The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. While there are several promising vaccine Nordihydroguaiaretic acid candidates in clinical trials approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both and within the family 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 α-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 replication complex. The lead candidate Nordihydroguaiaretic acid compound 14a is orally available and has good pharmacokinetic properties. Using a dengue mouse model we show that compound 14a suppressed peak viremia on day 3 postinfection (p.i.) LMO4 antibody even when treatment started 2 days after viral infection. Our results have pharmacologically validated that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. MATERIALS AND METHODS Cells compounds and antibodies. A549 cells (human alveolar epithelial cells) were maintained in F-12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. BHK-21 cells (baby hamster kidney cells) were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin. C6/36 mosquito cells were grown in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin. A549 cells containing a DENV-2 replicon were maintained in F-12 medium containing 10% FBS 20 μg/ml puromycin and 1% penicillin-streptomycin (11). Huh-7.5 cells Nordihydroguaiaretic acid containing a subgenomic replicon of hepatitis C virus (HCV) genotype 1b were licensed from Apath LLC (St. Louis MO) (34) and were maintained in DMEM containing 10% FBS 0.25 mg/ml Geneticin and 1% penicillin-streptomycin. A549 BHK-21 DENV-2 replicon and HCV replicon cell lines were incubated at 37°C. C6/36 cells were cultured at 28°C. All compounds were in-house synthesized. DENV-specific mouse monoclonal antibody 4G2 Nordihydroguaiaretic acid against the DENV envelope (E) protein was prepared from a hybridoma cell line purchased from the American Type Culture Collection (ATCC). DENV-2 and HCV replicon assays. A549 DENV-2 replicon cells were seeded at a density of 3 0 cells per well in a 384-well microplate. After incubation at 37°C with 5% CO2 overnight the cells were treated with compounds. After 48 h of incubation luciferase activities were measured by using the EndurRen live-cell substrate (Promega). Following luciferase activity measurement the CellTiter-Glo reagent (Promega) was added to each well to determine the cytotoxicity of the compounds. For the HCV replicon assay Huh-7.5 cells harboring the HCV replicon were seeded at a density of 20 0 cells per well in a 96-well microplate. At 48 h after compound treatment the cells were assayed for luciferase activity by using a Bright-Glo luciferase assay (Promega). As a quality control NITD-008 a nucleoside inhibitor of DENV and HCV (12) was included in our primary and secondary antiviral assays throughout the study. Viral titer reduction assay. The following viruses were used in the viral titer reduction assay: DENV-1 DENV-2 DENV-3 DENV-4 Japanese encephalitis virus (JEV) Powassan virus (POWV) Western equine encephalitis virus (WEEV) West Nile virus.