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13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。在一种情况下，我们已经开始了结构辅助药物开发的过程。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），我们正试图开发YopH的小分子抑制剂。到目前为止，我们已经鉴定了几种抑制YopH的化合物，其IC 50值在低微摩尔范围内，但是已经证明很难将这些化合物与酶共结晶。然而，我们已经确定了高分辨率的结构与不可水解的六肽底物类似物的复合物的YopH PTARY，为我们提供了另一个起点的抑制剂的发展。目前正在采用几种新的方法寻找YopH抑制剂。这些包括构建化合物库，其中各种官能团与磷酸酪氨酰模拟物连接，底物辅助筛选（SAS），以及用药物样“片段”的鸡尾酒浸泡YopH晶体以识别弱结合剂以进一步优化。我们最近扩大了结构研究的目标范围，包括来自其他生物恐怖主义潜在因子的毒力因子，包括天花病毒、土拉热弗朗西斯菌（土拉菌病的病原体）和委内瑞拉马脑炎病毒（VEEV）。最近已经确定了所有这三种来源的潜在分子靶点的晶体结构。其中一个靶点，即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。我们最近从我们的碎片鸡尾酒中获得了这种分子靶标与两种不同小分子复合的共晶结构（2009）。这两种配体都结合在酶的活性位点，可能是有前途的线索，进一步优化。VEEV编码的nsp 2蛋白酶也是治疗性抗病毒剂的潜在靶标。我们已经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。因此，正在努力修饰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 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 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 (2009). 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. 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.