Definition of structural organization and enzymology of the EBV protein kinase.

描述(申请人提供)：疱疹病毒编码的蛋白激酶(HPKs)最近引起了越来越多的关注，因为越来越多的证据表明它们参与了病毒生命周期的不同方面，使它们成为抗病毒治疗的吸引目标。这项建议的广泛目标是研究和开发病毒编码的靶点，寻找新的安全有效的抗病毒药物来对抗人类病原体EB病毒(EBV)。该项目采取了一种综合的方法：将利用分子生物学和生物化学技术研究EBV编码的蛋白激酶(EBV-PK)的结构和性质，并将探索小分子和基于多肽的策略来寻找抑制剂。第一个具体目的是通过诱变研究EBV-PK的结构，表征其对蛋白质底物和更昔洛韦(GCV)的活性，并建立一种快速有效地筛选抑制剂的方法。EBV-PK突变体的活性将在体外和细胞培养中进行研究。第二个特定目的是研究小分子和基于多肽的抑制剂对该激酶的抑制作用。选定的EBV-PK抑制剂将在细胞培养中进一步测试其抑制病毒复制的能力。因此，这项应用的目的是表征一种对EBV复制至关重要的独特的激酶，并鉴定将有效抑制该酶的化合物，从而潜在地抑制病毒。因此，它解决了一个一般性的生物学问题和一个制药问题。公共卫生相关性鉴于与EBV感染相关的疾病越来越多，其中许多疾病众所周知难以治疗，因此有必要开发和测试EBV靶向治疗方法，以尝试设计更好的治疗方法。这项提案中概述的研究将促进对EBV生物学的理解，并评估开发有效治疗EBV相关疾病的新目标。

DESCRIPTION (provided by applicant): Herpesvirus-encoded protein kinases (HPKs) have attracted increasing attention lately due to the growing evidence of their involvement in different aspects of the viral life cycle, making them appealing targets for antiviral therapy. The broad objectives of this proposal are to study and exploit a virally encoded target for novel safe and effective antiviral agents against the human pathogen Epstein-Barr virus (EBV). The project takes a combined approach: the structure and properties of the EBV-encoded protein kinase (EBV-PK) will be studied by molecular biology and biochemistry techniques, and in search for the inhibitors both small-molecule and peptide-based strategies will be explored. The first specific aim is to study EBV-PK structure by mutagenesis, characterize its activity in regard to protein substrates and ganciclovir (GCV) and develop an assay for fast and effective screening of inhibitors. The activity of EBV-PK mutants will be studied both in vitro and in cell culture. The second specific aim is designed to study inhibition of the kinase by small-molecule and peptide-based inhibitors. Selected EBV-PK inhibitors will be further tested in cell culture for their ability to inhibit viral replication. Thus, this application aims to characterize a unique kinase, which is crucial for EBV replication, and to identify compounds that will efficiently inhibit this enzyme, potentially inhibiting the virus. Therefore it addresses a general biological question and a pharmaceutical problem. PUBLIC HEALTH RELEVANCE Given the growing number of conditions associated with EBV infection, many of which are notoriously difficult to treat, the development and testing of EBV-targeting therapy approaches is warranted in attempts to devise better treatments. The studies outlined in this proposal will advance understanding of EBV biology and evaluate a novel target for the development of the effective treatment of EBV-associated diseases.