Re-engineering the arylomycins for antibiotic activity

说明书(申请人提供)：芳霉素是一系列联苯连接的大环脂多肽天然产物，在体外对细菌信号肽I(Spase)具有抑制作用，但与抗生素一样显示出较低的效力和光谱。Spase是所有细菌生存和致病所必需的酶。然而，在最初的一些兴趣之后，这些天然产物由于效力不足而被制药行业抛弃。这些天然产品抗生素的低效力可能是因为它们在亿万年的细菌战争中使用--大多数细菌已经进化出抗药性。我们已经合成了这类天然产物中的一员，芳霉素A2，在其生物学分析中，我们发现对人类重要病原体大肠杆菌和表皮葡萄球菌的抗药性机制是基于在Spase底物结合位点的保守区引入一个脯氨酸残基。对其他细菌中SPase的序列分析表明，芳霉素类抗药性与关键的“抗药性”脯氨酸残基的存在有显著的相关性。我们已经证明，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，而具有的细菌，通过去除它而变得敏感。这一数据表明，如果芳香菌素能够被重新设计成与Spase结合，而不考虑“授予耐药性”的脯氨酸，它们将再次成为有效的、广谱的抗生素。在这项R21探索性研究拨款中，我们建议进一步表征芳香霉素耐药的机制，并利用这一洞察力来确定如何重新设计芳香霉素以再次成为有效的抗生素。更具体地说，将表征来自铜绿假单胞菌Spase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组芳香霉素的光谱范围内。然后，将利用体外动力学数据和X射线结晶学从分子水平上阐明阿霉素类抗药性的机制。最后，在表征了芳霉素的作用机制之后，所有这些数据以及化学合成都将用于确定芳霉素支架的哪一部分最适合药物化学工作。我们的项目将提供对抗性分子进化的详细了解，重要的是，应该阐明芳香菌素可能如何重新设计以提高效力。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--发挥作用的事实，将重新激发制药行业对这些非凡的天然产品的兴趣。与公共卫生相关：芳霉素类天然产物抗生素的开发已被放弃，因为这些化合物对许多细菌没有足够的效力，这种抗生素通过抑制I型细菌信号肽酶和蛋白质运输的新机制杀死细菌。似乎在长期接触芳香菌素的情况下，细菌已经进化出抗药性。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共同的之后，我们提出了一些实验，以阐明如何重新改造芳香菌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.