Design and analysis of random copolymers with antimicrobial activity

描述(申请人提供)：病原性感染是对人类健康的持续威胁。对药物治疗的抗药性的快速发展创造了对新的抗感染药物的持续需求。建议的研究重点是开发以尼龙-3为主链的序列无规共聚物作为通用、廉价的抗菌剂。我们将合成和表征各种此类共聚物，旨在模拟已在植物、动物和人类中发现的天然宿主防御肽(HDPs)的活性。高密度脂蛋白在阻止革兰氏阴性和革兰氏阳性细菌生长的同时，使动物细胞基本不受影响，这一点值得注意。细菌对HDPs的相对抵抗力通常归因于一种涉及细菌细胞膜降解的一般作用模式。阳离子HDP结合到阴离子细胞膜上时，会形成全球两亲性螺旋(一侧为疏水侧链，另一侧为带电侧链和其他亲水侧链)。相反，感兴趣的尼龙-3共聚物包含随机序列的阳离子和疏水侧链；它们的长度也不同。初步工作表明，无规共聚物通过尼龙-3主链的柔性形成不规则的两亲性表面结构来攻击膜。几种无规尼龙-3共聚物具有与天然HDP相媲美的抑菌性能；它们只有在高浓度时才能攻击红细胞。这项工作将从根本上更好地了解这些共聚物是如何降解膜的，以便为设计改进提供信息。该方法既包括化学合成，也包括通过单细胞荧光成像详细分析功能。该合成方法能够控制共聚物的平均长度以及疏水、阳离子和极性侧链的类型和百分比。对于每个单独的细胞，分析方法能够实时地将细菌“症状”的发展与所吸收的抗菌聚合物的量和细胞生长停止直接相关。最初的工作将集中在具有代表性的革兰氏阴性和革兰氏阳性菌--大肠杆菌和枯草杆菌上。可观察到的症状包括外膜移位(OM)、OM裂解、细胞质移位(CM)和CM裂解。同样的技术将决定“存活细胞”异常抵抗攻击的特殊属性。恢复正常生长介质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。来自实验的详细机械数据将反馈到设计廉价而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功能的抗菌剂的所有努力中得到广泛应用。无规共聚物可能在抗真菌活性、抗疟原虫活性和肺表面活性方面有应用。 与公共卫生相关：细菌感染对抗生素疗法的抗药性越来越强。本工作探索了以尼龙-3为骨架的无规共聚物作为一类新的抗菌剂。来自详细实验工作的基本见解将为设计更有效的抗菌聚合物提供依据。这些化合物可以以较低的成本大量合成。

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. PUBLIC HEALTH RELEVANCE: Bacterial infections are increasingly resistant to antibiotic therapies. This work explores random copolymers based on the nylon-3 backbone as a new class of antibacterial agents. Fundamental insights from detailed experimental work will inform the design of more effective antibacterial polymers. These can be synthesized in large quantities at low cost.