various DENV proteins, including capsid (4, 5), envelope (6), protease (7, 8), nonstructural protein (NS) 4B (9, 10), methyltransferase (2, 11), and RNA-dependent RNA polymerase (12-16). Inhibition of host factors important for viral replication and of compounds with immunomodulation activities, including imino sugars (17), cholesterol inhibitors (18), chloroquine (19), and prednisolone (20), has also been pursued for potential treatment of DENV infections.Ribavirin is a drug with a...

The search for nucleoside/nucleotide analog inhibitors of dengue virus

“…Indeed, antiviral synergy could be achieved in DENV cell culture when a nucleoside analog is paired with an inhibitor of the synthesis pathway for the corresponding natural nucleoside. For example, synergy was observed between ribavirin (a guanosine analog with several antiviral mechanisms, one of which is to inhibit de novo biosynthesis of guanine nucleotide through direct binding to cellular IMP dehydrogenase (Malinoski and Stollar, 1981) and INX-08189 (a guanosine analog) in cell culture assay system for DENV (Yeo et al, 2015). Similar antiviral synergies were observed for combination therapy in HIV and HCV (Gao et al, 2000).…”

“…In particular, the 2'-C-methyl substitution was first reported to be active in HCV and it was soon found to have anit-DENV activity as well (Migliaccio et al, 2003). In addition, several other nucleoside analogs such as balapiravir and INX-189 were active in both DENV and HCV (Yeo et al, 2015). It is of interest to note that all DENV-active nucleosides are potent inhibitors of HCV.…”

Dengue Virus

“…Replication inhibitors targeting NS1, NS3 helicase, NS4A, NS4B, NS5 methyl transferase, and NS5 RdRp are being considered and many in vitro studies have proved their efficacy (Abdel-Magid, 2017; da Costa et al, 2013;Pelliccia et al, 2017;Yeo et al, 2015).…”

Dengue en el Perú: a un cuarto de siglo de su reemergencia

“…Estudios recientes han mostrado resultados alentadores utilizando un análogo de nucleósido, la 2'-C-metilcitidina en estudios en monos ha mostrado la replicación viral (54) , o una combinación de análogos de nucleósido con inhibidores de la síntesis de nucleósido (55) . En general, un mejor entendimiento de los mecanismos patogénicos en dengue proveerá herramientas valiosas para el desarrollo de antivirales y vacunas.…”