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的主要目的是使用机械酶学和X射线晶体学来了解两个蛋白酶SCLPRO和PLPRO的结构和功能，这对于SARS冠状病毒的毒力至关重要。结构和机械信息对于总体计划项目的基于结构的设计工作至关重要，该项目的目的是设计SCLPRO和PLPRO的有效抑制剂，最终将作为治疗药物开发。项目2的具体目的是：（1）确定SARS SCLPRO和PLPRO的动力学和化学机制，以及这些酶的底物特异性，并使用稳态动力学和定位定向的诱变方法来确定这些酶的底物特异性。 （2）确定与各种底物和抑制剂复合物中野生型和突变体SARS Sclpro酶的X射线晶体结构。 （3）结晶并确定SARS PLPRO酶的X射线结构； （4）开发用于SCLPRO和PLPRO蛋白酶的新型高通量荧光筛选（HTS）测定法，可以快速筛选从数千种化合物文库中的潜在抑制剂。一旦通过与项目3的合作开发了有效的抑制剂，我们将与项目1合作，使用分子生物学方法和X射线晶体学，以尝试鉴定SARS SCLPRO和PLP2序列中的氨基酸和PLP2序列，从而通过突变引起药物抗性。我们已经克隆和表达了Sclpro和PLPRO的主动构建体，我们已经结晶了，并且 确定了由项目3合成的三个抑制剂中SCLPRO的X射线结构，这些抑制剂在体内抑制了SARS-COV复制。我们已经为SCLPRO开发了一种连续的荧光测定法，这将使我们能够进行稳态动力学研究，并与工业合作伙伴Pharmoptima LLC建立了合作，该合作伙伴将帮助我们更全面地开发和优化荧光测定法，以便我们可以快速筛选大型化合物。我们提出的实验的结果将为程序项目提供重要信息，这对于新型抑制剂的发展至关重要，这些抑制剂可能用作减少SARS-COV复制和发病机理的治疗剂。