Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation

Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent

Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation

Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent

Pathogenic potential and growth kinetics of Muko virus in mice and human-derived cells

Background: Ticks have been long known as vectors of various pathogens, some of which can cause high fatality rates among infected individuals. Our enhanced tick surveillance around Nagasaki, Japan, led to the isolation and identification of a new strain of a recently identified Orbivirus, Muko virus (MUV). The orbiviruses have a wide host range, including humans, and is related to a spectrum of clinical outcomes. However, the zoonotic potential of some members of the genus, although reported, were not clearly elucidated. Hence, it is imperative to characterize newly isolated orbiviruses and investigate its ability to endanger public health. Methods: In this study, we explored the in vivo pathogenicity of a newly isolated MUV strain (MUV-Hay) using a mouse model and demonstrated its growth kinetics in human-derived cells. Results: Our results showed the ability of MUV-Hay to propagate in human neuronal and renal cells with some cytopathic effect. Furthermore, intracerebral inoculation of our new isolate caused high mortality in adult A129 mice. Conclusion: Our study provided a first step to experimentally test the hypothesis, that MUV can replicate and produce cytopathic effect in human cells and demonstrate virulence in adult mice

デング2型ウイルスプロテアーゼを標的とした新規抗ウイルス薬探索のための蛍光発色によるアッセイ法の開発

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 denguevirus 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 earlystages 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 saidobservation 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.長崎大学学位論文 学位記番号:博(医歯薬)甲第890号 学位授与年月日:平成28年9月20日Author: Gianne Eduard L. Ulanday, Kenta Okamoto and Kouichi MoritaCitation: Tropical Medicine and Health, 44, 22; 2016Nagasaki University (長崎大学)課程博