Enhancing In Vitro Antimicrobial Activity of Common Antibiotics with Cyclopeptide

描述（由申请方提供）：革兰氏阳性和阴性病原体的抗菌药物耐药性在全球范围内以越来越快的速度传播，成为一个具有挑战性的全球健康问题。即使在美国，医院和社区获得耐甲氧西林金黄色葡萄球菌（MRSA）变得比它们的前辈更具毒性，且可引起更大范围的疾病。特别是，MRSA已被发现表现出惊人的能力，以打击其他健康的人。预防抗生素耐药性的一种战略方法是一次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增加组合的功效，又可以通过产生传递抗性的双重偶然突变来降低细菌存活的概率。我们建议研究两亲性阳离子环肽的潜力，这是能够破坏细菌膜，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，像更好地已知的聚阳离子肽，可以增强小的疏水分子进入细菌的摄取，并呈现协同效应。该项目的长期目标是设计可能导致新的抗菌治疗途径的快速作用的抗菌剂，例如，它们可以在联合抗菌治疗中提供协同作用或作为抗生素靶向的支架。本项目的目标是（i）对阳离子环八肽与细菌膜及其相关的二价金属离子的相互作用有更清楚的了解（ii）通过固态研究鉴定环八肽上的供体原子与Ca 2+或Mg 2+之间的结合特异性的来源，以及（iii）利用这些理解来开发有效的抗菌剂。在实现这些项目目标的工作中，我们将通过微波辅助固相肽合成一系列两亲性阳离子环肽及其双类似物，并通过进行最小抑菌浓度（MIC）测定和细菌杀伤测定来评估其对几种细菌的体外固有抗菌活性。通过等温滴定量热法（ITC）、圆二色性和X射线衍射法研究了它们与模拟膜洗涤剂和稳定细菌膜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）测定和棋盘滴定法评估协同、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，并证明它们在增强某些抗生素的功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卫生。 公共卫生相关性：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蔓延，成为一个具有挑战性的全球健康问题。该项目的目标是设计快速作用的抗菌剂，可能导致新的抗菌治疗途径。这些基础研究有望证明细菌膜破坏分子在增强某些抗生素的功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.