Design and analysis of random copolymers with antimicrobial activity

具有抗菌活性的无规共聚物的设计与分析

• 批准号: 8513354
• 负责人: SAMUEL H. GELLMAN
• 金额: $ 30.58万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8513354
• 关键词: Adopted; Adoption; Affect; Amino Acids; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Antibiotic Resistance; Antibiotic Therapy; Antifungal Agents; Back; Bacteria; Bacterial Infections; Behavior; Belief; Benign; Binding; Biological; Cell Adhesion; Cell membrane; Cells; Charge; Chemicals; Culture Media; Cytolysis; Data; Development; Dissection; Drug resistance; Erythrocytes; Escherichia coli; Eukaryotic Cell; Extravasation; Gram-Positive Bacteria; Grant; Growth; Health; Host Defense; Human; Image; Imaging Techniques; Individual; Infection; Left; Length; Lipid Bilayers; Measurement; Membrane; Methodology; Molecular Conformation; Nucleic Acids; Nylons; Peptides; Pharmacotherapy; Phase; Plants; Polymers; Prokaryotic Cells; Property; Proteins; Proteolysis; Pulmonary Surfactants; Recovery; Relative (related person); Research; Resistance; Resistance development; S Phase; Side; Solid; Solutions; Structure; Surface; Survivors; Symptoms; Techniques; Therapeutic Agents; Time; Toxic effect; Variant; Vertebral column; Vesicle; Work; amphiphilicity; analytical method; antimicrobial; antimicrobial drug; arginyllysine; base; cell growth; cell killing; cellular imaging; chemical synthesis; copolymer; cost; design; feeding; flexibility; fluorescence imaging; innovation; insight; interest; killings; novel; prevent; research study; restoration; segregation; stereochemistry; tool

DESCRIPTION (provided by applicant): Pathogenic infections represent a persistent threat to human health. The rapid development of resistance to drug therapies creates a continuing need for new anti-infective agents. The proposed research focuses on the development of sequence-random copolymers having a nylon-3 backbone as general, inexpensive antibacterial agents. We will synthesize and characterize a variety of these copolymers designed to mimic the activity of natural host-defense peptides (HDPs), which have been discovered in plants, animals, and humans. HDPs are remarkable for their general ability to halt growth of both Gram negative and Gram positive bacteria while leaving animal cells largely unaffected. The relative inability of bacteria to resist HDPs is usually attributed to a general mode of action involving degradation of bacterial cell membranes. Cationic HDPs are known to form globally amphipathic helices (hydrophobic side chains on one side, charged and other hydrophilic side chains on the opposite side) when they bind to anionic cell membranes. In contrast, the nylon-3 copolymers of interest contain a random sequence of cationic and hydrophobic side chains; they also vary in length. Preliminary work suggests that the random copolymers attack membranes by forming irregular amphipathic surface structures enabled by the flexibility of the nylon-3 backbone. Several of the random nylon-3 copolymers have bacteriostatic properties rivaling those of natural HDPs; they hemolytically attack red blood cells only at high concentration. This work will obtain a better fundamental understanding of how these copolymers degrade membranes in order to inform design improvements. The approach includes both chemical synthesis and detailed analysis of function by single-cell fluorescence imaging. The synthetic methodology enables control of mean copolymer length and the type and percentage of hydrophobic, cationic, and polar side chains. For each individual cell, the analytical methods enable direct correlation in real time of the development of bacterial "symptoms" with the amount of antimicrobial polymer absorbed and the halting of cell growth. The initial work will focus on E. coli and B. subtilis as representative Gram negative and Gram positive species. Observable symptoms include translocation across the outer membrane (OM), lysing of the OM, translocation across the cytoplasmic membrane (CM) and lysing of the CM. The same techniques will determine the special properties of "survivor cells" that are unusually resistant to attack. Time lapse observations after restoration of normal growth medium will reveal which short-term symptoms are sufficient to kill cells, i.e. to prevent subsequent recovery and growth. Detailed mechanistic data from the experiments will feed back into the effort to design cheap and effective antimicrobial polymers. The novel techniques and concepts developed in this work will find wide application in all efforts to design antimicrobial agents involving membrane-based functions. Random copolymers may find applications in the context of antifungal activity, antiplasmodial activity, and lung-surfactant activity as well.

描述（由申请人提供）：致病感染代表了对人类健康的持续威胁。对药物疗法的抗药性的快速发展产生了对新的抗感染剂的持续需求。拟议的研究重点是具有尼龙3主链作为一般，廉价抗菌剂的序列随机共聚物的发展。我们将合成并表征各种这些共聚物，旨在模仿在植物，动物和人类中发现的自然宿主防御肽（HDP）的活性。 HDP因其在革兰氏阴性和革兰氏阴性细菌的一般能力中的一般能力而引人注目，同时使动物细胞在很大程度上不受影响。细菌抗HDP的相对无法归因于涉及细菌细胞膜降解的一般作用方式。 已知阳离子的HDP在与阴离子细胞膜结合时，会形成全球两亲螺旋（一侧的疏水侧链，另一侧充电和其他亲水侧链）。相反，感兴趣的尼龙-3共聚物包含阳离子和疏水侧链的随机序列。它们的长度也有所不同。初步工作表明，随机共聚物通过形成不规则的两亲性表面结构来攻击膜，这是由于尼龙-3骨架的柔韧性所启用的。 几种随机尼龙-3共聚物具有与天然HDP相匹配的抑菌特性。它们仅在高浓度下溶血攻击红细胞。这项工作将对这些共聚物如何降解膜以为设计改进提供更好的理解。该方法包括化学合成和通过单细胞荧光成像对功能的详细分析。合成方法可以控制平均共聚物长度以及疏水，阳离子和极性侧链的类型和百分比。对于每个单个细胞，分析方法可以实时发展细菌“症状”与吸收的抗菌聚合物的量和细胞生长的停止。最初的工作将集中于大肠杆菌和枯草芽孢杆菌作为代表性的革兰氏阴性和革兰氏阳性物种。可观察到的症状包括跨膜（OM）的易位，OM的裂解，跨细胞质膜（CM）的易位以及CM的裂解。相同的技术将确定异常抵抗攻击的“幸存者细胞”的特殊特性。恢复正常生长培养基后的延时观察将揭示哪些短期症状足以杀死细胞，即防止随后的恢复和生长。来自实验的详细机械数据将反馈到设计廉价有效的抗菌聚合物的努力。 这项工作中开发的新技术和概念将在设计涉及基于膜功能的抗菌剂的所有努力中找到广泛的应用。随机共聚物也可能在抗真菌活性，抗白皮病活性和肺表面活性剂活性的背景下找到应用。