A chemical genetic approach to inhibiting T-ag assembly on the viral origin

描述（由申请方提供）：许多DNA病毒复制过程中的关键事件之一是“起始蛋白”与复制起点的结合。一旦与复制起点结合，病毒编码的起始蛋白经常组装成更高的寡聚复合物。我们的长期目标是开发一种系统的方法来分离抑制剂，选择性地阻止复制起点上病毒启动子的组装。这些抑制剂将作为探索生物活性的有用试剂和药物设计的先导化合物。我们将利用的系统方法是最近描述的化学遗传学方法的扩展。该技术的一个关键组成部分是使用基于断裂内含肽的载体在E. coli细胞。这是一种非常经济有效的方法，用于生成复杂但非常稳定的库。使用简单的遗传筛选，将在文库中搜索能够破坏称为猿猴病毒40 T抗原的原型病毒起始物组装的成员。选择阻断T抗原寡聚化的环肽抑制剂的优点包括以下事实：已经确定了大部分T-ag的结构，并且非常详细地了解了T-ag与猿猴病毒40起源的相互作用。此外，T-ag寡聚化的抑制剂是临床相关的。例如，SV 40 T-ag可能是人类健康问题;尽管解释仍不确定，但数据已将其与多种人类癌症（包括间皮瘤和非霍奇金淋巴瘤）联系起来（综述于Vilchez和Butel（2004）Clinical Microbiology Reviews 17：495-508）。此外，SV 40病毒与两种人类病毒BK和JC病毒密切相关。这些病毒在人类中诱发许多疾病，包括癌症。此外，JC病毒可诱导进行性多灶性白质脑病（PML）;这是一种发生在细胞免疫受损患者中的疾病。事实上，大约5%的艾滋病患者患有PML。因此，我们建议使用的方法来分离抑制剂的T-Ag组装可能会识别针对BK和JC病毒的先导化合物。更重要的是，这些实验将有助于证明，我们所描述的化学遗传方法是一种通用的方法，用于分离抑制剂对病毒引发剂。一旦证明该方法的可行性，将用其他DNA病毒编码的病毒起始物进行类似的实验，例如单纯疱疹病毒和不同株的人乳头瘤病毒编码的病毒起始物。因此，本文所述的方法可能有助于分离具有广泛临床相关性的化合物。1 DNA病毒是一种重大的健康风险;然而，目前治疗这些病原体的选择很少。本文描述了一种用于分离抗DNA肿瘤病毒的抑制剂的化学遗传学方法的建议。因此，该建议可能具有广泛的临床意义。

DESCRIPTION (provided by applicant): One of the critical events during the replication of many DNA viruses is the binding of "initiator proteins" to origins of replication. Once bound to the origins of replication, the virally encoded initiator proteins frequently assemble into higher oligomeric complexes. Our long-term goal is to develop a systematic approach for the isolation of inhibitors that will selectively block the assembly of viral initiators on origins of replication. These inhibitors will serve as useful reagents for exploring biological activity and as lead compounds for drug design. The systematic approach that we will utilize is an extension of recently described chemical genetics methods. A critical component of this technique is the use of split intein based vectors to generate diverse cyclic-peptide libraries in E. coli cells. This is a very cost effective method for generating complex, but very stable, libraries. Using straightforward genetic screens, the libraries will be searched for members that are able to disrupt the assembly of a prototype viral initiator termed Simian Virus 40 T-antigen. The advantages of selecting cyclic peptide inhibitors that block T-antigen oligomerization include the fact that much of T-ag's structure has been determined and T-ag's interaction with the Simian Virus 40 origin is understood in great detail. Furthermore, inhibitors of T-ag oligomerization are clinically relevant. For example, SV40 T-ag may be a human health issue; although interpretations remain uncertain, data have linked it to a variety of human cancers including mesothelioma and non-Hodgkin's lymphomas (reviewed in Vilchez and Butel (2004) Clinical Microbiology Reviews 17: 495-508). Furthermore, SV40 virus is closely related to two human viruses, BK and JC virus. These viruses induce a number of diseases in humans, including cancer. In addition, JC virus induces progressive multifocal leukoencephalopathy (PML); a disease that occurs in patients whose cellular immunity has been impaired. Indeed, approximately 5% of patients with AIDS have PML. Therefore, the approach that we propose to use to isolate inhibitors of T-ag assembly may identify lead compounds against BK and JC viruses. More importantly, these experiments will serve to demonstrate that the chemical genetic approach that we describe is a general method for the isolation of inhibitors against viral initiators. Once the feasibility of the approach is demonstrated, similar experiments will be conducted with the viral initiators encoded by other DNA viruses, such as those encoded by Herpes simplex virus and different strains of human papillomavirus. Thus, the approach described herein might facilitate the isolation of compounds with broad clinical relevance. 1 DNA viruses are a significant health risk; however, there are currently few options for treating these pathogens. Describe herein is a proposal for a chemical genetics approach for the isolation of inhibitors against DNA tumor viruses. Thus the proposal is likely to have broad clinical significance.