Anthrax Toxin Lethal Factor Inhibition Study

描述（申请人提供）：炭疽毒素致死因子（LF）酶直接使炭疽芽孢杆菌逃避宿主免疫机制，导致循环休克和死亡。在炭疽感染的任何阶段，很少（如果有的话）可用的治疗方案来对抗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，并在此基础上开发新的相关生化工具，用于确定与lf相关的细胞死亡和组织损伤的机制。在这个特殊的应用目的是评估新的，有前途的分子支架的功效，以对抗lf介导的毒性。核心假设是，在体外和基于细胞的实验中，与新的谷氨酸支架结构相关的小分子将有效抑制LF蛋白酶的活性。提出这项研究的基本原理是，提高对LF抑制的关键结构特征的理解，将反过来导致对炭疽发病机制中毒素参与的理解，并可能为治疗暴露后炭疽提供有前途的策略。因此，拟议的研究与NIH的使命有关，即发展基础知识，这些知识可以应用于显著提高国家保护和改善健康的能力。在强有力的初步数据指导下，中心假设将通过以下四个具体目标进行验证：1)设计、合成和评估新的针对炭疽毒素致死因子活性位点的谷氨酸文库；2)通过高通量筛选确定LF探针设计的新支架；3)鉴定具有保护巨噬细胞免受lf介导的细胞毒性能力的探针化合物；4)通过x射线晶体学鉴定LF酶活性位点的关键结合模式。在第一个目标下，围绕候选支架的文库将在计算机上生成；化合物将使用分子建模进行优先排序；选定的化合物将被合成并评估其体外抗LF活性。在第二个目标中，将进行大规模的高通量筛选，采用新颖的分类和触及探针技术，以确定用于探针设计的其他潜在支架；从hit支架中提纯将遵循设计、合成、纯化和筛选的迭代、系统循环。在第三个目标中，将对探针进行测试，以评估其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功效；在第四个目标中，优先化合物将与LF共结晶，以便实验鉴定关键的LF配体-受体相互作用。该方法具有创新性，因为它针对的是尚未被充分研究的特定LF结构特征，并结合了最近开发的、尚未应用于LF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，并以此推进和扩大对炭疽毒血症复杂机制的理解。公共卫生相关性：拟议的工作涉及炭疽发病机制的重要和未充分研究的分子结构和机制。拟议的研究与公共卫生有关，因为尽管武器化炭疽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可以对抗lf介导的细胞死亡，这项工作的发现最终有望指导设计有效的治疗方法，以帮助那些在紧急情况下接触过或怀疑接触过炭疽孢子的人。

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.