Design of New Antimicrobials

描述（由申请人提供）：对传统抗生素具有抗性的许多相关细菌菌株的显著且不断增加的出现现在是人类健康的主要问题。由于经典抗生素的广泛临床使用，抗生素耐药性已经出现。因此，在最好的情况下，抗生素的疗效逐渐下降;在最坏的情况下，无法治疗的感染，如多重耐药结核病和耐万古霉素肠球菌菌株激增。因此，在美国，通过延长住院时间和越来越积极的治疗来治疗医院感染的成本已经上升到估计300亿美元。此外，必须应对生物恐怖主义的威胁，即能够轻而易举地设计对人类造成致命后果的新细菌菌株。与现有的抗生素相比，抗菌肽作为一种全新的结构类型的抗生素显示出巨大的潜力，具有新颖的作用模式以及不同的细胞靶点。预期不会对唯一靶点为细胞质膜的膜活性肽产生耐药性，因为这将需要微生物细胞膜的脂质组成发生实质性变化。阳离子抗菌肽的优点是它们能够快速杀死靶细胞，它们不寻常的广谱活性和它们对临床分离的一些更严重的耐药病原体的活性。使用抗微生物肽作为抗生素的主要障碍是它们的毒性或裂解真核细胞的能力。我们已经采取了一种方法，系统地改变两种不同类型的抗菌肽的两亲性，疏水性和结构的单一L-和D-氨基酸取代。通过这种方法，我们能够将抗真核活性与抗微生物活性分离，即增加抗微生物活性并显著降低或消除对正常细胞的毒性（如通过溶血活性所测量的）。我们已经在两种不同结构类别的抗菌肽中发现了先导化合物，14个残基的环状2-折叠肽和26个残基的1-螺旋肽具有作为广谱抗生素的临床潜力。这两类化合物的同时开发将最迅速地推进我们对这些肽的作用机制和对微生物膜的选择性的共同要求的认识。需要进一步优化我们重新设计的先导化合物，以确保我们获得最佳的抗微生物活性，同时保持高治疗指数。主要目标：1）确定在非极性面的中心的带正电荷的残基的最佳类型，以增强对环状2-折叠和1-螺旋肽的特异性; 2）已经选择了最佳带正电荷的残基以消除溶血活性，我们可以增加疏水性以改善抗微生物活性，同时保持高治疗指数; 3）证明我们的肽对一系列人类细胞系是无毒的; 4）通过结构测定显示（固态NMR）我们的两类抗微生物肽在膜中的位置; 5）在两种动物模型中证明我们的肽对铜绿假单胞菌攻击的体内功效; 6）将我们的研究扩展到其他严重细菌感染的动物模型。 公共卫生相关性：对传统抗生素具有抗性的许多相关菌株的戏剧性和不断增加的出现现在是人类健康的主要问题。由于经典抗生素的广泛临床使用，抗生素耐药性已经出现。因此，在美国，通过延长住院时间和越来越积极的治疗来治疗医院感染的成本已经上升到估计300亿美元。因此，存在采用新型抗生素的经济激励。

DESCRIPTION (provided by applicant): The dramatic and ever-increasing emergence of many relevant strains of bacteria resistant to traditional antibiotics is now a major issue in human health. Antibiotic resistance has arisen due to the extensive clinical use of classical antibiotics. Thus, at best, antibiotics are progressively demonstrating decreased efficacy; at worst, there has been an upsurge of untreatable infections, such as multi-resistant tuberculosis and vancomycin-resistant Enterococcus strains. Consequently, the cost of treating nosocomial infections through extended hospitalization and increasingly aggressive therapy has risen to an estimated $30 billion in the U.S. Therefore, there is an economic incentive to adopt novel antibiotics. In addition, the threat of bioterrorism, that is the ability to easily engineer new strains of bacteria with deadly consequences to humans, must be dealt with. Compared to existing antibiotics, antimicrobial peptides show great potential as a radically new structural class of antibiotics, with both novel modes of action as well as different cellular targets. The development of resistance to membrane active peptides whose sole target is the cytoplasmic membrane is not expected since this would require substantial changes in the lipid composition of cell membranes of microorganisms. The advantages of cationic antimicrobial peptides are their ability to kill target cells rapidly, their unusual broad spectrum activity and their activity against some of the more serious antibiotic-resistant pathogens isolated in clinics. The major barrier to the use of antimicrobial peptides as antibiotics has been their toxicity or ability to lyse eukaryotic cells. We have taken an approach of systematic alteration in the amphipathicity, hydrophobicity and structure of two different classes of antimicrobial peptides by single L- and D- amino acid substitutions. With this approach we were able to dissociate anti-eukaryotic activity from antimicrobial activity, i.e. increase the antimicrobial activity and dramatically reduce or eliminate toxicity to normal cells (as measured by hemolytic activity). We have discovered lead compounds in two different structural classes of antimicrobial peptides, a 14-residue cyclic 2-sheet peptide and a 26-residue 1-helical peptide with clinical potential as broad spectrum antibiotics. The simultaneous development of both classes of compounds will most rapidly advance our knowledge of the mechanism of action of these peptides and the common requirements for selectivity for microbial membranes. Further optimization of our de novo designed lead compounds is required to ensure we obtain the best antimicrobial activity while maintaining a high therapeutic index. Key goals: 1) to determine the best type of positively-charged residue in the center of the non-polar face to enhance specificity on the cyclic 2- sheet and 1-helical peptides; 2) having selected the best positively-charged residue to eliminate hemolytic activity, can we increase hydrophobicity to improve antimicrobial activity while maintaining a high therapeutic index; 3) to demonstrate that our peptides are non-toxic to a series of human cell lines; 4) to show by structural determination (solid-state NMR) the location of our two classes of antimicrobial peptides in the membrane; 5) to demonstrate in vivo efficacy of our peptides against Pseudomonas aeruginosa challenge in two animal models; 6) to extend our studies to animal models of other serious bacterial infections. PUBLIC HEALTH RELEVANCE: The dramatic and ever-increasing emergence of many relevant strains of bacteria resistant to traditional antibiotics is now a major issue in human health. Antibiotic resistance has arisen due to the extensive clinical use of classical antibiotics. Consequently, the cost of treating nosocomial infections through extended hospitalization and increasingly aggressive therapy has risen to an estimated $30 billion in the U.S. Thus, there is an economic incentive to adopt novel antibiotics.