Mechanistic & Crystallographic Studies of SARS Proteases

机械论

• 批准号: 6940584
• 负责人: ANDREW D MESECAR
• 金额: $ 27.24万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6940584
• 关键词: SARS virus; X ray crystallography; antiviral agents; combinatorial chemistry; drug resistance; endopeptidases; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme substrate; fluorescent dye /probe; gene mutation; high throughput technology; protease inhibitor; protein sequence; protein structure function; site directed mutagenesis; virulence; virus genetics; virus protein

The major objective of Project 2 is to use mechanistic enzymology and x-ray crystallography to understand the structure and function of two proteinases, SCLpro and PLpro, that are essential for the virulence of the SARS coronavirus. Structural and mechanistic information will be critical to the structure-based design efforts of the overall Program Project that has the goal of designing potent inhibitors of SCLpro and PLpro that will eventually be developed as therapeutic drugs. The specific aims of Project 2 are: (1) To determine the kinetic and chemical mechanisms of SARS SCLpro and PLpro as well as the substrate specificity of these enzymes using steady-state kinetics and site-directed mutagenesis approaches. (2) To determine the x-ray crystal structures of wild-type and mutant SARS SCLpro enzyme in complex with various substrates and inhibitors. (3) To crystallize and determine the x-ray structure of the SARS PLpro enzyme; and (4) develop novel high-throughput fluorescence screening (HTS) assays for SCLpro and PLpro proteinases that will allow for rapid screening of potential inhibitors from libraries of thousands of compounds. Once potent inhibitors are developed via collaboration with Project 3, we will then in collaboration with Project 1, use molecular biology approaches and x-ray crystallography in an attempt to identify the amino acids in the SARS SCLpro and PLP2 sequences that could give rise to drug resistance via mutation. We have already cloned and over-expressed active constructs of both SCLpro and PLpro, and we have crystallized and determined the x-ray structures of SCLpro in complex with three inhibitors, synthesized by Project 3, that inhibit SARS-CoV replication in vivo. We have developed a continuous fluorescence assay for SCLpro that will allow us to conduct our steady-state kinetic studies, and we have established a collaboration with an industrial partner, PharmOptima LLC, who will help us more fully develop and optimize our fluorescence assays so that we can rapidly screen large compound libraries. The results from our proposed experiments will provide important information to the Program Project that will be crucial for the development of novel inhibitors that may be used as therapeutic agents to reduce SARS-CoV replication and pathogenesis.

项目2的主要目标是使用机械酶学和X射线结晶学来了解两种对SARS冠状病毒毒力至关重要的蛋白酶SCLPro和PLPro的结构和功能。结构和机械信息将是整个计划项目基于结构的设计工作的关键，该计划的目标是设计最终将被开发为治疗药物的SCLPro和PLPro的有效抑制剂。项目2的具体目标是：(1)利用稳态动力学和定点突变方法，确定SARS、SCLPro和PLPro的动力学和化学机制以及这些酶的底物专一性。(2)测定野生型和突变型SARS SCLPro酶与不同底物和抑制剂形成的复合体的X射线晶体结构。(3)结晶并确定SARS PLPro酶的X射线结构；以及(4)开发SCLPro和PLPro蛋白酶的新型高通量荧光筛选(HTS)分析方法，这将使人们能够从数千种化合物库中快速筛选潜在的抑制剂。一旦通过与项目3的合作开发出有效的抑制剂，我们将与项目1合作，使用分子生物学方法和X射线结晶学，试图识别SARS SCLpro和PLP2序列中可能通过突变导致耐药性的氨基酸。我们已经克隆并过度表达了SCLPro和PLPro的活性结构，我们已经结晶和 测定了由Project 3合成的三种抑制SARS-CoV体内复制的抑制剂与SCLpro络合物的X射线结构。我们已经为SCLpro开发了一种连续荧光分析，这将使我们能够进行稳态动力学研究，我们还与工业合作伙伴PharmOptima LLC建立了合作关系，后者将帮助我们更全面地开发和优化我们的荧光分析，以便我们能够快速筛选大型化合物文库。我们建议的实验结果将为该计划项目提供重要信息，这将对开发新的抑制剂至关重要，这些抑制剂可能被用作治疗药物，以减少SARS-CoV的复制和发病。