Structural Proteomics of the Yersinia Yop Virulon

我们成功地生产了大量结晶级蛋白质，这导致了一个小规模的结构基因组学项目，旨在解决鼠疫病原体鼠疫杆菌III型分泌物中涉及的蛋白质的三维结构。由于III型分泌系统(T3SS)对毒力是必不可少的，由此产生的结构信息可以用来开发有效的对策来对付这种潜在的生物恐怖主义病原体。我们已经解决了15个新结构，并正在解决更多的新结构，包括几个蛋白质-蛋白质复合体。然而，由于各种原因，这个项目的结构基因组学方面正在被逐步淘汰。我们目前的重点已经转移到结构辅助药物开发的过程中。耶尔森氏菌通过T3SS注入哺乳动物细胞的细胞毒效应蛋白之一YopH是一种有效的真核样蛋白酪氨酸磷酸酶(PTPase)。YopH使真核细胞中与局部黏附相关的几种蛋白质去磷酸化，从而使细菌能够避免巨噬细胞的吞噬和破坏。与小特伦斯·伯克博士合作。在药物化学实验室(CCR)和Robert Ulrich博士(USAMRIID)的共同努力下，我们最近开发出一种高度特异和有效的YopH抑制剂，它非混杂、对哺乳动物细胞无毒，并且非常有效地杀灭巨噬细胞中的细菌。目前正在计划在鼠疫感染的动物模型中测试这种化合物。委内瑞拉马脑炎病毒(VEEV)编码的NSP2蛋白是治疗抗病毒药物的潜在靶点。我们对该酶的催化域进行了结晶，并确定了其结构。然而，由于各种原因，我们获得的晶体不太适合药物开发项目。最近，我们设计了一种新的酶晶体形式，通过表面熵还原突变，更适合于药物开发工作，并以1.3埃的分辨率确定了其结构。我们目前正在进行计算机筛选，以确定与酶共结晶的铅分子，并进一步优化。

Our success in producing large quantities of crystallization-grade proteins led to a small-scale structural genomics project aiming to solve the three-dimensional structures of proteins involved in Type III secretion in Yersinia pestis, the causative agent of plague. Because the Type III secretion system (T3SS) is essential for virulence, the resulting structural information could be used to develop effective countermeasures for this potential agent of bioterrorism. We have already solved 15 novel structures and are in the process of solving more of them, including several protein-protein complexes. However, the structural genomics aspect of this project is being phased out for a variety of reasons. Our current focus has shifted o the process of structure-assisted drug development. One of the cytotoxic effector proteins that Yersinia injects into mammalian cells via the T3SS, YopH, is a potent eukaryotic-like protein tyrosine phosphatase (PTPase). YopH dephosphorylates several proteins associated with the focal adhesion in eukaryotic cells, thereby enabling the bacterium to avoid phagocytosis and destruction by macrophages. In collaboration with Dr. Terrence Burke Jr. (Laboratory of Medicinal Chemistry, CCR) and Dr. Robert Ulrich (USAMRIID), we have recently developed a highly specific and potent inhibitor of YopH that is non promiscuous, nontoxic to mammalian cells and very effective at killing the bacterium in macrophages. Plans are underway to test this compound in animal models of plague infection. The nsp2 protease encoded by Venezuelan Equine Encephalitis virus (VEEV) is a potential target for therapeutic antiviral agents. We crystallized the catalytic domain of the protease and determined its structure. However, for a variety of reasons, the crystals we obtained were less than ideal for a drug development project. Recently we have engineered a new crystal form of the enzyme, by surface entropy reduction mutagenesis, that is much better suited for drug development efforts and determined its structure at a resolution of 1.3 Angstroms. We are currently performing in silico screening to identify lead molecules for co-crystallization with the enzyme and further optimization.