Mechanistic & Crystallographic Studies of SARS Proteases

项目2的主要目标是利用机械酶学和X射线晶体学来了解两种蛋白酶SCLpro和PLpro的结构和功能，这两种蛋白酶对SARS冠状病毒的毒力至关重要。结构和机制信息对于整个计划项目的基于结构的设计工作至关重要，该计划项目的目标是设计最终将被开发为治疗药物的SCLpro和PLpro的强效抑制剂。项目2的具体目标是：（1）使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特异性。(2)确定野生型和突变型SARS SCLpro酶与各种底物和抑制剂复合物的X射线晶体结构。(3)结晶和确定SARS PLpro酶的X射线结构;（4）开发新的SCLpro和PLpro蛋白酶的高通量荧光筛选（HTS）分析，这将允许从数千种化合物的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射线晶体学，试图确定SARS SCLpro和PLP 2序列中可能通过突变引起耐药性的氨基酸。我们已经克隆并过表达了SCLpro和PLpro的活性构建体，并且我们已经结晶并 确定了SCLpro与项目3合成的三种抑制剂复合物的X射线结构，这些抑制剂抑制SARS-CoV在体内的复制。我们已经开发了一种用于SCLpro的连续荧光测定法，这将使我们能够进行稳态动力学研究，我们已经与工业合作伙伴PharmOptima LLC建立了合作关系，他们将帮助我们更全面地开发和优化我们的荧光测定法，以便我们能够快速筛选大型化合物库。从我们提出的实验结果将提供重要的信息，该计划的项目，将是至关重要的新的抑制剂，可用作治疗剂，以减少SARS冠状病毒的复制和发病机制的发展。

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.