The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC 50 ], 10 to 80 nM) but not DENV-1 and -4 (EC 50 , >20 M). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial "hit" (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2-and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions Emerging infectious pathogens represent a major threat to public health. Vaccines and therapeutics are two key countermeasures against these pathogens. Many viruses of the genus Flavivirus within the family Flaviviridae are arthropod-borne human pathogens, among which the four serotypes of dengue virus (DENV) alone cause 390 million human infections each year (1). Several promising DENV vaccines are currently in clinical development (2). The most advanced vaccine (CYD-TDV) exhibited good efficacy against DENV-1, -3, and -4 but weak protection against DENV-2 (3-5). For antiviral development, four compounds have been tested in dengue clinical trials, including balapiravir (a nucleoside inhibitor) (6), celgosivir (a cellular ␣-glucosidase inhibitor) (7), chloroquine (a malaria drug with antiviral and immunomodulatory activities) (8), and prednisolone (a corticosteroid drug) (9). None of them showed any antiviral activity or clinical benefits in dengue patients. Notably, all these compounds were repurposed from existing drugs or compounds previously developed for other viruses. Bona fide inhibitors specifically designed for DENV have never advanced to clinical trials (10).In this paper, we report the identification of a novel class of small-molecule anti-DENV agents, the spiropyrazolopyridones, using phenotypic screening. These inhibitors block DENV replication by targeting nonstructural protein 4B (NS4B), a nonenzymatic transmembrane protein functioning as an essential component of the viral r...

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...

No abstract