抗單純皰疹病毒藥物篩選及其作用機制之探討

Screening of Anti-HSV Agents and Study of Their Antiviral Mechanisms

蘇峻霆(Chun-Ting Su)

指導教授 ： 張淑媛

國立臺灣大學/醫學院/醫事技術學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10198

摘要

單純皰疹病毒（Herpes Simplex Virus；HSV）分為HSV-1及HSV-2兩型，常引起口腔及生殖器部位的皮膚或黏膜感染，在初次感染後會潛伏在神經系統中，待宿主免疫力低下時再間歇性地復發。除了皮膚及黏膜的症狀，偶而亦可能導致失明或致命。由於其流行率高，具高度傳染性，且不易研發預防性的疫苗，故發展治療性藥物相對地十分重要。目前針對HSV感染最有效的治療方式為使用無環鳥苷（acyclovir）或與其相似的藥物，此類藥物屬於核苷酸相似物，透過病毒的胸腺嘧啶激酶（Thymidine kinase；TK）催化可以在受感染的細胞裡抑制病毒的DNA聚合酶，進而抑制病毒的複製；但近年來已逐漸有抗藥病毒株的產生，造成治療上的困難，故發展新的抗病毒藥物確有其必要性；目前已是許多研究單位致力的方向。在本篇論文中，我們首先對於九百多種候選化合物進行抗單純皰疹病毒活性之篩選，發現其中73種化合物可能具有抗病毒之活性。之後再利用溶菌斑減少實驗，對於篩選出來的藥物進行藥效確認，結果有14種化合物在1 μM的濃度可抑制50%以上的病毒複製。其中最有潛力的候選藥物12-2 8G被選出，利用MTT試驗進一步分析候選藥物的細胞毒性，計算50%細胞毒性濃度（CC50）及選擇性指標（SI），藉此評估藥物實際使用在治療上的可能性。我們嘗試尋找12-2 8G之抗病毒之機制，並發現藥物在病毒吸附、穿透時加入或作用於細胞及病毒前處理皆不會抑制病毒的複製。此外，12-2 8G與傳統藥物無環鳥苷間沒有明顯的協同作用。藥物作用時間點試驗則顯示12-2 8G作用在病毒複製的中期或後期。12-2 8G對病毒DNA複製無明顯的抑制作用。之後將利用RT-PCR偵測病毒基因量表現，以進一步瞭解藥物抗病毒活性的機制。希望藉由這些實驗結果可對於抗病毒藥物的研發與設計提供更多資訊。

關鍵字

單純皰疹病毒 ； 抗病毒藥物

並列摘要

Herpes Simplex Virus (HSV) types 1 and 2 infections are the cause of cold sores and genital herpes as well as life-threatening or sight-impairing disease mainly in immunocompromized patients, pregnant women and newborns. After primary infections, HSV can establish persistent infection in nervous system and may reactivate intermittently upon appropriate stimuli. Because of the wide popularity, high ability to transmit and the difficulty to develop prophylactic vaccines, development of chemotherapy is comparatively important. To date, the most widely used and successful chemotherapy are nucleoside analogue agents such as acyclovir (ACV), which inhibits viral DNA polymerase after being phosphorylated by HSV thymidine kinase (TK). However, development of nucleoside analogue-resistant HSV strains has been reported in immunocompromised individuals. Thus, there is a need to develop novel anti-HSV agents to substitute for or to complement conventional anti-HSV chemotherapy. In this study we first screened a total of 960 candidate chemicals for their antiviral activity. Seventy-three of these candidate chemicals were further confirmed to have definite antiviral activity by plaque reduction assay. Among these 73 chemicals, 14 have a 50% effective concentration (EC50) lower than 1 μM. The cytotoxicity concentration (CC50) of these chemicals was subsequently determined by MTT assay and the selective index (SI) for each chemical was thus calculated. One potential drug, 12-2 8G, with SI=17 was further analyzed. By in vitro assay, HSV-1 replication was not significantly inhibited when 12-2 8G was added at viral entry, virus pretreatment or cell pretreatment. In the time-of-addition assay, 12-2 8G was shown to inhibit HSV-1 replication between 6 and 12 hours after infection. It is likely that 12-2 8G block HSV-1 infection at early or late stage. However, there was no interaction between 12-2 8G and ACV based on isobologram analysis. 12-2 8G did not significantly inhibit HSV DNA replication. We will perform time-of-addition assay and RT-PCR to further clarify the target(s) of 12-2 8G. Such information will be helpful in the development and designing of antiviral agents in the future.

並列關鍵字

HSV ； antiviral drug

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