Design of New Antimicrobials

描述（由申请人提供）：许多对传统抗生素具有耐药性的相关细菌菌株的急剧和不断增加的出现，现在是人类健康的一个主要问题。由于临床广泛使用经典抗生素，抗生素耐药性已经出现。因此，抗生素在最好的情况下逐渐显示出疗效下降；在最坏的情况下，出现了无法治疗的感染激增，例如耐多药结核病和耐万古霉素肠球菌菌株。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费用估计已上升到300亿美元。因此，采用新型抗生素具有经济动机。此外，必须应对生物恐怖主义的威胁，即能够轻易地制造出对人类造成致命后果的新菌株。与现有的抗生素相比，抗菌肽作为一种具有全新结构的抗生素，具有新的作用方式和不同的细胞靶点，具有巨大的潜力。对膜活性肽（其唯一目标是细胞质膜）的耐药性的发展是不被期望的，因为这将需要微生物细胞膜的脂质组成发生实质性的变化。阳离子抗菌肽的优势在于其快速杀死靶细胞的能力，其不同寻常的广谱活性以及对临床分离的一些更严重的耐药病原体的活性。使用抗菌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。我们采用了一种方法，系统地改变了两种不同类型的抗菌肽的两亲性、疏水性和结构，通过单个的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菌活性分离开来，即增加抗菌活性并显着降低或消除对正常细胞的毒性（通过溶血活性测量）。我们已经在两种不同结构的抗菌肽中发现了先导化合物，一种是含有14个残基的环状2片肽，另一种是含有26个残基的1螺旋肽，它们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选择性的共同要求的认识。我们需要进一步优化我们从头设计的先导化合物，以确保我们在保持高治疗指数的同时获得最佳的抗菌活性。关键目标：1)确定非极性面中心带正电残基的最佳类型，以增强对环状2-片和1-螺旋肽的特异性；2)选择最佳带正电残基消除溶血活性，可以在保持高治疗指数的同时增加疏水性，提高抗菌活性；3)证明我们的肽对一系列人类细胞系无毒；4)通过结构测定（固态核磁共振）显示我们的两类抗菌肽在膜中的位置；5)在两种动物模型上验证我们的肽对铜绿假单胞菌攻击的体内有效性；6)将我们的研究扩展到其他严重细菌感染的动物模型。

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.