Antimicrobial Peptides and their Synthetic Mimics - Investigating the Mechanism o

说明(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泛的杀菌活性。最近，由于它们在治疗由于抗菌素耐药性而无法用常规抗生素治愈的疾病方面的出色潜力，它们成为越来越受关注的问题。AMPS要么导致膜损伤，这对目标细菌来说是致命的事件，要么与细菌细胞质区域的几个靶点结合。所有证据表明，AMP的作用不涉及立体特异性的蛋白受体识别，因为AMP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、病毒被膜或天然质膜脂质相互作用的性质在很大程度上决定了它们的裂解能力。作为我们研究的一部分，我们将开发新型的平面仿生膜，包括气-水界面和固-液界面。这将允许使用高度敏感的结构实验技术，而这种技术既不能用于囊泡系统，也不能用于真实细胞。此外，除了AMP之外，我们还计划研究它们的合成肽类模拟物(安培类)的膜相互作用，这些模拟物具有抗蛋白酶的优势，同时显示出作为抗菌剂的高效力和选择性。在这项高度跨学科的提案中，我们计划使用尖端同步加速器X射线散射技术，结合原子力显微镜和荧光研究，将产生近原子分辨率的肽-脂相互作用。这些数据将被用来促进对AMP和安培类药物作用模式的理解，这些数据可以用来制定AMP和抗菌肽模拟物的合理设计策略，以促进高效药物的开发，这些药物甚至可以有效地对抗多重耐药细菌和病毒。本项目的具体目标是：(1)利用同步辐射掠入射X射线衍射、X射线反射率、荧光显微镜和互补使用的AFM，研究安培类和天然AMP与代表红细胞膜外层和细菌细胞壁表层的脂单分子层的相互作用模式。(2)设计了一种新型的气-水界面流体双层膜，并用其研究了AMPs和安替比特与双层膜的两个小叶之间的相互作用。(3)设计具有细胞骨架成分的新型胆固醇栓系双层脂膜(TBLM)。研究AMPs和安培类化合物与这些tBLM的相互作用机制，并阐明细胞骨架在它们相互作用中的作用。拟议研究的更广泛影响是推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具有免疫力。抗菌肽及其合成的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，而且与细胞膜的脂基质相互作用，而细胞膜的脂组成极不可能因细菌突变而改变。在分子水平上更好地了解抗菌肽和类肽的作用方式，可以促进当今使用的传统抗生素和抗病毒药物的有效替代品的设计和开发。

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.