Antimicrobial Peptides and their Synthetic Mimics - Investigating the Mechanism o

描述（由申请人提供）：抗菌肽（AMP）形成先天免疫系统的第一道防线，对各种革兰氏阴性和革兰氏阳性细菌、真菌、原生动物甚至包膜病毒具有广谱的杀微生物活性。最近，由于它们在治疗由于抗菌素耐药性而不能被常规抗生素治愈的疾病方面具有良好的潜力，它们成为越来越感兴趣的问题。AMPs要么诱导膜损伤，这对目标细菌是致命的事件，要么结合细菌细胞质区域的几个目标。所有证据表明，AMPs的作用不涉及立体特异性蛋白质受体识别，因为AMPs与其靶标的相互作用通常被认为是非特异性的。因此，AMP与细菌细胞壁脂质、病毒包膜或天然浆细胞膜脂质相互作用的特性在很大程度上决定了它们的溶解潜力。作为我们研究的一部分，新型平面仿生膜，在空气-水和固体-液体界面将被开发。这将允许使用高度敏感的结构实验技术，这些技术不能用于囊泡系统或真实细胞。此外，除了AMP，我们还计划研究它们合成的类肽模拟物（ampetoids）的膜相互作用，这些类肽模拟物具有蛋白酶抗性的优势，同时显示出作为抗菌药物的高效力和选择性。在这个高度跨学科的建议中，我们计划使用尖端的同步加速器x射线散射技术，与原子力显微镜和荧光研究一起，将产生接近原子分辨率的肽-脂相互作用。这些数据将用于促进对AMP和ampetoids作用模式的理解，可用于制定AMP和抗菌肽模拟物的合理设计策略，以推进高效药物的开发，甚至对多重耐药细菌和病毒有效。本项目的具体目标是：(1)利用同步加速器掠射x射线衍射、x射线反射率、荧光显微镜和原子力显微镜互补的方式，研究类足肽和天然类足肽与代表红细胞膜外层和细菌细胞壁表层的脂质单层的相互作用模式。(2)在空气-水界面处设计新型流体双层膜，用它们来研究AMPs和ampe仿体与双层膜两个小叶的相互作用。(3)设计具有细胞骨架成分的新型胆固醇系固双层脂质膜（tBLM）。研究amp和类amp与这些tblm相互作用的机制，阐明细胞骨架在它们相互作用中的作用。拟议的研究的更广泛影响是促进新型抗生素和抗病毒药物的开发，这些药物将对细菌和病毒突变免疫。抗菌肽及其合成模拟物在细菌耐药性方面具有巨大的潜力，因为它们不仅与特定的膜蛋白受体相互作用，而且与细胞膜的脂质基质相互作用，而细胞膜的脂质组成极不可能因细菌突变而改变。更好地了解抗菌肽和类肽在分子水平上的作用模式，可以促进设计和开发有效的替代目前使用的传统抗生素和抗病毒药物。

DESCRIPTION (provided by applicant): Antimicrobial peptides (AMP) form a first line of defense of the innate immune system and have a broad spectrum of microbicidal activity against a wide range of Gram-negative and Gram-positive bacteria, fungi, protozoa, and even enveloped viruses. Recently they became a matter of increasing interest because of their excellent potential in treating diseases which cannot be cured by conventional antibiotics due to antimicrobial resistance. AMPs either induce membrane damage that is a lethal event for target bacteria or bind to several targets in the cytoplasmic region of the bacteria. All the evidence indicates that the action of the AMPs does not involve stereospecific protein-receptor recognition, since the interactions of AMPs with their targets are generally considered to be nonspecific. Therefore, the character of AMP interaction with bacterial cell wall lipids, viral envelope, or native plasma cell membrane lipids largely determine their lytic potential. As part of our study, novel planar biomimetic membranes, both at air-water and solid-liquid interface will be developed. This will allow use of highly sensitive structural experimental techniques, which cannot be employed with vesicle systems nor with real cells. Furthermore, in addition to AMP we also plan to investigate membrane interactions of their synthetic peptoid mimics (ampetoids), which have an advantage of being protease-resistant, while showing high potency and selectivity as antimicrobial agents. In this highly interdisciplinary proposal we plan to use cutting edge synchrotron X-ray scattering techniques, which together with AFM and epifluorescence studies will yield near atomic resolution of peptide-lipid interaction. These data will be used to advance the understanding of AMP and ampetoids mode of action which can be used to develop rational design strategies for AMPs and antimicrobial peptide mimics to advance development of highly potent drugs that are effective even against multidrug resistant bacteria and viruses. Specific aims of this project are: (1) Examine the modes of interaction of ampetoids and natural AMPs with lipid monolayers representing an outer leaflet of red blood cell membranes and surface layer of bacterial cell wall using synchrotron grazing incidence X-ray diffraction, X-ray reflectivity, epifluorescence microscopy, and AFM used in complementary manner. (2) Design novel fluid bilayer membranes at the air-water interface, use them to examine the interaction of AMPs and ampetoids with both leaflets of bilayer membrane. (3) Design novel cholesterol tethered bilayer lipid membranes (tBLM) with cytoskeleton component. Examine mechanism of AMPs and ampetoids interaction with these tBLMs and elucidate role of cytoskeleton in their interactions. The broader impact of the proposed research is to advance development of novel antibiotic and antiviral drugs that will be immune to bacterial and viral mutations. Antimicrobial peptides and their synthetic mimics have enormous potential with regard to bacterial resistance because they interact not only with specific membrane protein receptors, but also with the lipid matrix of cell membranes, whose lipid composition is highly unlikely to change as a result of bacterial mutation. Better understanding of antimicrobial peptides and peptoids mode of action on molecular level could enhance the design and development of potent alternatives to the conventional antibiotics and antiviral drugs used today.