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13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。在一个案例中，我们已经开始了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，我们正在尝试开发YopH的小分子抑制剂。到目前为止，我们已经确定了几种抑制YopH的化合物，其IC50值在低微摩尔范围内，但事实证明，很难将这些化合物与酶共结晶。然而，我们已经确定了YopH PTPase与不可水解的六肽底物类似物复合物的高分辨率结构（1.5），为我们开发抑制剂提供了另一个起点。目前正在采用几种新方法（2009财年）来寻找YopH抑制剂。其中包括构建化合物文库，其中各种官能团连接到模拟磷酸酪氨酸，底物辅助筛选（SAS），以及用类似药物的“片段”鸡尾酒浸泡YopH晶体以识别弱结合物以进一步优化。我们最近扩大了结构研究的目标范围，包括来自其他潜在生物恐怖主义病原体的毒力因子，包括大天花病毒、土拉菌弗朗西斯菌（土拉菌病的病原体）和委内瑞拉马脑炎病毒（VEEV）。这三种来源的潜在分子靶标的晶体结构最近已被确定。其中一个靶点是由天花病毒编码的双特异性H1磷酸酶，目前是另一个基于结构的药物开发项目的重点。我们最近从我们的片段鸡尾酒中获得了这种分子靶点与两种不同小分子复合物的共晶结构（FY2009）。这两种配体都结合在酶的活性位点上，可能是进一步优化的有希望的线索。由VEEV编码的nsp2蛋白酶也是治疗性抗病毒药物的潜在靶点。我们已经结晶了蛋白酶的催化结构域并确定了其结构（FY2009）。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。因此，人们正在努力修改酶的氨基酸序列，以改善晶体的性质。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13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。在一个案例中，我们已经开始了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，我们正在尝试开发YopH的小分子抑制剂。到目前为止，我们已经确定了几种抑制YopH的化合物，其IC50值在低微摩尔范围内，但事实证明，很难将这些化合物与酶共结晶。然而，我们已经确定了YopH PTPase与不可水解的六肽底物类似物复合物的高分辨率结构（1.5），为我们开发抑制剂提供了另一个起点。目前正在采用几种新方法（2009财年）来寻找YopH抑制剂。其中包括构建化合物文库，其中各种官能团连接到模拟磷酸酪氨酸，底物辅助筛选（SAS），以及用类似药物的“片段”鸡尾酒浸泡YopH晶体以识别弱结合物以进一步优化。我们最近扩大了结构研究的目标范围，包括来自其他潜在生物恐怖主义病原体的毒力因子，包括大天花病毒、土拉菌弗朗西斯菌（土拉菌病的病原体）和委内瑞拉马脑炎病毒（VEEV）。这三种来源的潜在分子靶标的晶体结构最近已被确定。其中一个靶点是由天花病毒编码的双特异性H1磷酸酶，目前是另一个基于结构的药物开发项目的重点。我们最近从我们的片段鸡尾酒中获得了这种分子靶点与两种不同小分子复合物的共晶结构（FY2009）。这两种配体都结合在酶的活性位点上，可能是进一步优化的有希望的线索。由VEEV编码的nsp2蛋白酶也是治疗性抗病毒药物的潜在靶点。我们已经结晶了蛋白酶的催化结构域并确定了其结构（FY2009）。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。因此，人们正在努力修改酶的氨基酸序列，以改善晶体的性质。

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 13 novel structures and are in the process of solving more of them, including several protein-protein complexes. In one case, we have already begun 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 are attempting to develop small molecule inhibitors of YopH. Thus far we have identified several compounds that inhibit YopH with IC50 values in the low micromolar range, but it has proven very difficult to co-crystallize these compounds with the enzyme. However, we have determined a high-resolution structure (1.5 ) of the YopH PTPase in complex with a nonhydrolyzable hexapeptide substrate analog, providing us with yet another starting point for the development of inhibitors. Several new approaches are currently being employed (FY2009) in the search for YopH inhibitors. These include the construction of libraries of compounds in which various functional groups are tethered to a phosphotyrosyl mimetic, substrate-assisted screening (SAS), and soaking YopH crystals with cocktails of drug-like "fragments" to identify weak binders for further optimization. We have recently expanded our range of targets for structural studies to include virulence factors from other potential agents of bioterrorism, including the variola major (smallpox) virus, Francisella tularensis, the causative agent of tularemia, and Venezuelan Equine Encephalitis Virus (VEEV). Crystal structures of potential molecular targets from all three of these sources have recently been determined. One target, the dual specificity H1 phosphatase encoded by smallpox virus, is currently the focus of another structure-based drug development project. We have recently obtained co-crystal structures of this molecular target in complex with two different small molecules from our fragment cocktails (FY2009). Both ligands are bound in the active site of the enzyme and may be promising leads for further optimization. The nsp2 protease encoded by VEEV is also a potential target for therapeutic antiviral agents. We have crystallized the catalytic domain of the protease and determined its structure (FY2009). However, for a variety of reasons, the crystals we obtained are less than ideal for a drug development project. Accordingly, efforts are underway to modify the amino acid sequence of the enzyme in order to improve the properties of the crystals.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 13 novel structures and are in the process of solving more of them, including several protein-protein complexes. In one case, we have already begun 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 are attempting to develop small molecule inhibitors of YopH. Thus far we have identified several compounds that inhibit YopH with IC50 values in the low micromolar range, but it has proven very difficult to co-crystallize these compounds with the enzyme. However, we have determined a high-resolution structure (1.5 ) of the YopH PTPase in complex with a nonhydrolyzable hexapeptide substrate analog, providing us with yet another starting point for the development of inhibitors. Several new approaches are currently being employed (FY2009) in the search for YopH inhibitors. These include the construction of libraries of compounds in which various functional groups are tethered to a phosphotyrosyl mimetic, substrate-assisted screening (SAS), and soaking YopH crystals with cocktails of drug-like "fragments" to identify weak binders for further optimization. We have recently expanded our range of targets for structural studies to include virulence factors from other potential agents of bioterrorism, including the variola major (smallpox) virus, Francisella tularensis, the causative agent of tularemia, and Venezuelan Equine Encephalitis Virus (VEEV). Crystal structures of potential molecular targets from all three of these sources have recently been determined. One target, the dual specificity H1 phosphatase encoded by smallpox virus, is currently the focus of another structure-based drug development project. We have recently obtained co-crystal structures of this molecular target in complex with two different small molecules from our fragment cocktails (FY2009). Both ligands are bound in the active site of the enzyme and may be promising leads for further optimization. The nsp2 protease encoded by VEEV is also a potential target for therapeutic antiviral agents. We have crystallized the catalytic domain of the protease and determined its structure (FY2009). However, for a variety of reasons, the crystals we obtained are less than ideal for a drug development project. Accordingly, efforts are underway to modify the amino acid sequence of the enzyme in order to improve the properties of the crystals.