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

描述(申请人提供)：许多DNA病毒复制过程中的一个关键事件是“启动子蛋白”与复制起点的结合。一旦结合到复制的起始点，病毒编码的启动子蛋白经常组装成更高的寡聚复合体。我们的长期目标是开发一种系统的方法来分离抑制物，这种方法将选择性地阻止病毒启动子在复制起点上的组装。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。我们将使用的系统方法是最近描述的化学遗传学方法的扩展。这项技术的一个关键组成部分是使用基于内含素的分裂载体在大肠杆菌细胞中产生不同的环肽文库。对于生成复杂但非常稳定的库来说，这是一种非常经济高效的方法。使用简单的基因筛查，将搜索文库中能够扰乱被称为猿猴病毒40T抗原的原型病毒启动子组装的成员。选择阻止T抗原寡聚的环肽抑制剂的优点包括：T-ag的大部分结构已经确定，并且对T-ag与猿猴病毒40起源的相互作用有了更详细的了解。此外，T-Ag寡聚的抑制物在临床上也是相关的。例如，SV40T-ag可能是一个人类健康问题；尽管解释仍然不确定，但数据已经将它与包括间皮瘤和非霍奇金淋巴瘤在内的各种人类癌症联系在一起(在Vilchez和Butel(2004)临床微生物学评论17：495-508中进行了审查)。此外，SV40病毒与两种人类病毒BK和JC病毒关系密切。这些病毒会导致人类患上多种疾病，包括癌症。此外，JC病毒还会导致进行性多灶性白质脑病(PML)；这种疾病发生在细胞免疫受损的患者身上。事实上，大约5%的艾滋病患者患有PML。因此，我们建议用来分离T-Ag组装抑制剂的方法可能会鉴定出抗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学方法是分离针对病毒启动子的抑制物的一般方法。一旦证明了该方法的可行性，将对其他DNA病毒编码的病毒启动子进行类似的实验，例如由单纯疱疹病毒和不同株的人乳头状瘤病毒编码的病毒启动子。因此，本文描述的方法可能有助于分离具有广泛临床意义的化合物。1 DNA病毒是一个重大的健康风险；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，该提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.