Novel Protease Inhibitors as SARS Therapeutics

描述(由申请人提供)：针对引起严重急性呼吸系统综合症(SARS)的病毒开发新的治疗药物显然是当务之急。自从2002年11月在广东省报告了SARS以来，SARS蔓延到了其他亚洲国家、北美和欧洲。到2003年7月，五大洲26个国家的8000多个病例，造成774人死亡和巨大的经济损失。最近发现的一种冠状病毒已被确定为SARS的病原体。到目前为止，还没有针对这种病毒的有效治疗方法。复制酶多蛋白被两种病毒蛋白水解酶--类木瓜蛋白水解酶2(PLPro)和3CLPro降解。蛋白水解性加工是产生功能性复制复合体的关键，因此SARS冠状病毒蛋白水解酶是开发抑制病毒复制的抗病毒药物的有吸引力的靶点。在参与该项目的研究人员之间建立良好的合作网络基础上，我们提出了一种开发新的抗病毒蛋白水解酶抑制剂的综合方法。我们将使用一系列策略，从表征复制过程组装所必需的蛋白酶和病毒蛋白开始，从蛋白酶的酶学表征和蛋白酶3D分子结构的X射线测定，利用不同的化学文库进行初步的先导发现，基于结构的药物设计，先导化合物的合成和优化，然后是细胞培养测试。应用的一个重要优势是参与者的广泛专业知识，包括病毒学、酶学、大分子结构生物学、计算机辅助药物设计、合成药物化学和细胞培养测试。项目1将描述PLPRO的特征，并评估PLPRO和3CLPRO在蛋白质降解过程中的作用。项目2已经开发了3CLPro的活性分析和3CLPro与抑制剂的结晶，并将继续开发3CLPro和PLP2的酶谱和X射线晶体结构以及它们与抑制剂的相互作用。项目3将利用基于结构的设计和高通量筛选来开发3CLPro和PLP2的抑制剂；最初的抑制剂对3CLPro显示出显著的活性。两个科学核心将支持这些项目：一个蛋白质表达核心将为目标评估和基于结构的设计提供蛋白质。计算和高通量筛选核心将为突变分析、酶特性表征、基于结构的设计和铅抑制剂筛选提供生物信息学支持。行政核心将提供财政管理和行政支助。

DESCRIPTION (provided by applicant): The development of new therapeutic drugs against the virus causing Severe Acute Respiratory Syndrome (SARS) is a clear imperative. Since its report in Guangdong Province, China, in November, 2002, SARS spread to other Asian countries, North America and Europe. By July 2003 there were more than 8,000 cases in 26 countries on five continents, with 774 deaths and enormous economic damage. A recently discovered coronavirus has been identified as the etiological agent for SARS. Thus far, no effective therapy exists for this virus. The replicase polyprotein is proteolytically processed by two viral proteases, papain-like protease 2 (PLpro) and 3CLpro. Proteolytic processing is essential for generating a functional replication complex, and thus the SARS-CoV proteases are attractive targets for the development of antiviral drugs that will inhibit viral replication. Building on a well-established collaborative network among the investigators participating in this project, we propose an integrated approach toward the development of new antiviral protease inhibitors. We will use a combination of strategies, beginning with characterization of proteases and viral proteins essential to assembly of the replication process, protease enzymatic characterization and X-ray determination of protease 3D molecular structures, utilization of diverse chemical libraries for initial lead discovery, structure-based drug design, synthesis of lead compounds and their optimization, followed by cell culture testing. An important strength of the application is the broad range of the participants' expertise, including virology, enzymology, structural biology of macromolecules, computer-assisted drug design, synthetic medicinal chemistry, and cell culture testing. Project 1 will characterize PLpro and evaluate the roles of PLpro and 3CLpro in proteolytic processing. Project 2 has already developed an activity assay for 3CLpro and crystallized 3CLpro with inhibitors, and will continue to develop enzymatic profiles and X-ray crystal structures of 3CLpro and PLP2 and their interactions with inhibitors. Project 3 will utilize structure-based design and high throughput screening to develop inhibitors of 3CLpro and PLP2; initial inhibitors exhibit significant activity against 3CLpro. Two scientific cores will support these projects: A protein expression core will provide proteins for target evaluation and structure-based design. A computational and high throughput screening core will provide bioinformatic support for mutational analysis, enzymatic characterization, computational support for structure based design and screening for lead inhibitors. An administrative core will provide fiscal management and administrative support.