Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities

Sveva Pelliccia,Yu-Hsuan Wu,Antonio Coluccia,Giuseppe La Regina,Chin-Kai Tseng,Valeria Famiglini,Domiziana Masci,John Hiscott,Jin-Ching Lee,Romano Silvestri

doi:10.1080/14756366.2017.1355791

Abstract

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes – NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

Highlights

Dengue virus (DENV) is responsible of worldwide arthropodborne viral infection, which globally represents a serious human health concern
NS2B serves as a cofactor of NS3 protease and forms complex with NS3; the central amino acid hydrophilic domain of NS2B is critical for cofactor activity[13]
DENV nonstructural protein 5 (NS5) RNA-dependent RNA polymerase (RdRp) has proven to be a promising target for direct-acting antiviral (DAA) drug development, because it is structurally conserved among the four DENV serotypes, and NS5 RdRp has no enzymatic counterpart in mammalian cells[15]

Summary

Introduction

Dengue virus (DENV) is responsible of worldwide arthropodborne viral infection, which globally represents a serious human health concern. PrM and E structural proteins are the primary antigenic targets of the humoural immune response in humans[9,10,11,12]. The N-terminal amino acids (residue 1–184) of NS3 is responsible for protease activity. DENV replication requires the nonstructural protein 5 (NS5) which is the essential RNA-dependent RNA polymerase (RdRp) activity[14]. Huh-7 cells were infected with DENV-2 and followed by RdRp inhibitors treatment for 3 days. The Huh-7 cells were transiently expressed p(þ)RLuc-(–)DV-UTRDC-FLuc and DENV NS5 expression vector pcDNA-NS5-Myc. fEC50 (enzyme-based DENV-2 NS5 RdRp): enzyme-based RdRp activity assay. Combination treatment with compounds, respectively, targeting NS5 RdRp polymerase and NS3 protease, demonstrated a synergistic inhibitory effect on DENV replication

Materials and methods

Ethics statement and experimental animals

Results and discussion

Conclusions

Disclosure statement