STRUCTURAL MECHANISM OF THE T GONDII UPRT, A TARGET FOR STRUCTURAL-BASED DRUG D

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。布伦南实验室的主要研究重点是了解蛋白质的三维结构与其生化和生物功能之间的关系。这些方法正被用来研究转录调控、蛋白质-核酸相互作用、多药识别和结合以及原生动物中嘌呤和嘧啶的挽救机制。我们还对基于结构的药物设计感兴趣。一种感兴趣的蛋白质是来自机会性寄生原虫刚地弓形虫的尿嘧啶磷酸核糖转移酶(UPRT)，它催化尿嘧啶向D-磷酸核糖焦磷酸(PRPP)的转移，释放焦磷酸并产生核苷酸一磷酸UMP。回收的UMP可用于多种代谢途径。在我们试图发现治疗这种机会主义寄生虫的新药时，这种酶是一个非常好的靶点。必须充分了解UPRT的底物识别和催化机理。我们实验室以前的研究已经为UPRT的完整结构和机械特性奠定了基础，我们正在开展下一组关键研究。具体地说，我们正在改进apo UPRT的超高分辨率(1.05？分辨率)结构，以及一些关键的二元和三元络合物的高分辨率结构。同时，我们正在对她的野生型蛋白和一些定点突变进行彻底的动力学评估。最后，这些信息开始开发结构参数，这些参数将指导我们未来的药物设计尝试。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The major research focus of the Brennan Laboratory is to understand the relationship between the 3-D structures of proteins and their biochemical and biological functions. These approaches are being used to study transcription regulation, protein-nucleic acid interaction, multidrug recognition and binding and the mechanisms of purine and pyrimidine salvage in protozoa. Our interest is also directed in structure-based drug design. One protein of interest is the uracil phosphoribosyltransferase (UPRT) from the opportunistic parasitic protozoan, Toxoplasma gondii, which catalyzes the transfer of uracil to D-phosphoribosyl pyrophosphate (PRPP) releasing pyrophosphate and creating the nucleotide monophosphate UMP. The salvaged UMP can then be used in a number of metabolic pathways. This enzyme presents a very good target to exploit in our attempts to discover new drugs against this opportunistic parasite. The substrate recognition and catalytic mechanisms of UPRT must be understood fully. Previous studies from our lab have set the groundwork for the complete structural and mechanistic characterization of UPRT and we are carrying out the next key set of studies. Specifically, we are working on the refinement of the ultra high resolution (1.05 ¿¿resolution) structure of apo UPRT and the high resolution structures of a number of key binary and ternary complexes. In parallel, we are carrying out a thorough kinetic evaluation of her wild type protein and a number of site directed mutants. Finally, this information starts to develop the structural parameters that will guide our future drug design attempts.