Ulanday Gianne Eduard L, Okamoto Kenta, Morita Kouichi
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.
Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523 Japan.
Trop Med Health. 2016 Aug 10;44:22. doi: 10.1186/s41182-016-0025-6. eCollection 2016.
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.
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.
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.
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.
登革热是世界上最重要的媒介传播疾病之一。登革热感染在世界许多地区的出现和再次出现每年影响数百万人，并继续给公共卫生系统带来负担，尤其是在低收入人群中。登革热疫苗研发取得了有前景的成果；然而，保护效果似乎并不理想。迄今为止，尚无针对登革热的许可化疗药物。在疫情爆发期间对高危个体进行联合疫苗接种和对患病者进行化疗的理想方案可能会弥补疫苗提供的微弱保护。登革热病毒蛋白酶对病毒复制很重要，因此已被确定为抗病毒药物的潜在靶点。因此，我们的目标是建立并优化一种适用于登革热药物研发早期阶段的筛选方法。
在本研究中，我们开发并优化了一种生化检测系统，用于筛选针对登革热病毒蛋白酶的化合物文库。我们用基于细胞的检测方法测试了所选的蛋白酶抑制剂，以确定其对病毒复制的抑制作用。
我们展示了底物动力学数据的直接图，显示在底物浓度过高时蛋白酶受到明显抑制。阐明了可能影响上述观察结果的最常见干扰源。最后，使用所开发的方法对现有的化合物文库进行了筛选。本研究中选择的化合物对重组登革热蛋白酶和基于细胞的感染性检测均显示出抑制活性。
我们的研究表明，定制的生化检测在药物发现的初始阶段寻找登革热病毒蛋白酶可能的抑制剂方面具有实用性。
