Anthrax Toxin Lethal Factor Inhibition Study

描述(申请人提供)：炭疽毒素致死因子(LF)酶直接使炭疽杆菌逃避宿主免疫机制，导致循环系统休克和死亡。在炭疽感染的任何阶段，几乎没有可用的治疗选择来对抗LF介导的细胞毒性。长期目标是更好地了解炭疽毒素的作用机制，并在此基础上开发可用于确定与LF相关的细胞死亡和组织损伤机制的新的和相关的生化工具。这一特殊应用的目的是评估新的、有希望的分子支架对抗LF介导的毒性的有效性。中心假设是，在体外和基于细胞的检测中，与新型谷氨酸为基础的支架结构相关的小分子将有效地抑制LF蛋白酶的活性。这项拟议研究的基本原理是，对有助于抑制LF的关键结构特征的更好理解将反过来导致对毒素参与炭疽发病机制的更好理解，并可能为治疗暴露后炭疽病提供一种有前途的策略。因此，拟议的研究与NIH任务中与开发基础知识相关的部分相关，这些基础知识可用于显著提高国家保护和改善健康的能力。在强劲的初步数据指导下，中心假设将通过追求四个具体目标来检验：1)设计、合成和评估针对炭疽毒素致命因子活性部位的新的谷氨酸文库；2)通过高通量筛选为LF探针设计寻找新的支架；3)鉴定能够保护巨噬细胞免受LF介导的细胞毒的探针化合物；以及4)通过X射线结晶学的手段确定与LF酶活性部位的关键结合模式。在第一个目标下，将在电子计算机中生成候选支架周围的库；将使用分子建模对化合物进行优先排序；并将合成选定的化合物，并在体外评估其对LF的活性。在第二个目标中，将进行大规模的高通量筛选，采用新颖的分诊和命中到探针技术，以确定更多用于探针设计的潜在支架；从命中支架提纯将遵循设计、合成、纯化和筛选的迭代、系统循环。在第三个目标中，将测试探针以评估它们在抑制小鼠巨噬细胞系对炭疽毒素反应的细胞死亡方面的相对保护效果；在第四个目标中，优先化合物将与LF共结晶，以实验确定关键的LF配体-受体相互作用。这种方法是创新的，因为它针对尚未充分研究的特定低频结构特征，并结合了最近开发的、尚未应用于低频系统的高精度计算和实验技术。这项拟议的研究意义重大，因为它有望为导致LF相关细胞死亡的途径提供进一步的分子见解，并通过这样做，促进和扩大对炭疽毒素血症涉及的复杂机制的理解。公共卫生相关性：拟议的工作涉及重要的和未被充分研究的分子结构和机制，这些分子结构和机制有助于炭疽病的发病。这项拟议的研究与公共健康相关，因为尽管武器化的炭疽病继续对社会构成威胁，但目前市场上还没有有效的治疗方法来对抗LF介导的细胞死亡，这项工作的发现最终有望指导有效疗法的设计，这些疗法可以在紧急情况下帮助那些接触或怀疑接触炭疽孢子的人。

DESCRIPTION (provided by applicant): The anthrax toxin lethal factor (LF) enzyme directly enables the Bacillus anthracis bacterium to evade host immunological mechanisms, leading to circulatory shock and death. Few, if any, therapeutic options are available to counteract LF-mediated cytotoxicity at any stage of anthrax infection. The long-term goal is to better understand the mechanisms by which anthrax toxins act, and, based upon that knowledge, to develop novel and relevant biochemical tools that can be utilized to define mechanisms of LF-related cell death and tissue damage. The objective in this particular application is to evaluate new, promising molecular scaffolds for efficacy against LF-mediated toxicity. The central hypothesis is that small molecules structurally related to a new glutamic acid-based scaffold will effectively inhibit LF protease activity in vitro and in cell-based assays. The rationale for the proposed research is that an improved understanding of key structural features that contribute to LF inhibition will, in turn, lead to an improved understanding of toxin involvement in anthrax pathogenesis, and will potentially provide a promising strategy for treating postexposure anthrax. Thus, the proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that can be applied to advance significantly the Nation's capacity to protect and improve health. Guided by strong preliminary data, the central hypothesis will be tested by pursuing four specific aims: 1) Design, synthesize and evaluate new glutamic acid-based libraries targeting the anthrax toxin lethal factor active site; 2) Identify new scaffolds for LF probe design using high-throughput screening; 3) Identify probe compounds with capacity to protect macrophages against LF-mediated cytotoxicity; and 4) Identify key binding modes to the LF enzyme active site by means of X-ray crystallography. Under the first aim, libraries around candidate scaffolds will be generated in silico; compounds will be prioritized using molecular modeling; and selected compounds will be synthesized and evaluated for activity against LF in vitro. In the second aim, a large-scale high-throughput screen with novel triage and hit-to-probe techniques will be conducted to identify additional potential scaffolds for probe design; refinement from hit scaffolds will follow an iterative, systematic cycle of design, synthesis, purification, and screening. In the third aim, probes will be tested to assess their relative protective efficacy in inhibiting cell death of a murine macrophage cell line in response to anthrax toxin; and in the fourth aim, prioritized compounds will be co-crystallized with LF in order to experimentally identify key LF ligand-receptor interactions. The approach is innovative, because it targets specific LF structural features that have not been fully investigated, and incorporates recently developed, highly accurate computational and experimental techniques not yet applied to the LF system. The proposed research is significant, because it is expected to provide further molecular insights into the pathways leading to LF-related cell death, and in so doing, to advance and expand understanding of the complex mechanisms involved in anthrax toxemia. PUBLIC HEALTH RELEVANCE: The proposed work addresses important and under-investigated molecular structures and mechanisms that contribute to anthrax pathogenesis. The proposed research has relevance to public health, because although weaponized anthrax continues to pose a threat to society, there is currently no effective therapeutic on the market that can counteract LF-mediated cell death, and the findings from this work are ultimately expected to guide the design of effective therapeutics that can aid persons who have been, or suspect they may have been, exposed to anthrax spores in an emergency situation.