Design and analysis of random copolymers with antimicrobial activity

描述（由申请人提供）：致病性感染是对人类健康的持续威胁。对药物治疗的快速发展产生了对新的抗感染药物的持续需求。提出的研究重点是开发具有尼龙-3骨架的无顺序共聚物作为通用的，廉价的抗菌剂。我们将合成并表征各种共聚物，这些共聚物旨在模仿天然宿主防御肽（HDPs）的活性，这些共聚物已在植物，动物和人类中发现。HDPs具有显著的抑制革兰氏阴性菌和革兰氏阳性菌生长的能力，而动物细胞基本不受影响。细菌抵抗HDPs的相对无能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，会形成全局两亲螺旋（一侧为疏水侧链，另一侧为带电和其他亲水侧链）。相反，所研究的尼龙-3共聚物含有阳离子和疏水侧链的随机序列；它们的长度也各不相同。初步研究表明，无规共聚物通过尼龙-3主链的柔韧性形成不规则的两亲表面结构来攻击膜。几种无规尼龙-3共聚物具有与天然HDPs相媲美的抑菌性能；它们只在高浓度时溶血攻击红细胞。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单细胞荧光成像的详细功能分析。该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。对于每个单独的细胞，分析方法可以实时将细菌“症状”的发展与抗菌聚合物的吸收量和细胞生长的停止直接相关。初步工作将集中在大肠杆菌和枯草芽孢杆菌作为革兰氏阴性和革兰氏阳性的代表性物种。可观察到的症状包括跨外膜易位、外膜溶解、跨细胞质膜易位和外膜溶解。同样的技术将决定“存活细胞”的特殊属性，这些细胞对攻击具有不同寻常的抵抗力。恢复正常生长培养基后的时间推移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。从实验中获得的详细机理数据将反馈到设计廉价有效的抗菌聚合物的努力中。在这项工作中开发的新技术和概念将在设计涉及膜基功能的抗菌剂的所有努力中得到广泛应用。随机共聚物可以在抗真菌活性，抗疟原虫活性和肺表面活性方面找到应用。

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.