CN1894277A

CN1894277A - Periodic antimicrobial peptides

Info

Publication number: CN1894277A
Application number: CNA2004800102993A
Authority: CN
Inventors: R·M·斯特罗姆; P·J·布龙达塞马
Original assignee: Dow Global Technologies LLC
Current assignee: Dow Global Technologies LLC
Priority date: 2003-02-24
Filing date: 2004-02-24
Publication date: 2007-01-10
Also published as: WO2005014639A2; WO2005014639A3; EP1599499A2

Abstract

One embodiment of the invention comprises a method of producing periodic peptides, which can have antimicrobial uses, and further comprises the peptides themselves. A preferred method comprises the synthesis of simple periodic peptides made from polymerizing identical monomer units of four or fewer amino acids, wherein the minimum length of active peptide is 15 or 16 residues and wherein the minimum percentage of cationic residues is at least 25%.

Description

periodic antimicrobial peptides

在先的相关申请prior related application

本发明要求2003年2月24日提交的美国专利申请US10/373,306的优先权。联邦赞助的研究报告This application claims priority to US Patent Application No. 10/373,306, filed February 24,2003. Federally Sponsored Research Reports

无。none.

发明领域field of invention

本发明包括一种合成周期肽(periodic peptide)的新方法，以及这种肽本身，和这些肽在如抗微生物剂、杀菌剂、抗病毒或抗肿瘤药剂等多种治疗用途，以及其它治疗、消毒、防腐之类中的应用。The present invention includes a novel method of synthesizing periodic peptides, as well as the peptides themselves, and the various therapeutic uses of these peptides such as antimicrobials, bactericides, antiviral or antineoplastic agents, and other therapeutic, Applications such as disinfection and antisepsis.

发明背景Background of the invention

在包括哺乳动物、鸟类、爬虫类、昆虫、植物和许多微生物的多种类型的生物体的天然防御机制中，抗微生物肽是常见的成分。天然生成的抗微生物肽是长度为大约10到50个氨基酸的单一序列。这些肽一般富含碱性氨基酸(赖氨酸和精氨酸)，因此通常为阳离子化的(cationic)。它们实际上也常常是两亲性的(即分子的一部分是亲水性的，而另一部分是疏水性的)。Antimicrobial peptides are common components in the natural defense mechanisms of many types of organisms including mammals, birds, reptiles, insects, plants and many microorganisms. Naturally occurring antimicrobial peptides are single sequences approximately 10 to 50 amino acids in length. These peptides are generally rich in basic amino acids (lysine and arginine) and are therefore usually cationic. They are also often amphiphilic in fact (ie, one part of the molecule is hydrophilic and the other is hydrophobic).

尽管经过了广泛的研究，抗微生物肽的作用方式仍然是科学界争论的主题。在许多情况下，数据表明这些两亲性肽在膜上组织起来形成孔或通道(Durell(1992))。在其他试验中，抗微生物肽表现为通过与膜缔合形成“地毯状物”而使膜破裂(Gazit(1995))。任意一种机理都通过令细胞膜去极化和基本细胞成分流失的方法使细胞破裂并杀死细胞。Despite extensive research, the mode of action of antimicrobial peptides remains a subject of debate in the scientific community. In many cases, data suggest that these amphiphilic peptides organize in membranes to form pores or channels (Durell (1992)). In other experiments, antimicrobial peptides have been shown to disrupt membranes by associating with them to form "carpets" (Gazit (1995)). Either mechanism ruptures and kills the cell by depolarizing the cell membrane and losing essential cellular components.

微生物选择性源于哺乳动物和微生物的细胞之间关于膜的脂类组成的差别。哺乳动物细胞膜的外层几乎完全由主要为卵磷脂、鞘磷脂和胆固醇的电中性的两性离子磷脂组成。相反，细菌膜主要由诸如磷脂酰甘油和双磷脂酰甘油带负电荷的磷脂组成。因此，细菌细胞对带阳离子的抗微生物肽敏感，而哺乳动物细胞却无此现象。也有一些迹象表明，癌细胞的膜成分与普通的哺乳动物细胞不同，这令肿瘤细胞对抗微生物肽敏感。Microbial selectivity arises from differences in the lipid composition of membranes between mammalian and microbial cells. The outer layer of mammalian cell membranes is composed almost entirely of electrically neutral, zwitterionic phospholipids mainly lecithin, sphingomyelin, and cholesterol. In contrast, bacterial membranes are mainly composed of negatively charged phospholipids such as phosphatidylglycerols and diphosphatidylglycerols. Thus, bacterial cells are sensitive to cationic antimicrobial peptides, whereas mammalian cells are not. There are also some indications that cancer cells have a different membrane composition than normal mammalian cells, which sensitizes tumor cells to antimicrobial peptides.

人们还认为，抗微生物肽具有抗HIV、单纯疱疹和细胞肥大病毒之类病毒的功效。但是，其机制略有不同。由于最外层的蛋白外壳，病毒通常对膜破坏机制免疫，但是多种抗微生物肽显示出通过阻碍病毒与宿主细胞壁融合(从而阻止病毒的遗传物质传递到宿主细胞内)，或者一旦宿主细胞壁被破坏时抑制病毒复制的机制而抗病毒的活性。Antimicrobial peptides are also believed to have efficacy against viruses such as HIV, herpes simplex and cytomegalovirus. However, the mechanism is slightly different. Viruses are normally immune to membrane disruption mechanisms due to the outermost protein coat, but a variety of antimicrobial peptides have been shown to work by impeding fusion of the virus with the host cell wall (thus preventing delivery of the virus' genetic material into the host cell), or once the host cell wall is Antiviral activity by inhibiting the mechanism of viral replication when disrupted.

考虑到病原体具有抗药性的普遍现象，如果能够经济地生产抗微生物肽，人们对采用抗微生物肽作为传统的小分子药物的替代品很感兴趣。但是，大量生产抗微生物肽是昂贵的，这实际上限制了抗微生物肽的大规模使用。例如，由于肽具有单一序列，因此肽的合成非常昂贵。必须将每一个氨基酸添加到生长的肽链上，其效率通常达不到最佳。因此，随着链长度的增加，其产率在下降。Considering the prevalence of drug resistance in pathogens, there is great interest in adopting antimicrobial peptides as alternatives to traditional small molecule drugs if they can be produced economically. However, mass production of antimicrobial peptides is expensive, which actually limits the large-scale use of antimicrobial peptides. For example, peptides are very expensive to synthesize because they have a single sequence. Every amino acid must be added to the growing peptide chain, often with suboptimal efficiency. Therefore, as the chain length increases, its yield decreases.

蛋白质的重组生产相对于固相合成具有一些优点，包括序列忠实性、方便、成本低，以及能够生产较长的蛋白质。但是，重组技术不能普遍应用，而且由于抗微生物肽易于杀死多种宿主细胞，重组生产抗微生物肽尤其困难。甚至当合成为非活性融合蛋白时，蛋白前体必须被切割以释放活性肽，且通常需要进一步纯化。这些附加的步骤提高了成本并降低了重组蛋白质的产率。Recombinant production of proteins has several advantages over solid-phase synthesis, including sequence fidelity, convenience, low cost, and the ability to produce longer proteins. However, recombinant technology is not universally applicable, and recombinant production of antimicrobial peptides is particularly difficult due to the tendency of antimicrobial peptides to kill a variety of host cells. Even when synthesized as inactive fusion proteins, the protein precursor must be cleaved to release the active peptide, and further purification is often required. These additional steps increase the cost and reduce the yield of recombinant protein.

Pittsburgh，PA的Demegen公司拥有几种为医用而开发的肽。一种是以商品名DEMEGELTM开发的D2A21(FAKKFAKKFKKFAKKFAKFAFAF)。这种单一抗微生物肽是一种使用4和3个氨基酸群以保持极性和非极性面成直线(3.6残基/圈)的两亲性α-螺旋肽。其通过传统方法——每次一个氨基酸——合成。Demegen Corporation of Pittsburgh, PA has several peptides in development for medical use. One is D2A21 (FAKKFAKKFKKFAKKFAKFAFAF) developed under the trade name DEMEGEL(TM). This single antimicrobial peptide is an amphipathic α-helical peptide using groups of 4 and 3 amino acids to keep the polar and nonpolar faces aligned (3.6 residues/turn). It is synthesized by traditional methods - one amino acid at a time.

D2A21具有抵御多种细胞类型的活性，包括阴道毛滴虫(T.vaginalis)、沙眼衣原体(C.trachomatis)和铜绿假单胞菌(T.aeruginosa)。初步结果已经证实了在大鼠前列腺癌模型中的抗肿瘤活性，以及存活率由25％提高到75％，而且没有引起任何明显的毒性。尽管对该活性的基本原理还不确定，人们认为，肿瘤细胞膜与正常细胞的细胞膜完全不同，因此更容易被抗微生物肽溶解(Arlotti(2001))。最后，已经表明D2A21具有抵御单纯疱疹病毒(HSV)的活性。当与经过修饰的类脂辛基甘油混合时，D2A21比其它五种抵御HSV的肽(包括爪蟾抗菌肽和防卫素)更好。D2A21 is active against a variety of cell types, including T. vaginalis, C. trachomatis, and T. aeruginosa. Preliminary results have demonstrated antitumor activity in a rat prostate cancer model, with an increase in survival from 25% to 75%, without causing any significant toxicity. Although the rationale for this activity is uncertain, it is believed that tumor cell membranes are quite different from those of normal cells and are therefore more readily lysed by antimicrobial peptides (Arlotti (2001)). Finally, D2A21 has been shown to be active against herpes simplex virus (HSV). When mixed with the modified lipid octylglycerol, D2A21 protected against HSV better than five other peptides, including magainin and defensins.

尽管前途光明，必须每次加入一个氨基酸地合成类似D2A21的肽，其成本大约为每克50到500美元。作为另一个例子，乳链菌肽是一种用于加工乳制品的抗微生物肽，其活性肽的销售价格大约为每磅6000美元。Although promising, peptides like D2A21 must be synthesized one amino acid at a time, at a cost of about $50 to $500 per gram. As another example, nisin, an antimicrobial peptide used in processed dairy products, sells for about $6,000 per pound for the active peptide.

一种可供选择的方法是设计具有多个氨基酸重复单元的肽。可以比合成长链肽更为廉价地合成氨基酸的短序列，并将重复单元寡聚以获得完整的肽长度。近年来，采用这种方法的成果包括US5789542和Javadpour(1996)。这些参考文献教导了可聚合为14和21个残基肽的7个残基(七聚物)的重复单元(其)可以生成抗微生物肽的基本成分。通过使用该七聚物，生成了“模拟的”，每圈具有3.5个氨基酸的α-螺旋。但是，七聚物的合成仍过于昂贵，因此限制了这种方法。An alternative approach is to design peptides with multiple amino acid repeat units. Short sequences of amino acids can be synthesized less expensively than long chain peptides and the repeat units oligomerized to achieve full peptide length. Recent results using this approach include US5789542 and Javadpour (1996). These references teach that repeating units of 7 residues (heptamers) that can be polymerized into 14 and 21 residue peptides (which) can generate the building blocks of antimicrobial peptides. By using this heptamer, a "mock" alpha-helix with 3.5 amino acids per turn was generated. However, the synthesis of heptamers is still too expensive, thus limiting this approach.

合意地存在一种方法，能够廉价地生产具有可与单一肽相比的含有抗微生物活性的肽。更合意地，由此方法合成的抗微生物肽无需忠实于传统的α-螺旋结构，以至于少于7个残基的小型重复单元可以用于组成最终的肽。因其简易，可以廉价地合成这种肽，且这种肽仍具有显著的抗微生物活性。It would be desirable to have a method that would enable inexpensive production of peptides having antimicrobial activity comparable to that of a single peptide. More desirably, the antimicrobial peptides synthesized by this method need not be faithful to the traditional α-helical structure, so that small repeating units of less than 7 residues can be used to make up the final peptide. Due to its simplicity, this peptide can be synthesized cheaply and still possesses significant antimicrobial activity.

发明概述Summary of the invention

本发明包括一种抗微生物周期肽的合成方法，并进一步包括这种肽本身及其多种用途。The present invention includes a synthetic method of antimicrobial periodic peptide, and further includes the peptide itself and its multiple uses.

在一种优选实施方案中，由4个或更少氨基酸的单体单元合成简单的肽。同样的单体单元首尾相连，直到达到大约15-16个氨基酸的最小尺寸。通过设计仅仅使用四聚物(4mer)、三聚物(3mer)或二聚物(2mer)单体的周期肽，与传统的定制合成法相比充分降低了生产成本。此外，即使给定的肽在每剂量基本成分的性能方面活性略低，其明显较低的生产成本仍能降低每单位剂量的成本。In a preferred embodiment, simple peptides are synthesized from monomeric units of 4 or fewer amino acids. Identical monomer units are joined end to end until a minimum size of approximately 15-16 amino acids is reached. By designing periodic peptides using only tetramer (4mer), trimer (3mer) or dimer (2mer) monomers, production costs are substantially reduced compared to traditional custom synthesis. Furthermore, even if a given peptide is slightly less active in terms of the performance of the base ingredient per dose, its significantly lower production cost can still reduce the cost per unit dose.

可以通过合成的方法或通过微生物、病毒或酶表达法生产单体。该单体越小，制备的成本越低。二聚的单体单元可以以低价格商业购得因而特别优选。同样的单体可以逐一多聚，以控制最终的尺寸，或者多聚为混合物，随后选择尺寸。可供选择的是也可以使用不同尺寸的混合物，而且这是尤其优选的具体实施方式。Monomers can be produced synthetically or by microbial, viral or enzymatic expression. The smaller the monomer, the lower the cost of preparation. Dimeric monomeric units are commercially available at low cost and are therefore particularly preferred. The same monomers can be polymerized one by one to control the final size, or as a mixture with subsequent size selection. Alternatively, a mixture of different sizes may be used, and this is a particularly preferred embodiment.

每个单体应含有带正电荷的氨基酸，例如赖氨酸、精氨酸等等。该单体还应含有疏水性的氨基酸，例如丙氨酸、缬氨酸等等，优选含有至少一种具有大侧链的疏水性氨基酸，例如苯丙氨酸。但是，当针对水疗法比较肽活性时不能发现清晰的趋势。Each monomer should contain positively charged amino acids such as lysine, arginine, etc. The monomer should also contain hydrophobic amino acids, such as alanine, valine, etc., preferably at least one hydrophobic amino acid with a large side chain, such as phenylalanine. However, no clear trend could be found when comparing peptide activity against hydrotherapy.

优选至少25％的(以数量计，而不是重量)为带正电荷的氨基酸，优选至少为30％。对含有75％阳离子残基(cationic residues)的周期肽的抗微生物活性进行了检测。Preferably at least 25% (by number, not weight) are positively charged amino acids, preferably at least 30%. The antimicrobial activity of periodic peptides containing 75% cationic residues was tested.

所得肽的总链长优选为至少14到16个氨基酸，但是在小至4个残基的肽中仍检测到活性。在活性方面尺寸的上限还没有发现，但是即使是有活性的，据认为，非常大的多聚体容易遇到稳定性或内吸收输送问题。因此，我们建议实际限制为大约50、80或100个残基，而且初步结果表明长约80个残基的肽是有活性的。所得肽的总链长更优选为长约14到40、或16到36、或20到24个氨基酸。The total chain length of the resulting peptides is preferably at least 14 to 16 amino acids, but activity is still detected in peptides as small as 4 residues. An upper size limit in terms of activity has not been found, but even if active, it is believed that very large multimers are prone to stability or systemic delivery problems. We therefore suggest a practical limit of approximately 50, 80 or 100 residues, and preliminary results suggest that peptides as long as approximately 80 residues are active. More preferably, the total chain length of the resulting peptide is about 14 to 40, or 16 to 36, or 20 to 24 amino acids in length.

该肽可以含有具有上述性质的天然的或合成的氨基酸。可以用D型或L型氨基酸合成这些肽。用D型氨基酸合成的肽的优点在于不易被蛋白水解降解。混合的肽应主要为D型(80％)，以便利用此特性。也可以利用非肽键以提高“肽”的稳定性。此处测试的肽并未封端，但具有自由的氨基和羧基末端。但是，如果需要可以采用封端和衍生。The peptide may contain natural or synthetic amino acids having the properties described above. These peptides can be synthesized using D or L amino acids. The advantage of peptides synthesized with D-amino acids is that they are less prone to proteolytic degradation. Mixed peptides should be predominantly D-form (80%) in order to take advantage of this property. Non-peptide bonds can also be utilized to increase the stability of the "peptide". The peptides tested here were not capped but had free amino and carboxy termini. However, capping and derivatization can be employed if desired.

“抗微生物活性”指小于或等于125微克/毫升的IC50的水平的抵御细菌、酵母、真菌和其它原生动物的活性。类似地定义抗细菌和抗真菌活性。“生物杀伤活性”指24小时时3.5对数杀伤(“Biocidal activity”means having killing activityof less than or equal to 125 ppm for 3.5 log kill at 24 hr)等于或低于125ppm的杀伤活性。“抗病毒活性”指IC50低于5mM、优选低于1mM时抵御病毒的活性。“抗肿瘤活性”指低于或等于250微克/毫升的TX50或(50％中毒量)水平的抵御肿瘤细胞的活性。"Antimicrobial activity" refers to activity against bacteria, yeast, fungi and other protozoa at levels less than or equal to an IC50 of 125 micrograms/ml. Antibacterial and antifungal activity are similarly defined. "Biocidal activity" means 3.5 log kill at 24 hours ("Biocidal activity" means having killing activity of less than or equal to 125 ppm for 3.5 log kill at 24 hr) equal to or lower than 125 ppm killing activity. "Antiviral activity" refers to the activity against viruses when the IC50 is lower than 5 mM, preferably lower than 1 mM. "Anti-tumor activity" refers to the activity against tumor cells at a level of TX50 or (50% toxic dose) lower than or equal to 250 μg/ml.

按照通常的方法可以合成许多周期性抗微生物肽。单体的通式为P2N2、P3N、PN2、P2N和NP，其中P为任意的阳离子残基，N为任意的疏水性残基，且N和P残基为任意次序(就所有情况而论，在所给单体中，第一和第二个P或N残基可以相同或不同)。优选序列包括PNNP、NNPP、NPPN、PPNN、PNPN、NPNP、PNP、NPP、PPN、NPN、PNN、NNP、NP和PN。优选地，P可以是K(赖氨酸)、O(鸟氨酸)或R(精氨酸)的任何一种，N可以是A(丙氨酸)、F(苯丙氨酸)、G(甘氨酸)、L(亮氨酸)、I(异亮氨酸)、T(苏氨酸)、Y(酪氨酸)、W(色氨酸)、V(缬氨酸)或M(甲硫氨酸)的任何一种。Many periodic antimicrobial peptides can be synthesized according to usual methods. The general formula of the monomers is P2N2, P3N, PN2, P2N and NP, where P is any cationic residue, N is any hydrophobic residue, and the N and P residues are in any order (in all cases, In a given monomer, the first and second P or N residues may be the same or different). Preferred sequences include PNNP, NNPP, NPPN, PPNN, PNPN, NPNP, PNP, NPP, PPN, NPN, PNN, NNP, NP and PN. Preferably, P can be any one of K (lysine), O (ornithine) or R (arginine), N can be A (alanine), F (phenylalanine), G (glycine), L (leucine), I (isoleucine), T (threonine), Y (tyrosine), W (tryptophan), V (valine) or M (formazine) thionine) of any kind.

周期肽具有多种用途，包括农业的(在牧场、果园、葡萄园、花园等中使用，用于控制细菌、真菌和病毒的疫情)；收获后谷物、水果和蔬菜的处理；兽医用途；个人卫生产品；婴儿产品；沐浴露；坚硬表面消毒剂；治疗传染病和肿瘤的医药用途；皮肤治疗(头皮屑、痤疮、牛皮癣)；药物渗透性增强剂；医疗器材处理；眼部治疗(传染病控制、隐形眼镜消毒、隐形眼镜护理液的防腐剂)；药物防腐(例如疫苗)；个人护理产品保存；家用产品保存；食物加工；肉类加工消毒剂；饮用水、果汁和饮料的防腐剂；以及食物的防腐剂。它们可以是洗涤剂、牙膏、坚硬表面清洁剂和消毒剂；浴室和厨房的清洁剂；除臭剂、纺织品和皮肤护理物等等中的活性成分。Periodic peptides have a variety of uses, including agricultural (use in pastures, orchards, vineyards, gardens, etc. to control bacterial, fungal and viral outbreaks); post-harvest treatment of grains, fruits and vegetables; veterinary use; personal Hygiene products; baby products; body washes; hard surface disinfectants; medicinal uses in the treatment of infectious diseases and tumors; skin treatments (dandruff, acne, psoriasis); drug penetration enhancers; medical device processing; eye treatments (infectious diseases control, contact lens disinfection, contact lens care solutions); drug preservation (such as vaccines); personal care product preservation; household product preservation; food processing; meat processing disinfectants; preservatives for drinking water, juices and beverages; and food preservatives. They can be active ingredients in detergents, toothpastes, hard surface cleaners and disinfectants; bathroom and kitchen cleaners; deodorants, textile and skin care products, and more.

实施例1Example 1

由商业肽合成商那里定制周期肽用于最初的抗微生物测试。目前为止测试的抗微生物肽包括表1中列示的那些。该肽的末端未封闭(自由的H和OH)。Periodic peptides were custom-made from commercial peptide synthesizers for initial antimicrobial testing. Antimicrobial peptides tested so far include those listed in Table 1. The ends of the peptide are not blocked (free H and OH).

表1具体的周期肽序列序列ID号序列 1. KFAK KFAK KFAK KFAK 2. KFAK KFAK KFAK KFAK KFAK 3. KFAK KFAK KFAK KFAK KFAK KFAK 4. KFAK KFAK KFAK KFAK KFAK KFAK KFAK 5. KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK 6. KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK 7. KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAKKFAK KFAK KFAK KFAK KFAK 8. KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAKKFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK 9. RFAR RFAR RFAR RFAR RFAR RFAR 10. RFAR RFAR RFAR RFAR RFAR RFAR RFAR 11. RFAR RFAR RFAR RFAR RFAR RFAR RFAR RFAR 12. FAKK FAKK FAKK FAKK FAKK FAKK 13. AKKF AKKF AKKF AKKF AKKF AKKF 14. KKFA KKFA KKFA KKFA KKFA KKFA 15. LKKL LKKL LKKL LKKL LKKL 16. LKKL LKKL LKKL LKKL LKKL LKKL 17. LKKL LKKL LKKL LKKL LKKL LKKL LKKL 18. LKKL LKKL LKKL LKKL LKKL LKKL LKKL LKKL 19. KFAF KFAF KFAF KFAF KFAF KFAF KFAF 20. KFFK KFFK KFFK KFFK KFFK KFFK KFFK 21. KFAK KFAK KFAK KFAK KFAK KFAK KFAK 22. KAAK KAAK KAAK KAAK KAAK KAAK KAAK 23. KKAK KKAK KKAK KKAK KKAK KKAK KKAK 24. KFK KFK KFK KFK KFK 25. KFK KFK KFK KFK KFK KFK 26. KFK KFK KFK KFK KFK KFK KFK 27. KFK KFK KFK KFK KFK KFK KFK KFK 28. KFKKFKKFKKFKKFKKFKKFKKFKKFK 29. KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK 30. KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK 31. KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFKKFK 32. KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFKKFK KFK KFK KFK KFK KFK 33. FKA FKA 34. FKA FKA FKA FKA 35. FKA FKA FKA FKA FKA FKA 36. FKA FKA FKA FKA FKA FKA FKA 37. FKA FKA FKA FKA FKA FKA FKA FKA 38. FKA FKA FKA FKA FKA FKA FKA FKA FKA 39. FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA 40. FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKAFKA 41. FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKAFKA FKA FKA FKA FKA 42. LK LK 43. LK LK LK LK LK 44. LK LK LK LK LK LK LK LK 45. LK LK LK LK LK LK LK LK LK 46. LK LK LK LK LK LK LK LK LK LK 47. LK LK LK LK LK LK LK LK LK LK LK 48. LK LK LK LK LK LK LK LK LK LK LK LK 49. LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK 50. LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LKLK LK 51. LR LR LR LR LR LR LR 52. LR LR LR LR LR LR LR LR LR 53. LR LR LR LR LR LR LR LR LR LR LR 54. KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK 55. KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGKKGK 56. KTK KTK KTK KTK KTK KTK KTK Table 1 Specific Periodic Peptide Sequences Serial ID number sequence 1. KFAK KFAK KFAK KFAK 2. KFAK KFAK KFAK KFAK KFAK 3. KFAK KFAK KFAK KFAK KFAK KFAK 4. KFAK KFAK KFAK KFAK KFAK KFAK KFAK 5. KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK 6. KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK 7. KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAKKFAK KFAK KFAK KFAK KFAK 8. KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAKKFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK KFAK 9. RFAR RFAR RFAR RFAR RFAR RFAR 10. RFAR RFAR RFAR RFAR RFAR RFAR RFAR 11. RFAR RFAR RFAR RFAR RFAR RFAR RFAR RFAR 12. FAKK FAKK FAKK FAKK FAKK FAKK 13. AKKF AKKF AKKF AKKF AKKF AKKF 14. KKFA KKFA KKFA KKFA KKFA KKFA 15. LKKL LKKL LKKL LKKL LKKL 16. LKKL LKKL LKKL LKKL LKKL LKKL 17. LKKL LKKL LKKL LKKL LKKL LKKL LKKL 18. LKKL LKKL LKKL LKKL LKKL LKKL LKKL LKKL 19. KFAF KFAF KFAF KFAF KFAF KFAF KFAF 20. KFFK KFFK KFFK KFFK KFFK KFFK KFFK twenty one. KFAK KFAK KFAK KFAK KFAK KFAK KFAK twenty two. KAAK KAAK KAAK KAAK KAAK KAAK KAAK twenty three. KKAK KKAK KKAK KKAK KKAK KKAK KKAK twenty four. KFK KFK KFK KFK KFK 25. KFK KFK KFK KFK KFK KFK 26. KFK KFK KFK KFK KFK KFK KFK 27. KFK KFK KFK KFK KFK KFK KFK KFK 28. KFKKFKKFKKFKKFKKFKKFKKFKKFK 29. KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK 30. KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK 31. KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFKKFK 32. KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFK KFKKFK KFK KFK KFK KFK KFK 33. FKA FKA 34. FKA FKA FKA FKA 35. FKA FKA FKA FKA FKA FKA FKA 36. FKA FKA FKA FKA FKA FKA FKA FKA 37. FKA FKA FKA FKA FKA FKA FKA FKA FKA 38. FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA 39. FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA 40. FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKAFKA 41. FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKA FKAFKA FKA FKA FKA FKA FKA 42. LK LK 43. LK LK LK LK LK 44. LK LK LK LK LK LK LK LK LK 45. LK LK LK LK LK LK LK LK LK 46. LK LK LK LK LK LK LK LK LK LK 47. LK LK LK LK LK LK LK LK LK LK LK LK 48. LK LK LK LK LK LK LK LK LK LK LK LK 49. LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK 50. LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK LK 51. LR LR LR LR LR LR LR LR 52. LR LR LR LR LR LR LR LR LR LR 53. LR LR LR LR LR LR LR LR LR LR LR LR 54. KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK 55. KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGK KGKKGK 56. KTK KTK KTK KTK KTK KTK KTK

实施例2：抗微生物测试Example 2: Antimicrobial Testing

通过按照如下的National Committee for Clinical Laboratory Standard的指导方针的微量肉汤稀释法测定MIC和IC50值：在96孔组织培养盘中，将2X肉汤(如下定义)中固定体积的细菌悬液加入到浓度1,000到1微克/毫升之间改变的混合物或单一化合物中，该混合物或单一化合物通过使用无菌水进行系列二倍稀释而获得。测试的细菌为绿脓杆菌美国标准菌种收藏所编号(ATCC)10145、大肠杆菌ATCC 2592和金黄色葡萄球菌(耐甲氧西林)ATCC 33591。MIC and IC50 values were determined by the broth microdilution method following the guidelines of the National Committee for Clinical Laboratory Standard: In a 96-well tissue culture dish, a fixed volume of bacterial suspension in 2X broth (defined below) was added to In mixtures or single compounds varying in concentration between 1,000 and 1 μg/ml, the mixtures or single compounds were obtained by serial two-fold dilutions with sterile water. The bacteria tested were Pseudomonas aeruginosa American Type Culture Collection number (ATCC) 10145, Escherichia coli ATCC 2592, and Staphylococcus aureus (methicillin-resistant) ATCC 33591.

所有试验与最适生长条件(37℃、pH7.0、没有额外的盐、培养基-被称为标准生长条件)下的细菌生长比较。在每个盘中，检测无生长对照(仅有培养基)、阳性生长对照(细菌与非测试样品)、阳性抗微生物对照(具有已知抗微生物活性的试剂)和测试化合物。阳性对照包括非周期型的抗微生物肽D2A21(FAKKFAKKFKKFAKKFAKFAFAF)和D4E1(FKLRAKIKVRLRAKIKL)。All experiments were compared to bacterial growth under optimal growth conditions (37°C, pH 7.0, no additional salt, medium - referred to as standard growth conditions). In each plate, no growth control (medium only), positive growth control (bacteria vs. no test sample), positive antimicrobial control (agent with known antimicrobial activity) and test compound were tested. Positive controls included non-periodic antimicrobial peptides D2A21 (FAKKFAKKFKKFAKKFAKFAFAF) and D4E1 (FKLRAKIKVRLRAKIKL).

将盘培养过夜，使用TITERTEK MULTISKAN PLUS^TM在620纳米处测得的光密度(OD620)确定相对生长百分比。MIC被定义为导致98％的生长抑制的测试样品的最低浓度。采用S型曲线拟合软件程序(CRAPHPAD，^TMISI Software，^TMSan Diego，CA)计算IC50值。一式两份测试所有样品，每次化验重复至少两次。在下表中，KFAK为重复单元，并检测了KFAK的多聚体。以微克/毫升为单位报道IC50和MIC。Plates were incubated overnight and relative percent growth was determined using optical density at 620 nm (OD620) measured on a TITERTEK MULTISKAN PLUS ^™ . MIC was defined as the lowest concentration of test sample that resulted in 98% growth inhibition. IC50 values were calculated using a sigmoid curve fitting software program (CRAPHPAD, ^TM ISI Software, ^TM San Diego, CA). All samples were tested in duplicate with at least two replicates per assay. In the table below, KFAK is the repeating unit, and multimers of KFAK were detected. IC50 and MIC are reported in micrograms/ml.

表2.(KFAK)_n抵御绿脓杆菌的抗微生物活性： N 序列ID号 IC50 MIC D2A21 - 11.3 32-64 1 - ＞250 ＞250 2 - ＞250 ＞250 3 - ＞250 ＞250 4 1 116 ＞250 5 2 7.7 16-32 6 3 5.6 16-32 7 4 12 16-32 Table 2. Antimicrobial activity of (KFAK) _n against Pseudomonas aeruginosa: N Serial ID number IC50 Mic D2A21 - 11.3 32-64 1 - >250 >250 2 - >250 >250 3 - >250 >250 4 1 116 >250 5 2 7.7 16-32 6 3 5.6 16-32 7 4 12 16-32

出乎意料的是，这些周期肽的表现相当于或优于它们的单一对应物(D2A21)。受到此令人惊讶的结果的鼓舞，合成多种周期肽，并采用同样的实验方案测试其抗菌活性。结果列示在下文中。Unexpectedly, these periodic peptides performed as well as or better than their single counterpart (D2A21). Encouraged by this surprising result, various periodic peptides were synthesized and tested for their antibacterial activity using the same protocol. The results are listed below.

表3：周期肽和抗菌活性氨基酸数目 ID 序列ID号绿脓杆菌10145革兰氏阴性大肠杆菌2592革兰氏阴性金黄色葡萄球菌(耐甲氧西林)33591革兰氏阳性 IC50(微克/毫升) MIC(微克/毫升) IC50(微克/毫升) MIC(微克/毫升) IC50(微克/毫升) MIC(微克/毫升) D2A21 - 11.3 32-62 4.5 8-16 11.9 62-125 D4E1 - 6.2 8-16 3.3 8-16 6.3 32-62 4 KFAK(1) - ＞500 500 ＞250 ＞250 ＞500 ＞500 8 KFAK(2) - ＞250 250 ＞250 ＞250 ＞250 ＞250 12 KFAK(3) - ＞500 500 ＞250 ＞250 ＞500 ＞500 16 KFAK(4) 1 85 125 46 62-125 ＞250 ＞250 20 KFAK(5) 2 5.5 8 6 16-32 ＞250 ＞250 24 KFAK(6) 3 5.6 16 4 8-16 38.7 125-250 28 KFAK(7) 4 12.2 16 8 32-62 108.8 ＞250 32 KFAK(8) 5 19 20 12 16-32 40 125-250 24 RFAR(6) 9 25 32 12 31-62 15 31-62 28 RFAR(7) 10 20 62 8 32-62 31 125-250 32 RFAR(8) 11 30 62 12 32-62 20 125-250 24 FAKK(6) 12 21 62 11 17-32 108 ＞250 24 AKKF(6) 13 21 25 8 16-32 41 125-250 24 KKFA(6) 14 33 62 7 16-32 136 ＞250 20 LKKL(5) 15 27 62 49 62-125 69 ＞250 24 LKKL(6) 16 58 65 31 62-125 98 ＞250 28 LKKL(7) 17 96 125 41 62-125 252 ＞250 32 LKKL(8) 18 61 125 37 62-125 80 ＞250 28 KFAF(7) 19 250 ＞250 ＞1000 ＞1000 ＞500 ＞500 28 KFFK(7) 20 87 125 63 ＞125 133 ＞250 28 KFAK(7) 21 12.2 16 8 32-62 108.8 ＞250 28 KAAK(7) 22 ＞250 ＞250 261 500- ＞500 ＞500 28 KKAK(7) 23 99 125 26 31-62 60 0 18 KFK(6) 25 74 125 39 62-125 322 ＞500 21 KFK(7) 26 87 125 18 31-62 141 ＞500 24 KFK(8) 27 145 250 20 32-62 72 ＞250 27 KFK(9) 28 68 125 14 16-32 92 125-250 20 KFK(10) 29 31 62 10 12-16 59 ＞250 18 FKA(6) 35 79 100 29 32-62 ＞500 ＞500 21 FKA(7) 36 23 32 7 16-32 ＞500 ＞500 24 FKA(8) 37 19 25 7 16-32 74 ＞500 27 FKA(9) 38 16 20 4 31-62 17 31-62 30 FKA(10) 39 27 32 9 31-62 12 31-62 14 LK(7) - 111 125 37 45-62 78 250-500 16 LK(8) 44 125 130 65 ＞125 56 125-250 18 LK(9) 45 74 80 35 62-125 50 250-500 20 LK(10) 46 47 125 127 150-250 29 125-250 22 LK(11) 47 51 62 50 ＞125 111 ＞250 24 LK(12) 48 41 45 23 62-125 85 ＞250 14 LR(7) 51 109 125 15 31-62 90 250-500 18 LR(9) 52 ＞250 ＞250 70 ＞125 28 125-250 22 LR(11) 53 ＞250 ＞250 96 125-250 39 125-250 ^*PBF16a - ＞250 ＞250 / / / / ^**PBF16b - ＞250 ＞250 / / / / ^***PBF16c - ＞250 ＞250 / / / / Table 3: Periodic peptides and antibacterial activity amino acid number ID Serial ID number Pseudomonas aeruginosa 10145 Gram-negative E. coli 2592 Gram-negative Staphylococcus aureus (methicillin resistant) 33591 Gram positive IC50(μg/ml) MIC (microgram/ml) IC50(μg/ml) MIC (microgram/ml) IC50(μg/ml) MIC (microgram/ml) D2A21 - 11.3 32-62 4.5 8-16 11.9 62-125 D4E1 - 6.2 8-16 3.3 8-16 6.3 32-62 4 KFAK(1) - >500 500 >250 >250 >500 >500 8 KFAK(2) - >250 250 >250 >250 >250 >250 12 KFAK(3) - >500 500 >250 >250 >500 >500 16 KFAK(4) 1 85 125 46 62-125 >250 >250 20 KFAK(5) 2 5.5 8 6 16-32 >250 >250 twenty four KFAK(6) 3 5.6 16 4 8-16 38.7 125-250 28 KFAK(7) 4 12.2 16 8 32-62 108.8 >250 32 KFAK(8) 5 19 20 12 16-32 40 125-250 twenty four RFAR(6) 9 25 32 12 31-62 15 31-62 28 RFAR(7) 10 20 62 8 32-62 31 125-250 32 RFAR(8) 11 30 62 12 32-62 20 125-250 twenty four FAKK(6) 12 twenty one 62 11 17-32 108 >250 twenty four AKKF(6) 13 twenty one 25 8 16-32 41 125-250 twenty four KKFA(6) 14 33 62 7 16-32 136 >250 20 LKKL(5) 15 27 62 49 62-125 69 >250 twenty four LKKL(6) 16 58 65 31 62-125 98 >250 28 LKKL(7) 17 96 125 41 62-125 252 >250 32 LKKL(8) 18 61 125 37 62-125 80 >250 28 KFAF(7) 19 250 >250 >1000 >1000 >500 >500 28 KFFK(7) 20 87 125 63 >125 133 >250 28 KFAK(7) twenty one 12.2 16 8 32-62 108.8 >250 28 KAAK(7) twenty two >250 >250 261 500- >500 >500 28 KKAK(7) twenty three 99 125 26 31-62 60 0 18 KFK(6) 25 74 125 39 62-125 322 >500 twenty one KFK(7) 26 87 125 18 31-62 141 >500 twenty four KFK(8) 27 145 250 20 32-62 72 >250 27 KFK(9) 28 68 125 14 16-32 92 125-250 20 KFK(10) 29 31 62 10 12-16 59 >250 18 FKA(6) 35 79 100 29 32-62 >500 >500 twenty one FKA(7) 36 twenty three 32 7 16-32 >500 >500 twenty four FKA(8) 37 19 25 7 16-32 74 >500 27 FKA(9) 38 16 20 4 31-62 17 31-62 30 FKA(10) 39 27 32 9 31-62 12 31-62 14 LK(7) - 111 125 37 45-62 78 250-500 16 LK(8) 44 125 130 65 >125 56 125-250 18 LK(9) 45 74 80 35 62-125 50 250-500 20 LK(10) 46 47 125 127 150-250 29 125-250 twenty two LK(11) 47 51 62 50 >125 111 >250 twenty four LK(12) 48 41 45 twenty three 62-125 85 >250 14 LR(7) 51 109 125 15 31-62 90 250-500 18 LR(9) 52 >250 >250 70 >125 28 125-250 twenty two LR(11) 53 >250 >250 96 125-250 39 125-250 ^* PBF16a - >250 >250 / / / / ^** PBF16b - >250 >250 / / / / ^*** PBF16c - >250 >250 / / / /

^*PBF16a＝KFAKKFAKKFAKKAAK(非周期性) ^* PBF16a=KFAKKFAKKFAKKAAK (aperiodic)

^**PBF16b＝KFAKKFAKKAAKKAAK(非周期性) ^** PBF16b=KFAKKFAKKAAKKAAK (aperiodic)

^***PBF16c＝KFAKKAAKKFAKKAAK(非周期性) ^*** PBF16c=KFAKKAAKKFAKKAAK (aperiodic)

回顾这些结果，很容易浮现几种模式。首先，由小至二聚体的单体单元合成的周期肽显示出强的抗微生物活性(例如，LK(7-12))，这是很清楚的。考虑到大多数抗微生物肽教导必须保持螺旋结构，并为此使用重复的七聚体，而且几乎不存在具有β折叠的片状结构的抗微生物肽，这一点非常令人惊讶。Looking back at these results, several patterns readily emerge. First, it is clear that periodic peptides synthesized from monomeric units as small as dimers exhibit strong antimicrobial activity (eg, LK(7-12)). This is quite surprising, considering that most antimicrobial peptides teach that helical structure must be maintained, and use repeating heptamers for this purpose, and that there are few antimicrobial peptides with a β-sheet sheet-like structure.

其次，这表明，较长的肽比较小的肽更有效。例如，肽应至少为大约14到16个氨基酸以显示最佳效果(将KFAK(1-3)与KFAK(4-8)对比)Second, this suggests that longer peptides are more effective than smaller ones. For example, peptides should be at least approximately 14 to 16 amino acids to show optimal effect (compare KFAK(1-3) to KFAK(4-8))

第三，在许多情况下周期肽表现出比现有技术的单一肽(将D2A21和D4E1与FKAK(5)对比)更好的活性。由于这意味着可以用具有相等或更佳的功效的周期肽取代现有技术的单一肽而充分节约了成本，因此特别有用。Third, in many cases period peptides showed better activity than single peptides of the prior art (compare D2A21 and D4E1 with FKAK (5)). This is particularly useful since it means that prior art single peptides can be replaced by periodic peptides with equal or better efficacy at substantial cost savings.

第四，测试的具有相似残基含量的非周期肽未显示抗微生物活性(参见PBF16a-c)。Fourth, tested aperiodic peptides with similar residue content showed no antimicrobial activity (see PBF16a-c).

实施例3：抗真菌测试Example 3: Antifungal Test

由于周期肽的抗菌活性很有希望，采用进一步实验以确定周期肽是否也具有抗真菌活性。实验设计与上面的相似，为真菌生长的需要作出适应性调节，包括使用沙氏葡萄糖肉汤(SDB)和沙氏葡萄糖琼脂(SDA)斜面培养。Since the antibacterial activity of period peptides was promising, further experiments were undertaken to determine whether period peptides also possess antifungal activity. The experimental design was similar to that above, adapting to the needs of fungal growth, including the use of Sabouraud Dextrose Broth (SDB) and Sabouraud Dextrose Agar (SDA) slants.

表4：周期肽和抗真菌活性氨基 ID 序列ID号酵母-纯白假丝酵母-10231 真菌-新型隐球菌-32045 酸数 IC50(微克/毫升) MIC(微克/毫升) IC50(微克/毫升) MIC(微克/毫升) D2A21 - 121.5 ＞250 21.9 62-125 D4E1 - 70.6 80-125 1.8 8-16 4 KFAK(1) - 329 ＞500 400 ＞500 8 KFAK(2) - ＞250 ＞250 ＞250 ＞250 12 KFAK(3) - 262 280-500 22 125-250 16 KFAK(4) 1 70 90-125 5.3 6.0-8.0 20 KFAK(5) 2 66 90-125 5 8-16 24 KFAK(6) 3 72 80-125 5.8 16-32 28 KFAK(7) 4 82 100-125 9.1 32-62 32 KFAK(8) 5 105 125-250 4.1 8-16 24 RFAR(6) 9 114 125-250 35 62-125 28 RFAR(7) 10 172 250-500 45 62-125 32 RFAR(8) 11 144 250-500 32 62-125 24 FAKK(6) 12 110 125-250 7.6 16-32 24 AKKF(6) 13 225 250-500 11 16-32 24 KKFA(6) 14 107 125-250 12 16-32 20 LKKL(5) 15 211 ＞500 55 62-125 24 LKKL(6) 16 213 250-500 93 ＞125 28 LKKL(7) 17 199 250-500 85 100-125 32 LKKL(8) 18 179 250-500 56 62-125 28 KFAF(7) 19 ＞500 ＞500 67 125-250 28 KFFK(7) 20 183 250-500 55 62-125 28 KFAK(7) 21 82 100-125 9 32-62 28 KAAK(7) 22 262 ＞500 41 62-125 28 KKAK(7) 23 ＞500 ＞500 28 ＞63 18 KFK(6) 25 164 250-500 33 40-62 21 KFK(7) 26 175 250-500 26 40-62 24 KFK(8) 27 113 125-250 25 40-62 27 KFK(9) 28 194 250-500 39 62-125 20 KFK(10) 29 104 125-250 30 40-62 18 FKA(6) 35 208 250-500 2.8 4-8 21 FKA(7) 36 203 250-500 3.2 4-8 24 FKA(8) 37 199 250-500 3.9 16-32 27 FKA(9) 38 195 250-500 16 62-125 30 FKA(10) 39 182 250-500 20 32-62 14 LK(7) - 166 250-500 0.8 2-4 16 LK(8) 44 162 250-500 9.7 32-63 18 LK(9) 45 216 250-500 4.9 32-62 20 LK(10) 46 187 250-500 3.0 62-125 22 LK(11) 47 185 250-500 26 32-62 24 LK(12) 48 175 250-500 22 32-62 14 LR(7) 51 182 250-500 1.8 4-8 18 LR(9) 52 195 250-500 21 62-125 22 LR(11) 53 ＞500 ＞500 3.3 62-125 Table 4: Periodic peptides and antifungal activity Amino ID Serial ID number Yeast - Candida albicans - 10231 Fungi - Cryptococcus neoformans - 32045 acid number IC50(μg/ml) MIC (microgram/ml) IC50(μg/ml) MIC (microgram/ml) D2A21 - 121.5 >250 21.9 62-125 D4E1 - 70.6 80-125 1.8 8-16 4 KFAK(1) - 329 >500 400 >500 8 KFAK(2) - >250 >250 >250 >250 12 KFAK(3) - 262 280-500 twenty two 125-250 16 KFAK(4) 1 70 90-125 5.3 6.0-8.0 20 KFAK(5) 2 66 90-125 5 8-16 twenty four KFAK(6) 3 72 80-125 5.8 16-32 28 KFAK(7) 4 82 100-125 9.1 32-62 32 KFAK(8) 5 105 125-250 4.1 8-16 twenty four RFAR(6) 9 114 125-250 35 62-125 28 RFAR(7) 10 172 250-500 45 62-125 32 RFAR(8) 11 144 250-500 32 62-125 twenty four FAKK(6) 12 110 125-250 7.6 16-32 twenty four AKKF(6) 13 225 250-500 11 16-32 twenty four KKFA(6) 14 107 125-250 12 16-32 20 LKKL(5) 15 211 >500 55 62-125 twenty four LKKL(6) 16 213 250-500 93 >125 28 LKKL(7) 17 199 250-500 85 100-125 32 LKKL(8) 18 179 250-500 56 62-125 28 KFAF(7) 19 >500 >500 67 125-250 28 KFFK(7) 20 183 250-500 55 62-125 28 KFAK(7) twenty one 82 100-125 9 32-62 28 KAAK(7) twenty two 262 >500 41 62-125 28 KKAK(7) twenty three >500 >500 28 >63 18 KFK(6) 25 164 250-500 33 40-62 twenty one KFK(7) 26 175 250-500 26 40-62 twenty four KFK(8) 27 113 125-250 25 40-62 27 KFK(9) 28 194 250-500 39 62-125 20 KFK(10) 29 104 125-250 30 40-62 18 FKA(6) 35 208 250-500 2.8 4-8 twenty one FKA(7) 36 203 250-500 3.2 4-8 twenty four FKA(8) 37 199 250-500 3.9 16-32 27 FKA(9) 38 195 250-500 16 62-125 30 FKA(10) 39 182 250-500 20 32-62 14 LK(7) - 166 250-500 0.8 2-4 16 LK(8) 44 162 250-500 9.7 32-63 18 LK(9) 45 216 250-500 4.9 32-62 20 LK(10) 46 187 250-500 3.0 62-125 twenty two LK(11) 47 185 250-500 26 32-62 twenty four LK(12) 48 175 250-500 twenty two 32-62 14 LR(7) 51 182 250-500 1.8 4-8 18 LR(9) 52 195 250-500 twenty one 62-125 twenty two LR(11) 53 >500 >500 3.3 62-125

结果表明，一些周期肽能够非常有效地抵御真菌病原体(例如，KFAF(7)，KFK(10))，尽管大多数是种特异的。The results showed that some periodic peptides are very effective against fungal pathogens (eg, KFAF(7), KFK(10)), although most are species-specific.

实施例4：抗肿瘤测试Example 4: Antitumor Test

受到周期肽的强抗微生物活性的鼓舞，同样测试肿瘤细胞。采用红血球(RBC)确定周期肽不会杀灭正常的哺乳动物细胞，这明显限制了它们的效力。Encouraged by the strong antimicrobial activity of cyclic peptides, tumor cells were also tested. The use of red blood cells (RBC) to determine that cyclins do not kill normal mammalian cells significantly limits their potency.

RBC实验方案来自于Blondelle(2000)，通常描述如下：使用MTS(3-(4，5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑，钠盐)细胞减少测定法测定对HeLa细胞系的毒性。在Dulbecco PBS中(pH7.35)制备MTS(2毫克/毫升)，过滤，等分并在-20℃下贮藏。在96孔平底盘中，细胞悬液(每孔中6×10⁴细胞/毫升250微升，)在37℃下培养48小时(5％CO2恒温箱)。随即以通过系列二倍稀释法获得的不同浓度的肽(50微升)加入到细胞单层中(当各个孔中的培养基被抽吸掉，并接着加入50微升的Dulbecco’s Modified Eagles Medium之后)，然后将该盘在37℃下条件下培养24小时(5％CO2恒温箱)。随即在检测前以1∶20的比例将吩嗪硫酸甲酯溶液(PMS：在DPBS中0.92毫克/毫升)加入到MTS中，由此测定增殖。向每个孔中加入二十微升MTS-PMS溶液，并将该盘在37℃下培养1小时(5％CO2恒温箱)。比较490纳米时测得的含有每种肽的细胞的吸光度与没有肽的细胞的吸光度，由此确定毒性的相对百分比。使用S型曲线拟合软件(Graphpad Prism)计算TX50(50％毒性所需的浓度)。The RBC protocol is from Blondelle (2000) and is generally described as follows: using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-( 4-sulfophenyl)-2H-tetrazole, sodium salt) cytoreduction assay to determine toxicity to HeLa cell line. MTS (2 mg/ml) was prepared in Dulbecco's PBS (pH 7.35), filtered, aliquoted and stored at -20°C. In a 96-well flat bottom plate, the cell suspension (6×10 ⁴ cells/ml in 250 μl per well) was incubated at 37° C. for 48 hours (5% CO 2 incubator). Peptides (50 microliters) at different concentrations obtained by serial doubling dilutions were then added to the cell monolayer (after the medium in each well was aspirated, followed by the addition of 50 microliters of Dulbecco's Modified Eagles Medium ), and then the plate was incubated at 37°C for 24 hours (5% CO2 incubator). Proliferation was determined by adding phenazine methyl sulfate solution (PMS: 0.92 mg/ml in DPBS) at a ratio of 1:20 to MTS immediately prior to the assay. Twenty microliters of MTS-PMS solution was added to each well, and the plate was incubated at 37° C. for 1 hour (5% CO 2 incubator). The relative percentage of toxicity was determined by comparing the absorbance at 490 nm of cells containing each peptide to the absorbance of cells without the peptide. TX50 (concentration required for 50% toxicity) was calculated using sigmoid curve fitting software (Graphpad Prism).

表5：周期肽和抗肿瘤活性氨基酸数 ID 序列ID号 RBCs HeLa TX50(微克/毫升) 250微克/毫升时的杀灭百分比 TX50(微克/毫升) 500微克/毫升时的杀灭百分比 D2A21 64 52 93.1 16.9 7.1 D4E1 65 133.6 77.2 ＞500 55.2 4 KFAK(1) 57 ＞500 2.8％＞500 112 8 KFAK(2) 58 ＞250 2.2％＞500 83 12 KFAK(3) 59 ＞500 0.4％＞500 77 16 KFAK(4) 1 ＞250 0.1％＞500 76 20 KFAK(5) 2 ＞250 4.1％＞500 89 24 KFAK(6) 3 ＞250 7.3 457.3 30.5 28 KFAK(7) 4 ＞250 13.7 204.1 13.0 32 KFAK(8) 5 ＞500 48％ 249 18.2％ 24 RFAR(6) 9 176 72％ 159 22.1％ 28 RFAR(7) 10 20 100％ 106 12.4％ 32 RFAR(8) 11 17 100％ 71 9.5％ 24 FAKK(6) 12 ＞500 7％ 359 28.3％ 24 AKKF(6) 13 39 100％ 33 7.3％ 24 KKFA(6) 14 ＞500 2％＞500 76.4％ 20 LKKL(5) 15 9 100％ 31 -0.7％ 24 LKKL(6) 16 11 100％ 26 -1.3％ 28 LKKL(7) 17 9 100％ 25 -0.4％ 32 LKKL(8) 18 8 100％ 23 0.1％ 28 KFAF(7) 19 ＞500 4％＞500 76.8％ 28 KFFK(7) 20 12 100％ 30 -0.5％ 28 KFAK(7) 21 ＞250 13.7 204.1 13.0 28 KAAK(7) 22 ＞500 -6％＞500 90.0％ 28 KKAK(7) 23 ＞500 -6％＞500 ＞87.9％ 18 KFK(6) 25 ＞500 -7％＞500 77.8％ 21 KFK(7) 26 ＞500 -7％＞500 80.9％ 24 KFK(8) 27 ＞500 -6％＞500 84.2％ 27 KFK(9) 28 ＞500 -6％＞500 80.7％ 20 KFK(10) 29 ＞500 3％＞500 56.8％ 18 FKA(6) 35 496 28％＞500 110.8％ 21 FKA(7) 36 ＞500 11％＞500 103.3％ 24 FKA(8) 37 ＞500 10％＞500 60.4％ 27 FKA(9) 38 ＞500 24％ 389 20.9％ 30 FKA(10) 39 ＞500 51％ 234 15.0％ 14 LK(7) 60 ＞500 16％ 309 29.6％ 16 LK(8) 44 ＞500 13％ 122 43.5％ 18 LK(9) 45 ＞500 21％ 137 46.0％ 20 LK(10) 46 ＞500 27％＞500 65.0％ 22 LK(11) 47 ＞500 24％ 202 43.8％ 24 LK(12) 48 ＞500 35％ 49 60.0％ 14 LR(7) 51 ＞500 15％＞500 86.5％ 18 LR(9) 52 ＞500 24％＞500 96.7％ 22 LR(11) 53 450 41％＞500 66.6％ Table 5: Periodic peptides and antitumor activity amino acid number ID Serial ID number RBCs HeLa TX50(μg/ml) Percent kill at 250 μg/ml TX50(μg/ml) Percent kill at 500 μg/ml D2A21 64 52 93.1 16.9 7.1 D4E1 65 133.6 77.2 >500 55.2 4 KFAK(1) 57 >500 2.8% >500 112 8 KFAK(2) 58 >250 2.2% >500 83 12 KFAK(3) 59 >500 0.4% >500 77 16 KFAK(4) 1 >250 0.1% >500 76 20 KFAK(5) 2 >250 4.1% >500 89 twenty four KFAK(6) 3 >250 7.3 457.3 30.5 28 KFAK(7) 4 >250 13.7 204.1 13.0 32 KFAK(8) 5 >500 48% 249 18.2% twenty four RFAR(6) 9 176 72% 159 22.1% 28 RFAR(7) 10 20 100% 106 12.4% 32 RFAR(8) 11 17 100% 71 9.5% twenty four FAKK(6) 12 >500 7% 359 28.3% twenty four AKKF(6) 13 39 100% 33 7.3% twenty four KKFA(6) 14 >500 2% >500 76.4% 20 LKKL(5) 15 9 100% 31 -0.7% twenty four LKKL(6) 16 11 100% 26 -1.3% 28 LKKL(7) 17 9 100% 25 -0.4% 32 LKKL(8) 18 8 100% twenty three 0.1% 28 KFAF(7) 19 >500 4% >500 76.8% 28 KFFK(7) 20 12 100% 30 -0.5% 28 KFAK(7) twenty one >250 13.7 204.1 13.0 28 KAAK(7) twenty two >500 -6% >500 90.0% 28 KKAK(7) twenty three >500 -6% >500 >87.9% 18 KFK(6) 25 >500 -7% >500 77.8% twenty one KFK(7) 26 >500 -7% >500 80.9% twenty four KFK(8) 27 >500 -6% >500 84.2% 27 KFK(9) 28 >500 -6% >500 80.7% 20 KFK(10) 29 >500 3% >500 56.8% 18 FKA(6) 35 496 28% >500 110.8% twenty one FKA(7) 36 >500 11% >500 103.3% twenty four FKA(8) 37 >500 10% >500 60.4% 27 FKA(9) 38 >500 twenty four% 389 20.9% 30 FKA(10) 39 >500 51% 234 15.0% 14 LK(7) 60 >500 16% 309 29.6% 16 LK(8) 44 >500 13% 122 43.5% 18 LK(9) 45 >500 twenty one% 137 46.0% 20 LK(10) 46 >500 27% >500 65.0% twenty two LK(11) 47 >500 twenty four% 202 43.8% twenty four LK(12) 48 >500 35% 49 60.0% 14 LR(7) 51 >500 15% >500 86.5% 18 LR(9) 52 >500 twenty four% >500 96.7% twenty two LR(11) 53 450 41% >500 66.6%

如期待的那样，周期肽的一部分具有抗肿瘤活性，但不会破坏诸如红血球(RBCs)之类的正常细胞。尤其是二肽LK(8-9，12)表现出极好的前景；杀死HeLa细胞，而不是RBCs。但是，抗肿瘤活性的可预知性比抗微生物活性稍差，在使用前需要针对细胞范围对每种周期肽进行检测。As expected, a portion of the cyclic peptides had antitumor activity but did not destroy normal cells such as red blood cells (RBCs). Especially the dipeptide LK (8-9, 12) showed excellent promise; killing HeLa cells, but not RBCs. However, antitumor activity is less predictable than antimicrobial activity, and each cycle peptide needs to be tested on a cell scale before use.

实施例5：生物杀灭测试Example 5: Biocidal Test

由于IC50和MIC是易于实施的简单、普通的测试，因而在前面的实验中测量IC50和MIC。但是，这些测试事实上测量的是生物静态(biostat)活性，而不是真正的生物杀灭活性。因此，在本实施例中测量生物杀灭活性。Since IC50 and MIC are simple, common tests that are easy to implement, IC50 and MIC were measured in previous experiments. However, these tests actually measure biostatic (biostat) activity rather than true biocidal activity. Therefore, biocidal activity was measured in this example.

通过降低试样量、将检测材料组织为阵列结构、确定最大可能数量(MPN)以及使用多通道液体处理装置的方法，改善生物杀灭功效的实验方案。我们称这种新的方法为“高通量微量分析和快速定量”或“HMARQ”。HMARQ直接适用于现有的工业的药效测试，例如多循环保藏激发法或时程消毒测试。Protocols to improve biocidal efficacy by reducing sample size, organizing test materials into array structures, determining maximum probable numbers (MPNs), and using multichannel liquid handling devices. We call this new method "High Throughput Microanalysis and Rapid Quantification" or "HMARQ". HMARQ is directly applicable to existing industrial efficacy testing, such as multi-cycle preservation challenge or time-course disinfection testing.

在高通过量盘中实施HMARQ，例如96孔微滴定量培养盘。典型样品的体积已经降低到200至300微升，但是如果需要可以进一步降低。在这些实施例中，不超过总体积的10％包含生物杀灭剂和有机体溶液，对于所有样品来说，所有的非基质添加物均规格化。Implement HMARQ in high-throughput plates, such as 96-well microtiter plates. Typical sample volumes have been reduced to 200 to 300 microliters, but can be further reduced if required. In these examples, no more than 10% of the total volume contained biocide and organism solution, and all non-matrix additions were normalized for all samples.

首先将合意浓度的微生物接种到样品基质。随后将接种过的样品基质加入到含有待研究的生物杀灭剂的96孔化验模块上。每个样品模块含有生物杀灭剂处理过的样品和未处理过的对照试样(没有生物杀灭剂)。一旦样品制备，通过涡流将样品的整个模块混合，直到每个样品均为均质的。通常，混合一结束就开始研究，并根据分析的需要移动样品。当进行杀灭时间测试(生物杀灭剂活性的速度)时，考虑到快速混合和分析，在将生物杀灭剂加入到样品中之后，加入微生物。The sample matrix is first inoculated with the desired concentration of microorganisms. The inoculated sample matrix is then added to the 96-well assay block containing the biocide to be studied. Each sample block contained a biocide-treated sample and an untreated control sample (no biocide). Once the samples are prepared, the entire block of samples is mixed by vortexing until each sample is homogeneous. Typically, studies are started as soon as mixing is complete and samples are moved as needed for analysis. When performing a time-to-kill test (speed of biocide activity), the microorganisms are added after the biocide is added to the sample to allow for rapid mixing and analysis.

采用最大可能计数法(MPN)测定细菌浓度(CFU/毫升)。将污染的溶液顺序稀释，直到达到“无生长”的终点。终点代表MPN，并以细菌浓度的单位表示。一系列1∶10稀释可以产生1log的细菌浓度分解度，而且通过将处理过的样品中的有机体浓度与未处理的样品中的有机体浓度比较，由此确定对数降低值。例如，如果在细菌生长消失前，样品需要四次1∶10稀释，那么样品中细菌浓度的MPN小于或等于1×10⁴CFU/毫升(1E4)。如果在一系列8倍稀释中的每个孔显示细菌生长，那么MPN大于或等于1×10⁸CFU/毫升(1E8)。这种计算方法通常适于所有非丝状微生物。Bacterial concentration (CFU/ml) was determined by maximum probable number method (MPN). Contaminated solutions were serially diluted until an endpoint of "no growth" was reached. Endpoints represent MPN and are expressed in units of bacterial concentration. A series of 1:10 dilutions yielded a 1 log resolution of the bacterial concentration, and the log reduction was determined by comparing the concentration of the organism in the treated sample to the concentration of the organism in the untreated sample. For example, if the sample requires four 1:10 dilutions before bacterial growth disappears, then the MPN of the bacterial concentration in the sample is less than or equal to 1 x ¹⁰⁴ CFU/ml (1E4). If each well in a series of 8-fold dilutions shows bacterial growth, the MPN is greater than or equal to 1 x ¹⁰⁸ CFU/ml (1E8). This calculation method is generally applicable to all non-filamentous microorganisms.

培养基包括用于细菌的胰蛋白酶大豆肉汤(TSB)和用于真菌的沙氏葡萄糖肉汤(SDB)。其可以购得并按照制造指南制备。指示物培养基是用于细菌的TSB/R和用于真菌的SDB/R。将50微摩尔过滤消毒的刃天青加入到消毒并冷却的培养基中，由此制备指示物培养基。使用胰蛋白酶大豆琼脂盘(TSA)和SDA斜面以提供用于细菌和真菌接种培养。当存在细菌生长时指示物染料呈粉红色或白色。蓝色表明没有生长，紫色表明存在生长并将随附加的时间而溶解。Media included Tryptic Soy Broth (TSB) for bacteria and Sabouraud's Dextrose Broth (SDB) for fungi. It is commercially available and prepared according to the manufacturer's directions. Indicator media were TSB/R for bacteria and SDB/R for fungi. Indicator medium was prepared by adding 50 micromolar filter-sterilized resazurin to the sterilized and cooled medium. Tryptic soy agar plates (TSA) and SDA slants were used to provide cultures for bacterial and fungal inoculum. The indicator dye is pink or white when bacterial growth is present. Blue indicates no growth, purple indicates growth and will dissolve with additional time.

采用如上所述的HMARQ测试，生成周期肽并检测其杀灭活性。在本实验中，对较宽范围的肽进行了活性测试。15分钟或24小时时3.5对数杀灭需要以ppm(微克/毫升)表示单位，结果列示在下面。Periodic peptides were generated and tested for their killing activity using the HMARQ assay as described above. In this experiment, a broad range of peptides was tested for activity. A 3.5 log kill at 15 minutes or 24 hours is required in ppm (micrograms per milliliter) and the results are listed below.

表6：周期肽与生物灭杀活性氨基酸数 ID 序列ID号绿脓杆菌ATCC15442(革兰氏阴性) 金黄色葡萄球菌ATCC6538(革兰氏阳性) 15分钟 24小时 15分钟 24小时 32 KFAK(8) 5 31 31 ＞500 8 36 KFAK(12) 6 16 31 31(3log) ＞500 52 KFAK(17) 7 16 250 250 500 80 KFAK(20) - 8 16 4 4 28 KKAK(7) 23 16 16 ＞500 125 28 KFAF(7) 19 63 63 ＞500 125 24 RFAR(6) 9 31 31 ＞500 8 28 RFAR(7) 10 63 125 ＞500 4 32 RFAR(8) 11 ＞500 16 ＞500 125 24 FAKK(6) 12 ＞500 16 ＞500 250 24 AKKF(6) 13 ＞500 16 ＞500 125 24 KKFA(6) 14 ＞500 8 ＞500 63 20 LKKL(5) 15 ＞500 8 ＞500 125 24 LKKL(6) 16 ＞500 8 4 125 28 LKKL(7) 17 125 8 4 125 32 LKKL(8) 18 4 8 ＞500 125 28 KFFK(7) 20 16 8 4 63 28 KAAK(7) 22 ＞500 125 ＞500 250 9 KFK(3) - ＞500 125 ＞500 ＞500 12 KFK(4) - 500 125 ＞500 ＞500 18 KFK(6) 25 ＞500 125 ＞500 21 KFK(7) 26 ＞500 125 ＞500 24 KFK(8) 27 ＞500 ＞125 ＞500 500 27 KFK(9) 28 ＞500 63 ＞500 500 30 KFK(10) 29 8 8 4 500 36 KFK(12) 30 4 8 ＞500 250 48 KFK(16) 31 16 31 ＞500 16 63 KFK(21) 32 31 63 ＞500 250 6 FKA(2) 33 ＞500 125 16 500 12 FKA(4) 34 ＞500 125 4 250 18 FKA(6) 35 8 8 4 500 21 FKA(7) 36 125 63 500 500 24 FKA(8) 37 125 16 8 125 27 FKA(9) 38 4 4 8 125 30 FKA(10) 39 8 16 8 8 51 FKA(17) 40 16 16 4 8 63 FKA(21) 41 8 16 4 125 4 LK(2) 42 ＞500 250 ＞500 16 10 LK(5) 43 62 62 ＞500 31 14 LK(7) - 8 8 16 4 16 LK(8) 44 8 16 31 4 18 LK(9) 45 8 8 16 4 20 LK(10) 46 8 8.166 16或更低 16或更低 22 LK(11) 47 16 8 16或更低 4 24 LK(12) 48 16(3log) 4 4 8 36 LK(18) 49 16 31 8 16 48 LK(24) 50 16 63 ＞500 ＞500 14 LR(7) 51 ＞63 4 31 7.8 18 LR(9) 52 31(3log) 4 16 8 22 LR(11) 53 8 16 8 21 KGK(7) - 500 500 ＞500 500 33 KGK(11) 54 8 8 ＞500 8 45 KGK(15) 55 4 16 125 62 21 KTK(7) 56 31 31 250 8 Table 6: Periodic peptides and biocidal activity amino acid number ID Serial ID number Pseudomonas aeruginosa ATCC15442 (Gram-negative) Staphylococcus aureus ATCC6538 (Gram positive) 15 minutes 24 hours 15 minutes 24 hours 32 KFAK(8) 5 31 31 >500 8 36 KFAK(12) 6 16 31 31(3log) >500 52 KFAK(17) 7 16 250 250 500 80 KFAK(20) - 8 16 4 4 28 KKAK(7) twenty three 16 16 >500 125 28 KFAF(7) 19 63 63 >500 125 twenty four RFAR(6) 9 31 31 >500 8 28 RFAR(7) 10 63 125 >500 4 32 RFAR(8) 11 >500 16 >500 125 twenty four FAKK(6) 12 >500 16 >500 250 twenty four AKKF(6) 13 >500 16 >500 125 twenty four KKFA(6) 14 >500 8 >500 63 20 LKKL(5) 15 >500 8 >500 125 twenty four LKKL(6) 16 >500 8 4 125 28 LKKL(7) 17 125 8 4 125 32 LKKL(8) 18 4 8 >500 125 28 KFFK(7) 20 16 8 4 63 28 KAAK(7) twenty two >500 125 >500 250 9 KFK(3) - >500 125 >500 >500 12 KFK(4) - 500 125 >500 >500 18 KFK(6) 25 >500 125 >500 twenty one KFK(7) 26 >500 125 >500 twenty four KFK(8) 27 >500 >125 >500 500 27 KFK(9) 28 >500 63 >500 500 30 KFK(10) 29 8 8 4 500 36 KFK(12) 30 4 8 >500 250 48 KFK(16) 31 16 31 >500 16 63 KFK(21) 32 31 63 >500 250 6 FKA(2) 33 >500 125 16 500 12 FKA(4) 34 >500 125 4 250 18 FKA(6) 35 8 8 4 500 twenty one FKA(7) 36 125 63 500 500 twenty four FKA(8) 37 125 16 8 125 27 FKA(9) 38 4 4 8 125 30 FKA(10) 39 8 16 8 8 51 FKA(17) 40 16 16 4 8 63 FKA(21) 41 8 16 4 125 4 LK(2) 42 >500 250 >500 16 10 LK(5) 43 62 62 >500 31 14 LK(7) - 8 8 16 4 16 LK(8) 44 8 16 31 4 18 LK(9) 45 8 8 16 4 20 LK(10) 46 8 8.166 16 or lower 16 or lower twenty two LK(11) 47 16 8 16 or lower 4 twenty four LK(12) 48 16(3log) 4 4 8 36 LK(18) 49 16 31 8 16 48 LK(24) 50 16 63 >500 >500 14 LR(7) 51 >63 4 31 7.8 18 LR(9) 52 31(3log) 4 16 8 twenty two LR(11) 53 8 16 8 twenty one KGK(7) - 500 500 >500 500 33 KGK(11) 54 8 8 >500 8 45 KGK(15) 55 4 16 125 62 twenty one KTK(7) 56 31 31 250 8

结果表明，大多数周期肽具有真正的生物灭杀活性。令人惊讶的是，甚至太小以至于不能跨膜的肽也显示了生物灭杀活性(FKA(2)、FKA(4)、LK(2)、LK(4))。因此，周期肽的尺寸的下限实际上可以低至4个残基。The results showed that most of the periodic peptides have genuine biocidal activity. Surprisingly, even peptides too small to transmembrane showed biocidal activity (FKA(2), FKA(4), LK(2), LK(4)). Thus, the lower limit on the size of periodic peptides can actually be as low as 4 residues.

实施例6：病毒测试Example 6: Virus Test

前面的实验证明了杀菌和杀真菌活性，以及抗肿瘤活性。下一个预言性实验将证实抗病毒活性。Previous experiments demonstrated bactericidal and fungicidal activity, as well as antitumor activity. The next prophetic experiment will demonstrate antiviral activity.

唯一的抗微生物肽D2A21，其氨基酸含量类似于本文中展示的肽，已经显示出具有抗微生物、抗真菌、抗肿瘤和抗病毒的活性。类似地，两种充分表征的来自昆虫的天然抗微生物肽——蜂毒肽和天蚕素——已经表现出能够对人类免疫缺陷性病毒1(HIV-1)有效，对蜂毒肽而言其IC50值在0.9到1.5mM之间，对天蚕素而言其IC50值在2到3mM之间(Wachinger(1998))。因此，我们预言，上面描述的肽的集合也同样具有抗病毒活性。The only antimicrobial peptide, D2A21, with an amino acid content similar to the peptides presented here, has been shown to have antimicrobial, antifungal, antitumor and antiviral activities. Similarly, two well-characterized natural antimicrobial peptides from insects—melittin and cecropin—have been shown to be effective against human immunodeficiency virus 1 (HIV-1), and melittin IC50 values are between 0.9 and 1.5 mM, and for cecropin between 2 and 3 mM (Wachinger (1998)). Therefore, we predict that the collection of peptides described above will also have antiviral activity.

可以以多种方法测量抗病毒活性，但是一种简单的测定对反转录病毒(如HIV或FIV)效果的方法，是测量反转录病毒的降低的反转录酶(RT)活性，并测定50％抑制浓度，为了有效其应为大约1mM(Jia Ma(2002))。Antiviral activity can be measured in a number of ways, but a simple way to determine the effect on a retrovirus, such as HIV or FIV, is to measure the reduced reverse transcriptase (RT) activity of the retrovirus, and The 50% inhibitory concentration was determined and to be effective it should be approximately 1 mM (Jia Ma (2002)).

本文中引用的所有参考文献经此引用，全部并入本文。为方便起见，将参考文献列示在此。All references cited herein are hereby incorporated by reference in their entirety. For convenience, references are listed here.

1.Durell SR等人，Modeling the ion channel structure of cecropin，Biophys J.(1992年12月)63(6)：1623-31。1. Durell SR et al., Modeling the ion channel structure of cecropin, Biophys J. (December 1992) 63(6): 1623-31.

2.E.Gazit等人，Interaction of the Mammalian Antibacterial PeptideCecropin P1 with Phospholipid Vesicles，Biochemistry(1995年)34，11479。2.E.Gazit et al., Interaction of the Mammalian Antibacterial Peptide Cecropin P1 with Phospholipid Vesicles, Biochemistry (1995) 34, 11479.

3.Arlotti等人，Efficacy of a synthetic lytic peptide in the treatment ofProstate cancer，Urol Oncol.(2001年)6(3)：97-102。3. Arlotti et al., Efficacy of a synthetic lytic peptide in the treatment of Prostate cancer, Urol Oncol. (2001) 6(3): 97-102.

4.US57895424.US5789542

5.Javadpour等人，De Novo Antimicrobial Peptides with LowMammalian Cell Toxicity，J.Med.Chem.(1996年)39(16)：3107-3113。5. Javadpour et al., De Novo Antimicrobial Peptides with Low Mammalian Cell Toxicity, J. Med. Chem. (1996) 39(16): 3107-3113.

6.Watchinger等人，Antimicrobial peptides melittin and cecropin inhibitrepilcation of human immunodeficiency virus 1 by suppressing viralgene expression，J.Gen.Virol.(1998年)(79)：731-740。6.Watchinger et al., Antimicrobial peptides melittin and cecropin inhibitionrepilcation of human immunodeficiency virus 1 by suppressing viralgene expression, J.Gen.Virol.(1998)(79):731-740.

7.Jia Ma等人，Inhibitory Activity of Synthetic Peptide Antibiotics onFeline Immunodeficiency Virus Infectivity In Vitro，J.Virol.(2002年)76(19)：9952-9961。7. Jia Ma et al., Inhibitory Activity of Synthetic Peptide Antibiotics on Feline Immunodeficiency Virus Infectivity In Vitro, J. Virol. (2002) 76 (19): 9952-9961.

8.S.E.Blondelle和Karl Lohner，Biopolymer (Peptide Science)，(2000年)卷55，74-87。8. S.E. Blondelle and Karl Lohner, Biopolymer (Peptide Science), (2000) Vol. 55, 74-87.

序列表sequence listing

<110>陶氏化学公司<110> Dow Chemical Company

R·M·斯特罗姆R. M. Strom

P·J·布龙达塞马P.J. Bronda Sema

<120>周期性抗微生物肽<120> Periodic Antimicrobial Peptides

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<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

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<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

Lys Phe Ala LysLys Phe Ala Lys

2020

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<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

Lys Phe Ala Lys Lys Phe Ala LysLys Phe Ala Lys Lys Phe Ala Lys

2020

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<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala LysLys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys

20 2520 25

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<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

20 25 3020 25 30

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<211>48<211>48

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

20 25 3020 25 30

35 40 4535 40 45

<210>7<210>7

<211>68<211>68

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

20 25 3020 25 30

35 40 4535 40 45

50 55 6050 55 60

Lys Phe Ala LysLys Phe Ala Lys

6565

<210>8<210>8

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<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

20 25 3020 25 30

35 40 4535 40 45

50 55 6050 55 60

65 70 75 8065 70 75 80

Lys Phe Ala LysLys Phe Ala Lys

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<213>周期性肽<213> Periodic peptides

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Arg Phe Ala Arg Arg Phe Ala Arg Arg Phe Ala Arg Arg Phe Ala ArgArg Phe Ala Arg Arg Phe Ala Arg Arg Phe Ala Arg Arg Phe Ala Arg

1 5 10 151 5 10 15

Arg Phe Ala Arg Arg Phe Ala ArgArg Phe Ala Arg Arg Phe Ala Arg

2020

<210>10<210>10

<211>28<211>28

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>10<400>10

1 5 10 151 5 10 15

Arg Phe Ala Arg Arg Phe Ala Arg Arg Phe Ala ArgArg Phe Ala Arg Arg Phe Ala Arg Arg Phe Ala Arg

20 2520 25

<210>11<210>11

<211>32<211>32

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>11<400>11

1 5 10 151 5 10 15

20 25 3020 25 30

<210>12<210>12

<211>24<211>24

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>12<400>12

Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys LysPhe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys

1 5 10 151 5 10 15

Phe Ala Lys Lys Phe Ala Lys LysPhe Ala Lys Lys Phe Ala Lys Lys

2020

<210>13<210>13

<211>24<211>24

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>13<400>13

Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys PheAla Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe

1 5 10 151 5 10 15

Ala Lys Lys Phe Ala Lys Lys PheAla Lys Lys Phe Ala Lys Lys Phe

2020

<210>14<210>14

<211>24<211>24

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe AlaLys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala

1 5 10 151 5 10 15

Lys Lys Phe Ala Lys Lys Phe AlaLys Lys Phe Ala Lys Lys Phe Ala

2020

<210>15<210>15

<211>20<211>20

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>15<400>15

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1 5 10 151 5 10 15

Leu Lys Lys LeuLeu Lys Lys Leu

2020

<210>16<210>16

<211>24<211>24

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>16<400>16

1 5 10 151 5 10 15

Leu Lys Lys Leu Leu Lys Lys LeuLeu Lys Lys Leu Leu Lys Lys Leu

2020

<210>17<210>17

<211>28<211>28

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys LeuLeu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu

20 2520 25

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<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

20 25 3020 25 30

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<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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Lys Phe Ala Phe Lys Phe Ala Phe Lys Phe Ala Phe Lys Phe Ala PheLys Phe Ala Phe Lys Phe Ala Phe Lys Phe Ala Phe Lys Phe Ala Phe

1 5 10 151 5 10 15

Lys Phe Ala Phe Lys Phe Ala Phe Lys Phe Ala PheLys Phe Ala Phe Lys Phe Ala Phe Lys Phe Ala Phe

20 2520 25

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<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

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20 2520 25

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<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

20 2520 25

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<211>28<211>28

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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Lys Ala Ala Lys Lys Ala Ala Lys Lys Ala Ala Lys Lys Ala Ala LysLys Ala Ala Lys Lys Ala Ala Lys Lys Ala Ala Lys Lys Ala Ala Lys

1 5 10 151 5 10 15

Lys Ala Ala Lys Lys Ala Ala Lys Lys Ala Ala LysLys Ala Ala Lys Lys Ala Ala Lys Lys Ala Ala Lys

20 2520 25

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<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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Lys Lys Ala Lys Lys Lys Ala Lys Lys Lys Ala Lys Lys Lys Ala LysLys Lys Ala Lys Lys Lys Ala Lys Lys Lys Ala Lys Lys Lys Ala Lys

1 5 10 151 5 10 15

Lys Lys Ala Lys Lys Lys Ala Lys Lys Lys Ala LysLys Lys Ala Lys Lys Lys Ala Lys Lys Lys Ala Lys

20 2520 25

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<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

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1 5 10 151 5 10 15

Phe LysPhe Lys

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<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>26<400>26

1 5 10 151 5 10 15

Phe Lys Lys Phe LysPhe Lys Lys Phe Lys

2020

<210>27<210>27

<211>24<211>24

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

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1 5 10 151 5 10 15

Phe Lys Lys Phe Lys Lys Phe LysPhe Lys Lys Phe Lys Lys Phe Lys

2020

<210>28<210>28

<211>27<211>27

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>28<400>28

1 5 10 151 5 10 15

Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe LysPhe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys

20 2520 25

<210>29<210>29

<211>30<211>30

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>29<400>29

1 5 10 151 5 10 15

Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe LysPhe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys

20 25 3020 25 30

<210>30<210>30

<211>36<211>36

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>30<400>30

1 5 10 151 5 10 15

Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys PhePhe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe

20 25 3020 25 30

Lys Lys Phe LysLys Lys Phe Lys

3535

<210>31<210>31

<211>48<211>48

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>31<400>31

1 5 10 151 5 10 15

20 25 3020 25 30

Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe LysLys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys Lys Phe Lys

35 40 4535 40 45

<210>32<210>32

<211>63<211>63

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>32<400>32

1 5 10 151 5 10 15

20 25 3020 25 30

35 40 4535 40 45

50 55 6050 55 60

<210>33<210>33

<211>6<211>6

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>33<400>33

Phe Lys Ala Phe Lys AlaPhe Lys Ala Phe Lys Ala

1 51 5

<210>34<210>34

<211>12<211>12

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>34<400>34

Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys AlaPhe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala

1 5 101 5 10

<210>35<210>35

<211>18<211>18

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>35<400>35

Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala PhePhe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe

1 5 10 151 5 10 15

Lys AlaLys Ala

<210>36<210>36

<211>21<211>21

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>36<400>36

1 5 10 151 5 10 15

Lys Ala Phe Lys AlaLys Ala Phe Lys Ala

2020

<210>37<210>37

<211>24<211>24

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>37<400>37

1 5 10 151 5 10 15

Lys Ala Phe Lys Ala Phe Lys AlaLys Ala Phe Lys Ala Phe Lys Ala

2020

<210>38<210>38

<211>27<211>27

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>38<400>38

1 5 10 151 5 10 15

Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys AlaLys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala

20 2520 25

<210>39<210>39

<211>30<211>30

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>39<400>39

1 5 10 151 5 10 15

Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys AlaLys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala

20 25 3020 25 30

<210>40<210>40

<211>51<211>51

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>40<400>40

1 5 10 151 5 10 15

Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe LysLys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys

20 25 3020 25 30

Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys AlaAla Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala

35 40 4535 40 45

Phe Lys AlaPhe Lys Ala

5050

<210>41<210>41

<211>63<211>63

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>41<400>41

1 5 10 151 5 10 15

20 25 3020 25 30

35 40 4535 40 45

Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys AlaPhe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala Phe Lys Ala

50 55 6050 55 60

<210>42<210>42

<211>4<211>4

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>42<400>42

Leu Lys Leu LysLeu Lys Leu Lys

11

<210>43<210>43

<211>10<211>10

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>43<400>43

Leu Lys Leu Lys Leu Lys Leu Lys Leu LysLeu Lys Leu Lys Leu Lys Leu Lys Leu Lys

1 5 101 5 10

<210>44<210>44

<211>16<211>16

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>44<400>44

Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu LysLeu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys Leu Lys

1 5 10 151 5 10 15

<210>45<210>45

<211>18<211>18

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>45<400>45

1 5 10 151 5 10 15

Leu LysLeu Lys

<210>46<210>46

<211>20<211>20

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>46<400>46

1 5 10 151 5 10 15

Leu Lys Leu LysLeu Lys Leu Lys

2020

<210>47<210>47

<211>22<211>22

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>47<400>47

1 5 10 151 5 10 15

Leu Lys Leu Lys Leu LysLeu Lys Leu Lys Leu Lys

2020

<210>48<210>48

<211>24<211>24

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>48<400>48

1 5 10 151 5 5 10 15

Leu Lys Leu Lys Leu Lys Leu LysLeu Lys Leu Lys Leu Lys Leu Lys

2020

<210>49<210>49

<211>36<211>36

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>49<400>49

1 5 10 151 5 10 15

20 25 3020 25 30

Leu Lys Leu LysLeu Lys Leu Lys

3535

<210>50<210>50

<211>48<211>48

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>50<400>50

1 5 10 151 5 10 15

20 25 3020 25 30

35 40 4535 40 45

<210>51<210>51

<211>14<211>14

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>51<400>51

Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu ArgLeu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg

1 5 101 5 10

<210>52<210>52

<211>18<211>18

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>52<400>52

Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu ArgLeu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg Leu Arg

1 5 10 151 5 10 15

Leu ArgLeu Arg

<210>53<210>53

<211>22<211>22

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>53<400>53

1 5 10 151 5 10 15

Leu Arg Leu Arg Leu ArgLeu Arg Leu Arg Leu Arg

2020

<210>54<210>54

<211>33<211>33

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>54<400>54

Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys LysLys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys

1 5 10 151 5 10 15

Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys GlyGly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly

20 25 3020 25 30

LysLys

<210>55<210>55

<211>45<211>45

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>55<400>55

1 5 10 151 5 10 15

20 25 3020 25 30

Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly LysLys Lys Gly Lys Lys Gly Lys Lys Gly Lys Lys Gly Lys

35 40 4535 40 45

<210>56<210>56

<211>21<211>21

<212>PRT<212>PRT

<213>周期性肽<213> Periodic peptides

<400>56<400>56

Lys Thr Lys Lys Thr Lys Lys Thr Lys Lys Thr Lys Lys Thr Lys LysLys Thr Lys Lys Thr Lys Lys Thr Lys Lys Thr Lys Lys Thr Lys Lys

1 5 10 151 5 10 15

Thr Lys Lys Thr LysThr Lys Lys Thr Lys

2020

Claims

1. An antimicrobial peptide, comprising a periodic peptide with repeated identical monomer units of 2, 3 or 4 residues, n wherein the minimum length of the antimicrobial peptide is 4 residues, with 25-75 % cationic residues, and other residues are hydrophobic residues, and the IC50 of the antimicrobial peptide is ≤ 125 micrograms/ml.

2. The antimicrobial peptide according to claim 1, wherein said antimicrobial peptide has a maximum length of 80 residues and a minimum length of 14 residues.

3. The antimicrobial peptide according to claim 1, wherein said antimicrobial peptide is a mixture of antimicrobial peptides having different lengths.

4. The antimicrobial peptide according to claim 1, wherein said hydrophobic residue has a bulky side chain.

5. The antimicrobial peptide according to claim 1, wherein said antimicrobial peptide has a biocidal activity of < 125 ppm at 3.5 log kill at 24 hours.

6. The antimicrobial peptide according to claim 1, wherein the measured antiviral activity IC50 of said antimicrobial peptide is ≤ 5mM.

7. The antimicrobial peptide according to claim 1, wherein said monomer is a dimer, and the antitumor cell activity TX50 of the antimicrobial peptide is ≤ 250 μg/ml.

8. according to the antimicrobial peptide of claim 1, wherein said monomer is selected from PNNP, NNPP, NPPN, PPNN, PNPN, NPNP, PNP, NPP, PPN, NPN, PNN, NNP, NP and PN, wherein P is any cationic residue, N is any hydrophobic residue.

9. The antimicrobial peptide according to claim 1, wherein said monomer comprises P2N2, P3N, PN3, PN2, P2N and NP, wherein P is any cationic residue, N is any hydrophobic residue, N and P The residues are in any order.

10. An antimicrobial peptide comprising

a) a periodic peptide having repeats of the same monomer unit;

b) wherein the monomer is selected from P2N2, PN3, P3N, P2N, NP2 and NP, wherein P is any cationic residue, N is any hydrophobic residue, and wherein the N and P residues are in any order;

c) wherein the minimum length of the antimicrobial peptide is 4 residues;

d) The target cell IC50 of the antimicrobial activity of the antimicrobial peptide against target cells is ≤ 125 μg/ml.

11. The antimicrobial peptide according to claim 10, wherein said antimicrobial peptide has a maximum length of 80 residues and a minimum length of 14 residues.

12. The antimicrobial peptide according to claim 10, wherein said antimicrobial peptide has a biocidal activity of < 125 ppm at 3.5 log kill at 24 hours.

13. The antimicrobial peptide according to claim 10, wherein the anti-tumor cell activity TX50 of said antimicrobial peptide is ≤ 250 μg/ml.

14. The antimicrobial peptide of claim 10, wherein said monomer is selected from PNNP, NNPP, NPPN, PPNN, PNPN, NPNP, PNP, NPP, PPN, NPN, PNN, NNP, NP and PN, and P is K, Any one of O or R residues, N is any one of A, F, G, L, I, T, Y, W, V or M residues.

15. A peptide comprising

a) a periodic peptide having repeating monomeric units;

b) wherein the monomer is selected from PNNP, NNPP, NPPN, PPNN, PNPN, NPNP, PNP, NPP, PPN, NPN, PNN, NNP, NP and PN, and P is any one of K, O or R residues, N is any one of A, F, G, L, I, T, Y, W, V or M residues;

c) wherein the peptide has a minimum length of 14 residues and a maximum length of 80 residues;

d) The antimicrobial activity of the peptide against target cells with an IC50 of ≤ 125 μg/ml.

16. A peptide comprising a sequence selected from the group consisting of SEQ ID NOS: 1 to 56.

17. A peptide consisting essentially of a sequence selected from the group consisting of SEQ ID NOS: 1 to 56.

18. A peptide consisting of a sequence selected from the group consisting of SEQ ID NOS: 1 to 56.

19. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. A method for synthesizing periodic peptides, comprising: oligomerizing the same monomer units with 2, 3 or 4 residues by polycondensation to generate antimicrobial peptides;

a) The antimicrobial peptide has a length > 14 residues; at least 25% cationic residues, the remainder being hydrophobic, and an IC50 for target cells < 125 μg/ml.

21. A method of inhibiting the growth of a target cell comprising administering to the target cell an amount of the peptide according to any one of claims 1 to 18 effective to inhibit the growth of said target cell.

22. A method for killing target cells, comprising administering to target cells a dose of the peptide according to any one of claims 1 to 18 effective to kill the growth of said target cells.