Re-engineering the arylomycins for antibiotic activity

描述（由申请人提供）：芳霉素是一系列联苯连接的大环脂肽天然产物，在体外抑制细菌信号肽酶I（SPase），但作为抗生素的效力和谱较低。SPase是所有细菌生存力和毒力所必需的酶。然而，在一些最初的兴趣之后，这些天然产品由于效力不足而被制药业放弃。这些天然产物抗生素的低效力可能是由于它们在细菌战争中使用了20多年-大多数细菌已经进化出耐药性。我们已经合成了这类天然产物的一个成员，芳基霉素A2，并在其生物学分析过程中，我们发现，在重要的人类病原体大肠杆菌和表皮葡萄球菌的耐药机制是基于引入一个脯氨酸残基到保守区域的SPase底物结合位点。在其他细菌中的SPases序列分析揭示了arylomycin耐药性和关键的“抗性赋予”脯氨酸残基的存在之间的显着相关性。我们已经表明，不具有脯氨酸的细菌，包括重要的革兰氏阳性和革兰氏阴性人类病原体是敏感的，并且具有脯氨酸的细菌通过去除脯氨酸而变得敏感。该数据表明，如果芳基霉素可以被重新工程化以结合SPase而不管“赋予抗性”的脯氨酸，则它们将再次成为有效的广谱抗生素。在这项R21探索性研究资助中，我们建议进一步表征芳基霉素耐药性的机制，并利用这一见解来确定芳基霉素如何重新设计成为有效的抗生素。更具体地，将表征来自铜绿假单胞菌的SPase的底物结合位点中的脯氨酸的作用，以证明这种重要的病原体也将在重新工程化的芳基霉素的谱内。体外动力学数据和X-射线晶体学将用于阐明分子水平上的芳霉素耐药机制。最后，在表征芳霉素的作用机制之后，所有这些数据，沿着化学合成，将用于确定芳霉素支架的哪一部分最适合于药物化学工作。我们的项目将提供一个详细的了解耐药性的分子进化，重要的是，应该阐明如何芳基霉素可能重新设计的效力。我们希望，这一点，沿着事实，即这些化合物通过一种新的作用机制-抑制蛋白质转运-将重振制药工业对这些显着的天然产品的兴趣。公共卫生相关性：芳基霉素类天然产物抗生素通过抑制I型细菌信号肽酶和蛋白质转运的新机制杀死细菌，其开发已被放弃，因为这些化合物对许多细菌没有足够的效力。看来，在暴露于芳基霉素的10年内，细菌已经进化出耐药性。我们已经发现了这种耐药性的机制，在证明这种机制在不同的细菌物种中是常见的之后，我们提出了一些实验，这些实验将阐明如何重新设计芳基霉素，使其再次成为有效的抗生素。

DESCRIPTION (provided by applicant): The arylomycins are a series of biphenyl-linked macrocyclic lipopeptide natural products that inhibit bacterial signal peptidase I (SPase) in vitro, but show low potency and spectrum as antibiotics. SPase is an essential enzyme required for viability and virulence in all bacteria. However, after some initial interest, these natural products have been abandoned by pharmaceutical industry due to insufficient potency. The low potency of these natural product antibiotics is presumably due to their use in bacterial warfare over eons of time - most bacteria having already evolved resistance. We have synthesized one member of this class of natural products, arylomycin A2, and during its biological analysis we discovered that the mechanism of resistance in the important human pathogens Escherichia coli and Staphylococcus epidermidis is based on the introduction of a proline residue into a conserved region of the SPase substrate binding site. Sequence analysis of SPases in other bacteria reveals a remarkable correlation between arylomycin resistance and the presence of the critical 'resistance-conferring' proline residue. We have shown that bacteria that do not possess the proline, including important Gram-positive and Gram-negative human pathogens are sensitive, and that bacteria that do, are rendered sensitive by its removal. This data suggests that if the arylomycins could be re-engineered to bind SPase regardless of the 'resistance-conferring' proline, they would again be potent, broad spectrum antibiotics. In this R21 exploratory research grant, we propose to further characterize the mechanism of arylomycin resistance and use this insight to determine how the arylomycins might be re-engineered to again be potent antibiotics. More specifically, the role of the proline in the substrate binding site of SPase from P. aeruginosa will be characterized to demonstrate that this important pathogen would also be within the spectrum of a re-engineered arylomycin. In vitro kinetic data and X-ray crystallography will then be used to elucidate the mechanism of arylomycin resistance at the molecular level. Finally, after characterization of the arylomycins' mechanism of action, all of this data, along with chemical synthesis, will be used to determine which part of the arylomycin scaffold is best suited for medicinal chemistry efforts. Our project will provide a detailed understanding of the molecular evolution of resistance, and importantly, should elucidate how the arylomycins might re-engineer for potency. We hope that this, along with the fact that these compounds act via a novel mechanism of action - the inhibition of protein transport - will reinvigorate the pharmaceutical industries interest in these remarkable natural products. PUBLIC HEALTH RELEVANCE: The development of the arylomycin class of natural product antibiotics, which kill bacteria via the novel mechanism of inhibiting type I bacterial signal peptidases and protein transport, has been abandoned because these compounds are not sufficiently potent against many bacteria. It appears that over eons of being exposed to the arylomycins, bacteria have already evolved resistance. We have discovered the mechanism of this resistance, and after demonstrating that the mechanism is common among different bacterial species, we propose experiments that will elucidate how to re-engineer the arylomycins to again be potent antibiotics.