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，旨在解决鼠疫病原体鼠疫耶尔森菌III型分泌相关蛋白质的三维结构。由于III型分泌系统（T3SS）对毒力至关重要，因此所得的结构信息可用于制定有效的对策，以应对这种潜在的生物恐怖主义制剂。我们已经解决了15种新的结构，并且正在解决更多的结构，包括几种蛋白质-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退出。我们目前的重点已经转移到结构辅助药物开发的过程。耶尔森菌通过T3SS注入哺乳动物细胞的细胞毒性效应蛋白之一YopH是一种强效的真核样蛋白酪氨酸磷酸酶（PTPase）。YopH使真核细胞中与局灶黏附相关的几种蛋白去磷酸化，从而使细菌能够避免巨噬细胞的吞噬和破坏。在与Terrence Burke Jr.博士（药物化学实验室，CCR）和Robert Ulrich博士（USAMRIID）的合作下，我们最近开发了一种高度特异性和有效的YopH抑制剂，它是非混合的，对哺乳动物细胞无毒，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nsp2蛋白酶是治疗性抗病毒药物的潜在靶点。我们结晶了蛋白酶的催化结构域并确定了它的结构。然而，由于种种原因，我们获得的晶体对于药物开发项目来说并不理想。最近，我们通过表面熵减少诱变设计了一种新的酶晶体形式，这种形式更适合药物开发工作，并以1.3埃的分辨率确定了其结构。我们目前正在进行硅筛选，以确定与酶共结晶和进一步优化的铅分子。

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.