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: 8708892
• 负责人: SAMUEL H. GELLMAN
• 金额: $ 31.69万
• 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 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8708892
• 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）：病原体感染对人类健康构成持续威胁。药物治疗耐药性的迅速发展产生了对新型抗感染药物的持续需求。拟议的研究重点是开发具有尼龙 3 主链的无规序列共聚物作为通用、廉价的抗菌剂。我们将合成并表征各种旨在模拟天然宿主防御肽（HDP）活性的共聚物，这种肽已在植物、动物和人类中发现。 HDP 因其普遍能够阻止革兰氏阴性菌和革兰氏阳性菌生长而使动物细胞基本上不受影响而著称。细菌相对无力抵抗 HDP 通常归因于涉及细菌细胞膜降解的一般作用模式。 众所周知，当阳离子 HDP 与阴离子细胞膜结合时，会形成整体两亲性螺旋（一侧为疏水侧链，另一侧为带电侧链和其他亲水侧链）。相比之下，我们感兴趣的尼龙-3 共聚物含有随机序列的阳离子和疏水侧链；它们的长度也各不相同。初步研究表明，无规共聚物通过形成不规则的两亲表面结构（由尼龙 3 主链的柔性实现）来攻击膜。 几种无规尼龙 3 共聚物具有可与天然 HDP 相媲美的抑菌性能；它们仅在高浓度时才溶血攻击红细胞。这项工作将更好地了解这些共聚物如何降解膜，以便为设计改进提供信息。该方法包括化学合成和通过单细胞荧光成像对功能进行详细分析。该合成方法能够控制平均共聚物长度以及疏水性、阳离子性和极性侧链的类型和百分比。对于每个单独的细胞，分析方法能够将细菌“症状”的发展与吸收的抗菌聚合物的量和细胞生长的停止直接相关。初步工作将重点关注大肠杆菌和枯草芽孢杆菌作为革兰氏阴性和革兰氏阳性菌的代表性物种。可观察到的症状包括跨外膜 (OM) 易位、OM 裂解、跨细胞质膜 (CM) 易位和 CM 裂解。同样的技术将确定“存活细胞”的特殊特性，这些特性对攻击具有异常抵抗力。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，即阻止随后的恢复和生长。实验中的详细机械数据将反馈到设计廉价且有效的抗菌聚合物的工作中。 这项工作中开发的新技术和概念将广泛应用于设计涉及膜功能的抗菌剂的所有努力中。无规共聚物还可以在抗真菌活性、抗疟原虫活性和肺表面活性剂活性方面得到应用。