Structure and Function of Virulence Factors of Bacillus anthracis

炭疽杆菌毒力因子的结构和功能

基本信息

批准号：
8336253
负责人：
Stephen Leppla
金额：
$ 54.84万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8336253
关键词：
Adenylate Cyclase Affinity Animal Model Animals Anthrax disease Antibodies Antigens Bacillus anthracis Bacillus anthracis spore Bacterial Proteins Bacterial Toxins Binding Biochemical Genetics Biotechnology Blood capillaries Body Weight Calcium Calmodulin Cell Surface Receptors Cell surface Cells Cessation of life Chimeric Proteins Cleaved cell Collaborations Colon Carcinoma Complex Cultured Cells Cyclic AMP Cytosol Data Dose Drug or chemical Tissue Distribution Endocytosis Endothelium Extracellular Domain Fc domain Genetic Half-Life Human Immunoglobulin G In Vitro Infection Ion Channel Laboratories Lead Mammalian Cell Metalloproteases Methods Mitogen-Activated Protein Kinase Kinases Modeling Modification Molecular Target Morphogenesis Oryctolagus cuniculus Panthera Peptide Hydrolases Pharmaceutical Preparations Planets Plants Plasma Positioning Attribute Production Property Protease Inhibitor Proteinase-Activated Receptors Proteins Rattus Relative (related person)Research Resistance Role Serum Shock Structure Surface System Testing Therapeutic Agents Time Tissues Tobacco Toxin Tumor Markers Virulence Factors Virulent Weight Work anthrax lethal factor anthrax toxin capillary design edema factor experience follow-up improved in vitro testing in vivo inhibitor/antagonist mutant protective efficacy receptor residence response small molecule tumor

项目摘要

Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and LF and EF translocate to the cytosol. EF is a calcium- and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Entry of anthrax toxin into cells depends on two related cell surface receptors, tumor endothelium marker 8 (TEM8) and capillary morphogenesis gene product 2(CMG2). TEM8 was initially identified as a protein upregulated in colon cancers. CMG2 has substantial sequence similarity to this candidate tumor marker. The tissue distribution and the relative importance of the two toxin receptors in toxin action are not well understood. During 2011 we extended analysis of toxin receptor in a collaboration with Keith Wycoff of Planet Biotechnology, Inc. While antibodies to PA have proven efficacious, an alternative strategy is to create receptor decoys. The CMG2 extracellular domain has high affinity to PA, but it would not be expected to have a long residence time in plasma. To improve its pharmacological properties, this domain was fused to the Fc domain of human IgG. Furthermore, to assure that adequate amounts could be produced, the fusion protein was expressed in plants, specifically tobacco plants. In the best clone, nearly 0.1% of the plant's wet weight was the Fc-CMG2 fusion protein, meaning that almost unlimited amounts of protein could be produced. Administration of the purified protein at 2 mg/kg body weight protected rabbits against a lethal challenge with fully virulent anthrax spores. The plant-produced protein was glycosylated, but the presence of this modification did not alter serum half-life, which was approximately 5 days. These data show that this receptor decoy protein has considerable potential for production as a therapeutic agent for anthrax. In other collaborative research done during 2011, we worked to improve small molecule inhibitors directed to the protease activity of LF. Our colleagues at Panthera, Inc., used an LF inhibitor described by Merck as a point of departure in design of more potent compounds. A large number of compounds were synthesized and tested for in vitro protease inhibition. From among the most potent of these, compounds with properties suggesting they could be useful in vivo as drugs were tested at the LBD for protective efficacy in the rat lethal toxin (PA + LF) challenge model. In this model, rats injected intravenously with lethal toxin experience a rapid and highly reproducible shock response that would lead to death in 60-100 minutes. This system provides a quantitative method for assessing and comparing the in vivo efficacies of compounds. One of these compounds, which is active in vitro at subnanomolar concentrations, provided 100% protection in the rat lethal toxin model. In a subsequent follow-up study, additional compounds were synthesized to examine the roles of substituents at several positions on the core structure. This led to identification of an improved group of compounds having Ki values of 0.1-2.0 nM. The best of these compounds protected rats from lethal toxin challenge at drug doses of only 2.5 mg/kg. These are among the most potent LF protease inhibitors yet described.

炭疽毒素保护性抗原蛋白（PA，83 kDa）与哺乳动物细胞表面的受体结合，被细胞表面蛋白酶弗林蛋白酶切割，然后捕获其他两种毒素蛋白，致死因子（LF，90 kDa）或水肿因子（EF，89 kDa）。 PA-LF 和 PA-EF 复合物通过内吞作用进入细胞，并且 LF 和 EF 易位至胞质溶胶。 EF 是一种钙和钙调蛋白依赖性腺苷酸环化酶，可导致细胞内 cAMP 浓度大幅且不受调节地增加。 LF 是一种金属蛋白酶，可裂解多种丝裂原激活蛋白激酶激酶 (MEK)。炭疽毒素进入细胞取决于两种相关的细胞表面受体，肿瘤内皮标记物8（TEM8）和毛细血管形态发生基因产物2（CMG2）。 TEM8 最初被鉴定为结肠癌中表达上调的蛋白质。 CMG2 与该候选肿瘤标志物具有显着的序列相似性。两种毒素受体在毒素作用中的组织分布和相对重要性尚不清楚。 2011 年，我们与 Planet Biotechnology, Inc. 的 Keith Wycoff 合作扩展了毒素受体的分析。虽然 PA 抗体已被证明有效，但另一种策略是创建受体诱饵。 CMG2 胞外结构域对 PA 具有高亲和力，但预计其在血浆中的停留时间不会很长。为了改善其药理特性，该结构域与人 IgG 的 Fc 结构域融合。此外，为了确保能够产生足够的量，融合蛋白在植物中表达，特别是在烟草植物中。在最好的克隆中，近 0.1% 的植物湿重是 Fc-CMG2 融合蛋白，这意味着可以产生几乎无限量的蛋白质。以 2 毫克/千克体重施用纯化蛋白可以保护兔子免受全毒力炭疽孢子的致命攻击。植物产生的蛋白质被糖基化，但这种修饰的存在并没有改变血清半衰期，大约为 5 天。这些数据表明，这种受体诱饵蛋白具有作为炭疽治疗剂生产的巨大潜力。在 2011 年进行的其他合作研究中，我们致力于改进针对 LF 蛋白酶活性的小分子抑制剂。我们 Panthera, Inc. 的同事使用默克 (Merck) 描述的 LF 抑制剂作为设计更有效化合物的出发点。合成了大量化合物并测试了体外蛋白酶抑制作用。其中最有效的化合物的特性表明它们可能在体内有用，因为在 LBD 中测试了药物在大鼠致死毒素 (PA + LF) 攻击模型中的保护功效。在该模型中，静脉注射致命毒素的老鼠会经历快速且高度可重复的休克反应，在 60-100 分钟内导致死亡。该系统提供了评估和比较化合物体内功效的定量方法。其中一种化合物在体外亚纳摩尔浓度下具有活性，在大鼠致死毒素模型中提供 100% 的保护。在随后的后续研究中，合成了其他化合物来检查核心结构上多个位置的取代基的作用。这导致鉴定出一组改进的 Ki 值为 0.1-2.0 nM 的化合物。这些化合物中最好的药物剂量仅为 2.5 毫克/千克，即可保护大鼠免受致命毒素的攻击。这些是迄今为止描述的最有效的 LF 蛋白酶抑制剂之一。