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)，但作为抗生素表现出低效价和低谱。酶是所有细菌生存和毒力所必需的酶。然而，在最初的一些兴趣之后，这些天然产品由于效力不足而被制药行业所放弃。这些天然产物抗生素的效力较低，可能是由于它们在细菌战中被使用了亿万年——大多数细菌已经进化出了耐药性。我们已经合成了这类天然产物的一个成员，arylomycin A2，在其生物学分析中我们发现，在重要的人类病原体大肠杆菌和表皮葡萄球菌中，抗性的机制是基于在SPase底物结合位点的保守区域引入脯氨酸残基。对其他细菌中SPases的序列分析显示，阿霉素耐药性与关键的“赋予耐药性”脯氨酸残基的存在之间存在显著的相关性。我们已经证明，不含脯氨酸的细菌，包括重要的革兰氏阳性和革兰氏阴性的人类病原体是敏感的，而含有脯氨酸的细菌，通过去除脯氨酸而变得敏感。这一数据表明，如果可以重新设计芳霉素，使其与SPase结合，而不考虑“赋予耐药性”的脯氨酸，它们将再次成为有效的广谱抗生素。在这项R21探索性研究中，我们建议进一步表征芳霉素耐药的机制，并利用这一见解来确定如何重新设计芳霉素，使其再次成为有效的抗生素。更具体地说，脯氨酸在铜绿假单胞菌SPase底物结合位点的作用将被表征，以证明这种重要的病原体也将在重组的芳霉素的光谱内。体外动力学数据和x射线晶体学将在分子水平上阐明阿霉素耐药机制。最后，在表征了芳霉素的作用机制后，所有这些数据以及化学合成将用于确定芳霉素支架的哪一部分最适合用于药物化学工作。我们的项目将提供对耐药性分子进化的详细了解，重要的是，应该阐明如何重新设计芳霉素的效力。我们希望这一点，以及这些化合物通过一种新的作用机制——抑制蛋白质运输——起作用的事实，将重新激发制药行业对这些非凡的天然产品的兴趣。公共卫生相关性：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的天然产物抗生素阿霉素类的开发已被放弃，因为这些化合物对许多细菌没有足够的效力。看来，在暴露于芳霉素的千万年中，细菌已经进化出了耐药性。我们已经发现了这种耐药性的机制，在证明了这种机制在不同的细菌物种中是共同的之后，我们提出了实验，将阐明如何重新设计芳霉素，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.