Structural Proteomics of the Yersinia Yop Virulon

鼠疫耶尔森氏菌是鼠疫的病原体，被认为是最有可能发动生物恐怖主义的工具之一。像许多革兰氏阴性细菌病原体一样，鼠疫杆菌利用III型(接触依赖型)分泌系统(TTSS)将细胞毒效应蛋白直接注入真核细胞，在那里它们干扰调节炎症和细胞骨架动力学的信号通路，从而使细菌能够避免被巨噬细胞和其他专业吞噬细胞吞噬和破坏。我们的目标是通过试图解决与鼠疫杆菌TTSS相关的关键毒力因子的三维结构来促进抗鼠疫治疗的发展，该毒力因子被称为YOP(耶尔森氏菌外蛋白)毒力因子。由于这些“分子恐怖分子”在致病机制中发挥了关键作用，因此很可能很难(如果不是不可能)设计出对抗病毒药物有抗药性但仍能致病的鼠疫杆菌的武器化变种。 TTSS由大约50种不同的蛋白质组成，几乎任何一种蛋白质的零突变都会消除毒力。因此，几乎所有这些蛋白质都是抗鼠疫治疗的潜在分子靶点。我们的方法是基因组驱动的、自下而上的目标选择方法。我们认为，当结构生物学和筛选方法可以结合使用时，药物发现过程最有效，我们的战略是首先确定哪些潜在靶点适合基于结构的方法，然后根据目前已知的生物学功能和作用机制对它们进行优先排序。在过去的四年中，已经确定了五种新的结构：YopM，一种功能未知的细胞毒剂；YopE，一种针对RhoA，rac1和CDc42的GTPase激活蛋白；SycE，YopE的同源分泌伴侣；YopH的N-末端结构域，一个依赖磷酸酪氨酸的蛋白质-蛋白质相互作用模块；以及LcrV(V抗原)，一种保护性抗原和III型分泌的关键调节因子。 从历史上看，酶一直是基于结构的药物设计最成功的治疗靶点。蛋白酪氨酸磷酸酶(PTPase)是一种信号酶，参与调节多种细胞功能，包括生长、有丝分裂、运动、细胞-细胞相互作用、新陈代谢、基因转录和免疫反应。由于PTPase的异常作用与癌症、糖尿病、骨质疏松症和免疫功能障碍等衰弱疾病有关，因此PTPase抑制剂的开发是制药行业非常活跃的研究领域。事实上，已经描述了一些哺乳动物PTPase的高度有效和特定的抑制剂，帮助建立了这一概念的原则证明。鼠疫杆菌通过TTSS将细胞毒效应蛋白YopH注入哺乳动物细胞，YopH是一种有效的类真核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。因为YopH的PTPase活性对毒力是必不可少的，而且这种酶很容易结晶，我们认为它是一个特别有希望的治疗干预靶点。因此，我们正在与特伦斯·伯克博士(药物化学实验室，NCI)合作，开发YopH PTPase抑制剂的抑制剂。我们的战略需要筛选Burke博士大量的PTPase抑制剂，以确定对YopH具有合理效力的先导化合物，将这些化合物与酶共结晶，然后利用产生的结构信息，通过基于结构的药物设计的迭代循环来提高抑制剂的效力。已鉴定了几种IC50值在一位数微摩尔范围内的化合物，并确定了YopH与非水解性六肽和三肽底物类似物的高分辨结构。

Yersinia pestis, the causative agent of plague, is considered to be one of the most likely instruments of bioterrorism. Like many Gram-negative bacterial pathogens, Y. pestis utilizes a type III (contact dependent) secretion system (TTSS) to inject cytotoxic effector proteins directly into eukaryotic cells where they interfere with signaling pathways that regulate inflammation and cytoskeleton dynamics, thereby enabling the bacteria to avoid engulfment and destruction by macrophages and other professional phagocytes. Our objective is to facilitate the development of anti-plague therapeutics by attempting to solve the three-dimensional structures of key virulence factors associated with the TTSS in Y. pestis, which has been termed the Yop (Yersinia outer protein) virulon. Because these "molecular terrorists" play a critical role in pathogenesis, it is likely to be difficult if not impossible to engineer weaponized variants of Y. pestis that are resistant to antivirulence drugs but that can still cause disease. The TTSS is comprised of approximately 50 different proteins, and a null mutation in almost any of them abrogates virulence. Accordingly, nearly all of these proteins are potential molecular targets for anti-plague therapeutics. Ours is a genome-driven, bottom-up approach to target selection. Believing that the drug discovery process works most effectively when structural biology and screening approaches can be used in concert, our strategy is first to determine which potential targets are amenable to structure-based approaches and then prioritize them on the basis of what is currently known about their biological functions and mechanisms of action. Five novel structures have been determined during the last four years: YopM, a cytotoxic agent of unknown function; YopE, a GTPase activating protein that specifically targets RhoA, Rac1 and Cdc42; SycE, the cognate secretion chaperone for YopE; the N-terminal domain of YopH, a phosphotyrosine-dependent protein-protein interaction module; and LcrV (V antigen), a protective antigen and key regulator of type III secretion. Historically, enzymes have been the most successful therapeutic targets for structure-based drug design. Protein Tyrosine Phosphatases (PTPases) are signaling enzymes that participate in the regulation of numerous cellular functions, including growth, mitogenesis, motility, cell-cell interactions, metabolism, gene transcription, and the immune response. Because the aberrant action of PTPases has been linked to debilitating diseases like cancer, diabetes, osteoporosis, and immune dysfunctions, the development of PTPase inhibitors is a very active area of research in the pharmaceutical industry. Indeed, highly potent and specific inhibitors of some mammalian PTPases have already been described, helping to establish proof-of-principal for the concept. One of the cytotoxic effector proteins that Y. pestis injects into mammalian cells via the TTSS, YopH, is a potent eukaryotic-like PTPase. YopH dephosphorylates several proteins associated with the focal adhesion in eukaryotic cells. Because the PTPase activity of YopH is essential for virulence and the enzyme crystallizes readily, we view it as a particularly promising target for therapeutic intervention. Accordingly, we are collaborating with Dr. Terrence Burke (Laboratory of Medicinal Chemistry, NCI), a chemist with considerable expertise in the field of PTPase inhibitor design, to develop inhibitors of the YopH PTPase. Our strategy entails screening Dr. Burke's extensive collection of PTPase inhibitors to identify lead compounds with reasonable potency against YopH, cocrystallizing these compounds with the enzyme, and then exploiting the resulting structural information to improve the potency of the inhibitors through iterative cycles of structure-based drug design. Several compounds with IC50 values in the single-digit micromolar range have already been identified, and high-resolution structures of YopH in complex with nonhydrolyzable hexapeptide and tripeptide substrate analogs have been determined.