Determinants and Mechanisms of Efficacy of Peptide Antibiotics as Novel Sepsis Therapy

项目摘要/摘要 该提议的前提是阳离子抗菌肽(AMPs)可以被设计成增强全身 基于杀灭细菌的独特特性的脓毒症治疗效果，而不考虑多重耐药 (MDR)，以及减轻内毒素刺激Toll样受体(TLR)4介导的炎症。 将使用一系列合理设计的新型多肽抗生素(PAX)文库来实现 确定抗菌剂效力的结构决定因素和宿主毒性的结构决定因素之间的区别。 脓毒症或全身炎症反应综合征(SIRS)被定义为“威胁生命的器官功能障碍” 这是由于宿主对感染的反应失调所致。与脓毒症相关的死亡率每年约为30%， 在更严重的情况下，这一比例可能高达50%。多类抗生素的发展趋势 20世纪中叶最初导致败血症相关死亡率急剧下降。然而，持续性脓毒症的发病率 而死亡率，即使使用了有效的抗生素，也表明免疫反应和MDR增强。 耐多药细菌的频率增加，迫切需要开发新的类别的 具有新的抗菌机制的抗生素。此外，脓毒症具有复杂的病理生理机制，这使得它 很难治疗。通过TLR4信号减轻炎症刺激，消除致病因子 (细菌)，AMPS有可能从根本上根除这个问题。在过去的十年里，我们发展了 设计和表征治疗多药耐药相关感染的阳离子AMP的新策略 细菌基于从头开始工程的阳离子抗菌肽(ECAPs)。色氨酸替代技术在我国的应用 疏水侧产生两个铅多肽(WLBU2和WR12)，它们在 生理盐水、酸性pH和人体血浆，表明不同氨基酸在AMP功能中的特定作用。两者都有 与活性相比，eCAP对89%-92%的不同ESKAPE(MDR)病原体有效 对这些菌株中只有50%由人AMP LL37和粘菌素显示。WLBU2还会显示 铜绿假单胞菌败血症模型的全身性疗效，消除了应该使用AMPS的观念 只是局部有效。单次全身剂量的WLBU2可保护注射了致死疫苗的小鼠 接种铜绿假单胞菌。然而，一个重要的问题是一个狭窄的治疗指数[TI=最大耐受性 剂量(MTD)/最小治疗剂量(MTD)]WLBU2 3-5(MTD为3-4 mg/kg，MTD为12-15 mg/kg)， 这将在这项拟议的研究中解决。我们假设AMP可以为应用而设计 通过阐明独特的结构-功能相关性的合理框架来治疗脓毒症 阳离子和疏水含量对抗菌选择性、内毒素中和和 寄主毒性。为了解决这一假设，我们将检验细菌性脓毒症和盲肠小鼠模型的疗效。 结扎穿刺术致多菌败血症。这项基础性和翻译性研究将产生最终的 选择单一有效药物用于脓毒症治疗的高级临床前和临床研究。

Project summary/Abstract The premise of the proposal is that cationic antimicrobial peptides (AMPs) can be designed to enhance systemic efficacy for sepsis treatment based on the unique properties to kill bacteria regardless of multidrug resistance (MDR) as well as to attenuate inflammation mediated by endotoxin stimulation of toll like receptor (TLR) 4. This will be accomplished using a series of rationally engineered libraries of novel peptide antibiotics (PAX) to establish the distinction between structural determinants of antimicrobial potency and those of host toxicity. Sepsis or SIRS (systemic inflammatory response syndrome) is defined as a “life-threatening organ dysfunction caused by a dysregulated host response to infection.” Sepsis-related fatality rate is approximately 30% annually, which may reach up to 50% in more severe cases. The development of multiple classes of antibiotics toward the mid-20th century initially led to a sharp decline in sepsis-related mortality. However, persistent sepsis morbidity and mortality, even with the use of effective antibiotics, is indicative of heightened immune responses and MDR. The increased frequency of MDR bacteria has created an urgent need for the development of novel classes of antibiotics with new antimicrobial mechanisms. In addition, sepsis has a complex pathophysiology that makes it difficult to treat. By mitigating stimulation of inflammation via TLR4 signaling and eliminating the causative agent (bacteria), AMPs has the potential to eradicate the problem by its root. Over the past decade we have developed novel strategies to design and characterize cationic AMPs for treatment of infections associated with MDR bacteria based on de novo engineered cationic antimicrobial peptides (eCAPs). The use of Trp substitution on the hydrophobic side results in two lead peptides (WLBU2 and WR12) that retain broad-spectrum activity in saline, acidic pH, and human plasma, indicating the specific roles of different amino acids in AMP function. Both eCAPs were effective against 89-92% of a diverse panel of ESKAPE (MDR) pathogens compared to activity against only 50% of these strains displayed by both the human AMP LL37 and colistin. WLBU2 also displays systemic efficacy in a P. aeruginosa septicemia model, which dispels the notion that AMPs ought to be used only topically to be effective. A single systemic dose of WLBU2 protects mice injected with an otherwise lethal inoculum of P. aeruginosa. However, an important concern is a narrow therapeutic index [TI = maximum tolerated dose (MTD)/minimum therapeutic dose (mTd)]) of 3-5 (mTd of 3-4mg/kg and MTD of 12-15mg/kg) of WLBU2, which will be addressed in this proposed research. We hypothesize that AMPs can be designed for application to sepsis treatment based on a rational framework for structure-function correlations by elucidating the unique contributions of the cationic and hydrophobic contents to antibacterial selectivity, endotoxin neutralization, and host toxicity. To address this hypothesis, we will examine efficacy in murine models of bacterial sepsis and cecal ligation puncture-induced polymicrobial sepsis. This basic and translational research will result in the final selection of a single effective drug for advanced pre-clinical and clinical studies for sepsis treatment.