title: Development and utility of an in vitro, fluorescence-based assay for the discovery of novel compounds against dengue 2 viral protease.
abstract: BACKGROUND
  Dengue disease is one of the most significant vector-borne illnesses in the world. The emergence and re-emergence of dengue infections in many parts of the world affect millions annually and continue to burden public health systems especially in low-income populations. Advances in dengue vaccine development showed promising results; however, protection seems to be suboptimal. There is no licensed chemotherapeutic agent against dengue to date. An ideal scenario of combinatorial vaccination of high-risk individuals and chemotherapy of the diseased during outbreaks may compensate for the meager protection offered by the vaccine. The dengue virus protease is important to viral replication and, as such, has been identified as a potential target for antivirals. It is, therefore, our objective to establish and optimize an appropriate screening method for use during the early stages of drug development for dengue.
  
  
  METHODS
  In this study, we developed and optimized a biochemical assay system for use in screening compound libraries against dengue virus protease. We tested the selected protease inhibitors with a cell-based assay to determine inhibition of viral replication.
  
  
  RESULTS
  We have presented direct plots of substrate kinetics data showing an apparent inhibition of the protease at excessive substrate concentrations. The most common sources of interference that may have affected the said observation were elucidated. Finally, a screen was done on an existing compound library using the developed method. The compounds selected in this study showed inhibitory activity against both the recombinant dengue protease and cell-based infectivity assays.
  
  
  CONCLUSIONS
  Our study shows the practicality of a customized biochemical assay to find possible inhibitors of dengue viral protease during the initial stages of drug discovery.
authors.affiliation: Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523 Japan ; Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Gianne Eduard L
authors.initials: GE
authors.lastname: Ulanday
authors.affiliation: Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523 Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Okamoto
authors.affiliation: Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523 Japan ; Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan ; Leading Graduate School Program, Nagasaki University, Nagasaki, Japan.
authors.forename: Kouichi
authors.initials: K
authors.lastname: Morita
keywords: Antiviral screening; Dengue protease; Dengue virus; NS2B-NS3pro