Development of Broad-Spectrum Cyclic Amphiphilic Peptides against Multidrug-Resistant Bacteria

摘要 对甲氧西林、万古霉素、头孢菌素等常见一线抗生素出现抗药性， 和碳青霉烯类，给数百万患者带来了全球公共卫生挑战。因此，关键是要发现和 商业化新的抗菌剂，可以成功地中和多药耐药细菌(MDRB)，以最少的 毒性。这项计划的目标是开发独特的一流两亲性环状抗菌肽(Amps)。 对临床相关病原体，如粪肠球菌、金黄色葡萄球菌、克雷伯氏菌有积极作用 肺炎、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌(ESKAPE病原体)。我们 建议开发含有天然和/或非天然疏水和带正电荷残基的AMP，用于其广泛的- 使用体外和体内试验，对特定MDR病原体的光谱活性和有效性。我们发现， 一种由色氨酸(W)和精氨酸(R)氨基酸组成的环状两亲性多肽[R4W4]对 各种细菌病原体，如耐甲氧西林金黄色葡萄球菌(MRSA)(MIC=2.7微克/毫升)， 铜绿假单胞菌(MIC=42.8微克/毫升)、肺炎克雷伯菌(MIC=16.0微克/毫升)、大肠埃希氏菌(MIC= 16.0微克/毫升)，与四环素、异烟肼和吡嗪酰胺对金黄色葡萄球菌有协同作用 结核分枝杆菌。基于这个模板，我们生成了一个新的多肽文库(&gt；200)，并增强了 抗菌活性。例如，IFX-027、IFX-135、IFX-145、IFX-146、IFX-154和IFX-301显示MIC=1.5-25 对革兰氏阳性菌和革兰氏阴性杆菌的抑菌作用。几种先导化合物显示出协同活性。 其他几种抗生素的分数抑制浓度(FIC)指数在0.3-0.5之间。我们的先导肽(IFX- 031、IFX-031-1和IFX-111)也减少了MRSA和铜绿假单胞菌的生物被膜形成。IFX-301被发现是 对小鼠无毒，剂量为50 mg/kg，对人红细胞(HRBC)无毒性 (Hc50&GT；500μg/mL)。在目标1中，我们将建立基于六个先导肽的构效关系(SAR)，以 获得对分子对细菌病原体选择性负责的结构决定因素的见解。这个 大多数有效的化合物将进一步评估它们的稳定性、细胞毒性和随着时间的推移对抗菌剂的发展。 抵抗。目标1的拟议里程碑是鉴定5个具有最低抑菌浓度≤5微克/毫升和最低抑菌浓度≤10的先导肽类似物 对革兰氏阳性菌和革兰氏阳性菌的抑制率分别为5%和5%，对20倍浓度的≤对人红细胞的溶血作用也不同 MIC值。在目标2中，我们将评估其体内疗效和毒性、初步药代动力学(例如，Cmax，Tmax， T1/2)，以及目标1中鉴定的2-3先导抗菌肽类似物在小鼠感染模型上对4种 病原菌。在第一阶段的成功完成后，治疗指数最大的最有效的化合物将 将进入第二阶段研究，并成为IND申请的重点。

ABSTRACT The emergence of antibacterial resistance to common frontline antibiotics, such as methicillin, vancomycin, cephalosporins, and carbapenem, have created a global public health challenge for millions of patients. It is therefore critical to discover and commercialize new antimicrobial agents that can successfully neutralize multidrug-resistant bacteria (MDRB) with minimal toxicity. The objective of this proposal is to develop unique first-in-class amphiphilic cyclic antimicrobial peptides (AMPs) that are active against clinically relevant pathogens like Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE pathogens). We propose to develop AMPs containing natural and/or unnatural hydrophobic and positively charged residues for their broad- spectrum activity and efficacy against specific MDR pathogens, using in vitro and in vivo assays. We have discovered that a cyclic amphipathic peptide [R4W4], which comprises tryptophan (W) and arginine (R) amino acids was effective against diverse bacterial pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) (MIC = 2.7 µg/mL), Pseudomonas aeruginosa (MIC = 42.8 µg/mL), Klebsiella pneumoniae (MIC = 16.0 µg/mL), and Escherichia coli (MIC = 16.0 µg/mL) and showed synergistic activity with tetracycline against MRSA, and isoniazid and pyrazinamide against Mycobacterium tuberculosis. Based on this template, we generated a new library of peptides (>200) with enhanced antimicrobial activities. For example, IFX-027, IFX-135, IFX-145, IFX-146, IFX-154, and IFX-301 showed MIC = 1.5-25 µg/mL against Gram+ve and Gram-ve bacteria. Several of the lead compounds demonstrated synergistic activity with several other antibiotics with fractional inhibitory concentration (FIC) indices ranging from 0.3-0.5. Our lead peptides (IFX- 031, IFX-031-1, and IFX-111) also reduced biofilm formation by MRSA and P. aeruginosa. IFX-301 was found to be nontoxic at a dose level of 50 mg/kg in mice, and all peptides were not toxic against human red blood cells (hRBC) (HC50>500 μg/mL). In Aim 1, we will establish a structure-activity relationship (SAR) based on the six lead peptides to obtain insights into the structural determinants responsible for the molecules’ selectivity towards bacterial pathogens. The most potent compounds will be further evaluated for their stability, cytotoxicity, and development over time to antimicrobial resistance. The proposed milestones for Aim 1 are to identify five lead peptide analogs with MIC ≤5 µg/mL and MIC ≤10 µg/mL respectively against Gram+ve and Gram-ve bacteria, and hRBC hemolysis of ≤5% at a concentration of 20 times the MIC value. In Aim 2, we will evaluate the in vivo efficacy and toxicity, preliminary pharmacokinetics (e.g., Cmax, tmax, t1/2), and efficacy of the 2-3 lead antimicrobial peptide analogs identified in Aim 1 on a murine infection model against four pathogenic bacteria. At the successful completion of Phase I, the most potent compound with a large therapeutic index will be advanced to Phase II studies and be the focus for an IND application.