開發以結構及細胞為基礎之魚類神經壞死症病毒抑制劑

Abstract

魚類神經壞死症病毒 (Nervous necrosis virus, NNV) 是一具有高度傳染性之病原體，在水產養殖產業中造成嚴重的損失。直到目前為止，仍無任何商品化之抗病毒藥物可用來防治魚苗早期發育階段因該病毒感染而產生之大量死亡現象。因此，為了加速抗NNV藥物之開發，建立藥物開發所需之工具顯得十分重要。本研究中，我們建立了以細胞存活率為基礎之藥物篩選平台，並針對NNV之RNA聚合酶 (RdRp) 進行以蛋白質結構為基礎之模擬分析。本研究首先進行藥物篩選試驗條件之最佳化，並進行篩選結果之強韌性確認，其中代表藥物篩選結果穩定性之Z’ factor值分布於0.70至0.94間。利用此篩選平台篩選2,000種小分子化合物之後，共有43種化合物之病毒抑制能力≥55%。其中，proadifen hydrochloride 屬於細胞色素 (cytochrome) P450抑制劑，其EC50及CC50分別為6.48μM及20.63μM，並在石斑魚鰭細胞株 (GF-1) 感染病毒五天後降低病毒RNA量達99.68%。除此之外，我們更發現這43種化合物中共有18種化合物屬於神經傳導物質藥劑 (neurotransmitter agents)。在病毒RdRp結構分析中，其蛋白質結構藉由SWISS-MODEL server進行同源模擬而建立。將該結構與口蹄疫病毒RdRp 蛋白質結晶結構(與ribavirin triphosphate結合) 進行結構比對的結果顯示其配體結合位置之結構具有高度保留性。由於ribavirin在過去被發現可以抑制多種病毒之RdRp，因此，本研究利用濃度相依實驗以及病毒RNA量測來確認ribavirin在GF-1細胞中抑制病毒之活性。實驗結果顯示ribavirin在濃度25 μM可抑制病毒引發之細胞病變效應(cytopathic effect) 達54.41%。同時病毒RdRp以及蛋白質外殼之RNA量在細胞感染病毒後48小時皆有顯著之下降，本結果顯示ribavirin可能藉由與NNV RdRp間之作用抑制病毒在GF-1細胞中之感染。總結本研究，以結構及細胞為基礎之魚類神經壞死症病毒抑制劑開發之工具已成功建立，本研究亦發現數種抗魚類神經壞死症病毒之抑制劑。本研究結果除了提供魚類神經壞死症病毒防治之候選藥物，亦可用以探討該病毒與宿主間之交互作用。

Nervous necrosis virus (NNV) is a highly contagious pathogen, responsible for severe losses incurred in the aquaculture industry. Currently, no commercially available antivirals against the virulence observed during very early stages of fish larvae development. Therefore, to facilitate the discovery of antivirals against NNV, establishing tools for discovering anti-NNV agents is important. In this study, we developed a novel cell viability-based screening assay as well as a structure-based survey of viral RNA-dependent RNA polymerase (RdRp). The cell viability-based screening assay conditions were optimized and the robustness of the assay was confirmed by a Z’ factor value ranging from 0.7 to 0.94. After screening a library of 2,000 small molecule compounds, 43 compounds with a virus inhibition capacity of ≥55% were identified. A cytochrome P450 inhibitor, proadifen hydrochloride, was validated with an EC50 value of 6.48 μM and a CC50 value of 20.63 μM. This compound reduced viral RNA level by 99.68% in grouper fin-1 cells (GF-1) at 5 days post-infection. Surprisingly, we found that 18 of 43 compounds act as neurotransmitter agents. For structure-based survey of NNV RdRp, the three-dimensional structure of NNV RdRp was modeled using the SWISS-MODEL server. Structure-based alignment of the modeled NNV RdRp and foot-and-mouth disease virus RdRp crystal structure (in complex with ribavirin triphosphate) indicated that the conformation of the ligand-binding pockets were highly conserved. Since ribavirin was shown to inhibit the RdRp from various viruses, it was thus tested for its ability to inhibit NNV infection by a dose-dependent assay and viral RNA determination in GF-1 cells. Ribavirin inhibited the virus-induced CPE by 54.41% at a concentration of 25 μM, and the viral RNA levels of both RdRp and capsid protein decreased significantly at 48 hours post-infection. These results indicate that ribavirin may interact with the RdRp of NNV to inhibit viral infection in GF-1 cells. In summary, novel tools for discovering anti-NNV agents with structure and cell-based assays have been established. The inhibitors of NNV were also revealed. These results may not only be used as antiviral therapies, but also help us to further understand the interactions between viruses and host cells.