The lead candidate, compound 14a, is orally available and has good pharmacokinetic properties. 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,C5). 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 replication complex. The lead candidate, Inosine pranobex 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.), 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 g/ml puromycin, and 1% penicillin-streptomycin (11). Huh-7.5 cells 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 37C. C6/36 cells were cultured at 28C. All compounds were in-house synthesized. DENV-specific mouse monoclonal antibody 4G2 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 3,000 cells per well in a 384-well microplate. After incubation at 37C 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 20,000 cells per well in a 96-well microplate. At 48 h after compound treatment, the cells were assayed for luciferase activity by using.***, 0.001 for comparison of binding of the compound to WT and V63I mutant NS4B proteins. where it was not previously endemic. While there are several promising vaccine 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,C5). 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 replication complex. The lead candidate, 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.), 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 g/ml puromycin, and 1% penicillin-streptomycin (11). Huh-7.5 cells 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 37C. C6/36 cells were cultured at 28C. All compounds were in-house synthesized. DENV-specific mouse monoclonal antibody 4G2 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 3,000 cells per well in a 384-well microplate. After incubation at 37C 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 20,000 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 (WNV), yellow fever virus (YFV), and vesicular stomatitis virus (VSV). The sources of these viruses were reported previously (13). Approximately 2 104 cells (A549 or Vero cells) were seeded into each well of 96-well plates. At 24 h postseeding, A549 cells were infected with DENV (multiplicity of infection [MOI] of 0.5); Vero cells were infected with JEV, POWV, WNV, YFV, WEEV, or VSV (MOI of 0.1). The infected cells were immediately treated with serial dilutions of the compound. Because of the difference in replication kinetics among the different viruses, culture fluids were collected at different time points p.i.: culture fluids from JEV, POWV, WNV, WEEV, and YFV infections were collected at 42.5B, bottom). 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,C5). 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 Inosine pranobex 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, 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.), 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 g/ml puromycin, and 1% penicillin-streptomycin (11). Huh-7.5 cells 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 37C. C6/36 cells were cultured at 28C. All compounds were in-house synthesized. DENV-specific mouse monoclonal antibody 4G2 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 3,000 cells per well in a 384-well microplate. After incubation at 37C with 5% CO2 overnight, the Rat monoclonal to CD8.The 4AM43 monoclonal reacts with the mouse CD8 molecule which expressed on most thymocytes and mature T lymphocytes Ts / c sub-group cells.CD8 is an antigen co-recepter on T cells that interacts with MHC class I on antigen-presenting cells or epithelial cells.CD8 promotes T cells activation through its association with the TRC complex and protei tyrosine kinase lck 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 20,000 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 (WNV), yellow fever virus (YFV), and vesicular stomatitis virus (VSV). The sources of these viruses were reported previously (13). Approximately 2 104 cells (A549 or Vero cells) were seeded into each well of 96-well plates. At 24 h postseeding, A549 cells were infected.Complete genome sequencing of the resistant viruses revealed nucleotide changes at positions 7012 and/or 7013 of the genomic RNA, leading to a V63A, V63L, V63M, V63S, or V63T amino acid substitution in the NS4B protein (Fig. 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,C5). 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 replication complex. The lead candidate, 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.), 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 g/ml puromycin, and 1% penicillin-streptomycin (11). Huh-7.5 cells 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 37C. C6/36 cells were cultured at 28C. All compounds were in-house synthesized. DENV-specific mouse monoclonal antibody 4G2 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 3,000 cells per well in a 384-well microplate. After incubation at 37C 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 look for the cytotoxicity of the compounds. For the HCV replicon assay, Huh-7.5 cells harboring the HCV replicon were seeded at a density of 20,000 cells per well in a 96-well microplate. At 48 h after compound treatment, the cells were assayed for luciferase activity with a Bright-Glo luciferase assay (Promega). As an excellent control, NITD-008, a nucleoside inhibitor of DENV and HCV (12), was contained in our primary and secondary antiviral assays through the entire study. Viral titer reduction assay. The next viruses were found 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 (WNV), yellow fever virus (YFV), and vesicular stomatitis virus (VSV). The resources of these viruses were reported previously (13). Approximately 2 104 cells (A549 or Vero cells) were seeded into each well of 96-well plates. At 24 h postseeding, A549 cells were infected with DENV (multiplicity of infection [MOI] of 0.5); Vero cells were infected with JEV, POWV, WNV, YFV, WEEV, or VSV (MOI of 0.1). The infected cells were immediately treated with serial dilutions of the compound. Due to the difference in replication kinetics among the various viruses, culture fluids were collected at different.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 that your four serotypes of dengue virus (DENV) alone cause 390 million human infections every year (1). family are arthropod-borne human pathogens, among that your four serotypes of dengue virus (DENV) alone cause 390 million human infections every year (1). Several promising DENV vaccines are in clinical development (2). The innovative vaccine (CYD-TDV) exhibited good efficacy against DENV-1, -3, and -4 but weak protection against DENV-2 (3,C5). For antiviral development, four compounds have already 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). non-e of these showed any antiviral activity or clinical benefits in dengue patients. Notably, each one of these compounds were repurposed Inosine pranobex from existing drugs or compounds previously developed for other viruses. Real inhibitors specifically created for DENV haven’t 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 non-structural protein 4B (NS4B), a non-enzymatic transmembrane protein functioning as an important element of the viral replication complex. The lead candidate, compound 14a, is orally available and has good pharmacokinetic properties. Utilizing a dengue mouse model, we show that compound 14a suppressed peak viremia on day 3 postinfection (p.i.), even though treatment started 2 days after viral infection. Our results have pharmacologically validated that inhibitors of NS4B may 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 g/ml puromycin, and 1% penicillin-streptomycin (11). Huh-7.5 cells 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 37C. C6/36 cells were cultured at 28C. All compounds were in-house synthesized. DENV-specific mouse monoclonal antibody 4G2 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 3,000 cells per well in a 384-well microplate. After incubation at 37C with 5% CO2 overnight, the cells were treated with compounds. After 48 h of incubation, luciferase activities were measured utilizing the EndurRen live-cell substrate (Promega). Following luciferase activity measurement, the CellTiter-Glo reagent (Promega) was put into each well to look for the cytotoxicity of the compounds. For the HCV replicon assay, Huh-7.5 cells harboring the HCV replicon were seeded at a density of 20,000 cells per well in a 96-well microplate. At 48 h after compound treatment, the cells were assayed for luciferase activity with a Bright-Glo luciferase assay (Promega). As an excellent control, NITD-008, a nucleoside inhibitor of HCV and DENV.