Enhancing In Vitro Antimicrobial Activity of Common Antibiotics with Cyclopeptide

描述(申请人提供)：革兰氏阳性和阴性病原体的抗菌素耐药性在全球范围内以越来越快的速度传播，提出了一个具有挑战性的全球卫生问题。即使在美国，医院和社区获得的耐甲氧西林金黄色葡萄球菌(MRSA)也变得更加毒力，比它们的前身能引起更广泛的疾病。特别是，MRSA已被发现显示出一种令人震惊的能力，可以攻击其他健康的人。预防抗生素耐药性的一个战略方法是同时使用一种以上的药物治疗细菌感染。相互作用的协同作用既可以提高组合的效率，又可以通过产生传递抗药性的双重偶然突变来降低细菌存活的可能性。我们建议研究能够破坏细菌膜的两亲性阳离子环肽，以提高一些临床使用的抗生素对几种类型的革兰氏阳性和阴性细菌的疗效。这些两亲性阳离子环肽，就像更广为人知的聚阳离子多肽一样，可以增强小分子疏水分子对细菌的吸收，并呈现协同效应。该项目的长期目标是设计快速有效的抗菌药，这些抗菌药可能导致新的抗菌治疗路线，例如，它们可以在联合抗菌治疗中提供协同作用，或者作为抗生素靶向的支架。本项目的目标是(I)通过结构和结合相关性研究，更清楚地了解阳离子环八肽与细菌膜及其相关的二价金属离子(钙和镁)的相互作用；(Ii)通过固态研究，确定环八肽上供体原子与钙或镁离子结合专一性的来源，以及(Iii)利用这些理解来开发有效的抗菌剂。为了实现这些项目目标，我们将通过微波辅助固相多肽合成来合成一系列两亲性阳离子环肽及其双类似物，并通过最小抑菌浓度(MIC)试验和细菌杀灭试验来评价它们对几种细菌的体外内在抗菌活性。用等温滴定热法(ITC)、圆二色谱和X射线衍射法研究了它们与仿膜洗涤剂和与稳定细菌膜有关的二价金属离子的相互作用。我们还将研究它们与一些临床常用抗生素的相互作用，并使用最小抑菌浓度(MIC)分析和棋盘滴定来评估它们的协同、相加或拮抗作用。这些基础研究将促进我们对这些抗菌环肽与细菌膜及其相关金属离子的作用机制的理解，并展示它们在提高某些抗生素的疗效和克服抗生素耐药性方面的潜力。因此，这些研究结果可能有助于改善公共健康。 公共卫生相关性：革兰氏阳性和革兰氏阴性病原体的抗菌素耐药性在全球范围内以越来越快的速度传播，提出了一个具有挑战性的全球健康问题。该项目的目标是设计可能导致新的抗菌治疗路线的快速作用的抗菌药。这些基础研究有望证明细菌膜破坏分子在提高一些抗生素的疗效和克服抗生素耐药性方面的潜力。

DESCRIPTION (provided by applicant): Antimicrobial resistance for both Gram-positive and -negative pathogens has spread worldwide at an increasing rate, presenting a challenging global health problem. Even in the U.S., hospital- and community- acquired methicillin-resistant Staphylococcus aureus (MRSA) have became more virulent and can cause a greater spectrum of illness than their predecessors. In particular, MRSA has been found to exhibit an alarming ability to strike otherwise healthy people. One strategic approach to preventing antibiotic resistance is to treat bacterial infections with more than one drug at a time. Synergies of interaction can both increase the efficacy of the combination and reduce the probability of the bacterium's surviving by developing a double fortuitous mutation conveying resistance. We propose to investigate the potential of amphipathic cationic cyclopeptides, which are capable of disrupting the bacterial membranes, to elevate the efficacy of some clinically used antibiotics against several types of Gram-positive and -negative bacteria. These amphipathic cationic cyclopeptides, like the better-known polycationic peptides, could enhance the uptake of small hydrophobic molecules into the bacterium and present synergistic effects. The long-term goal of this project is to design-fast acting antimicrobials that may lead to novel antimicrobial therapeutic routes, for example, they can provide synergism in combination antimicrobial therapy or act as a scaffold for antibiotic targeting. The objectives of this project are (i) to develop a clearer understanding of the interaction of cationic cyclooctapeptides with bacterial membranes and their associated divalent metal ions (Ca2+ and Mg2+) by a structure-and-binding correlation study, (ii) to identify the origins of the binding specificity between the donor atoms on the cyclooctapeptide and Ca2+ or Mg2+ by solid state studies, and (iii) to utilize these understandings to develop effective antimicrobials. In working towards these project objectives, we will synthesize a series of amphipathic cationic cyclopeptides and their bis-analogs by microwave-assisted solid-phase-peptide synthesis and evaluate their in vitro intrinsic antimicrobial activities against several strains of bacteria by conducting minimum inhibitory concentration (MIC) assays and bacterial killing assays. Their interactions with membrane-mimicking detergents and divalent metal ions associated with stabilizing bacterial membrane will be studied by isothermal titration calorimetry (ITC), circular dichroism and X-ray diffraction methods. We will also investigate their interactions with some common clinically used antibiotics and assess for synergistic, additive, or antagonistic effects using minimum inhibitory concentration (MIC) assays and checkerboard titrations. These fundamental studies will advance our understanding of the mechanism of action of these antimicrobial cyclopeptides with bacterial membranes and their associated metal ions, and demonstrate their potential in enhancing the efficacy of some antibiotics and in prevailing over antibiotic resistance. Consequently, these research findings could be useful in improving public health. PUBLIC HEALTH RELEVANCE: Antimicrobial resistance for both Gram-positive and -negative pathogens has spread worldwide at an increasing rate, presenting a challenging global health problem. The goal of this project is to design fast-acting antimicrobials that may lead to novel antimicrobial therapeutic routes. These fundamental studies are expected to demonstrate the potential of bacterial membrane-disrupting molecules in enhancing the efficacy of some antibiotics and in prevailing over antibiotic resistance.