Structural Proteomics of the Yersinia Yop Virulon

我们在生产大量结晶级蛋白质方面的成功导致了一个小规模的结构基因组学项目，旨在解决鼠疫耶尔森氏菌（鼠疫的病原体）中参与III型分泌的蛋白质的三维结构。由于III型分泌系统（T3 SS）是必不可少的毒力，由此产生的结构信息可以用来制定有效的对策，这种潜在的生物恐怖主义的代理。我们已经解决了15个新的结构，并正在解决更多的问题，包括几个蛋白质-蛋白质复合物。然而，由于各种原因，该项目的结构基因组学方面正在逐步淘汰。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T3 SS注入哺乳动物细胞的细胞毒性效应蛋白之一YopH是一种有效的真核生物样蛋白酪氨酸磷酸酶（PTP 3）。YopH使真核细胞中与粘着斑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Terrence Burke Jr博士合作。（药物化学实验室，CCR）和Robert Ulrich博士（USAMRIID），我们最近开发了一种高度特异性和有效的YopH抑制剂，该抑制剂对哺乳动物细胞无毒性，并且非常有效地杀死巨噬细胞中的细菌。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nsp 2蛋白酶是治疗性抗病毒药物的潜在靶标。我们结晶的蛋白酶的催化结构域，并确定其结构。然而，由于各种原因，我们获得的晶体对于药物开发项目来说并不理想。最近，我们已经设计了一种新的晶体形式的酶，通过表面熵减少诱变，这是更好地适合于药物开发的努力，并确定其结构的分辨率为1.3埃。我们目前正在进行计算机筛选，以确定与酶共结晶的先导分子，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.