Structure and Function of Virulence Factors of Bacillus anthracis

炭疽杆菌毒力因子的结构和功能

• 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
• 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

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上的受体结合，被细胞表面蛋白酶呋喃裂解，然后捕获两个其他两种毒素蛋白，致死因子（LF，90 kDa）或Edema因子或Edema因子（EF，89 KDA）。 PA-LF和PA-EF复合物通过内吞作用进入细胞，LF和EF转移到细胞质。 EF是一种钙和钙调蛋白依赖性腺苷酸环化酶，在细胞内cAMP浓度中导致大量和不受监管的增加。 LF是一种金属蛋白酶，可裂解几种有丝分裂原激活的蛋白激酶激酶（MEKS）。 炭疽毒素进入细胞中的进入取决于两个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最初被鉴定为在结肠癌中上调的蛋白质。 CMG2与该候选肿瘤标记具有很大的序列相似性。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。 在2011年期间，我们在与Planet Biotechnology，Inc。的Keith Wycoff合作中扩展了对毒素受体的分析。尽管PA的抗体已被证明有效，但另一种策略是创建受体诱饵。 CMG2细胞外结构域对PA具有很高的亲和力，但预计血浆中的停留时间不会很长。 为了提高其药理特性，该结构域与人IgG的FC结构域融合在一起。此外，为确保可以产生足够的量，融合蛋白在植物（特别是烟草植物）中表达。 在最好的克隆中，植物的湿重近0.1％是FC-CMG2融合蛋白，这意味着几乎可以生产几乎无限量的蛋白质。 用2 mg/kg体重保护兔子的纯化蛋白质保护兔子，以防止用完全毒性炭疽孢子的致命挑战。 植物生产的蛋白是糖基化的，但是这种修饰的存在并没有改变血清半衰期，约为5天。 这些数据表明，该受体诱饵蛋白作为炭疽治疗剂具有很大的生产潜力。 在2011年的其他协作研究中，我们致力于改善针对LF蛋白酶活性的小分子抑制剂。 我们在Panthera，Inc。的同事使用默克描述的LF抑制剂作为设计更有效化合物的设计。 合成了大量化合物并测试了体外蛋白酶抑制作用。 从其中最有效的情况下，具有特性的化合物表明它们在体内可能很有用，因为在LBD上测试了药物在大鼠致命毒素（PA + LF）挑战模型中的保护效率。 在此模型中，大鼠对致命的毒素进行了静脉注射，经历了快速且高度可重现的休克反应，这将在60-100分钟内导致死亡。 该系统提供了一种定量方法，用于评估和比较化合物的体内功效。 这些化合物之一是在亚植物浓度下在体外活性的，在大鼠致命的毒素模型中提供了100％的保护。 在随后的随访研究中，合成了其他化合物，以检查取代基在核心结构上的多个位置的作用。 这导致鉴定具有0.1-2.0 nm的Ki值的一组改进的化合物。这些化合物中最好的最好的大鼠免受了仅2.5 mg/kg的药物剂量的致命毒素挑战。 这些是尚未描述的最有效的LF蛋白酶抑制剂之一。