Structural determinants of activity and mechanism of cationic peptide antibiotic activity against colistin-resistant bacteria

抽象的。抗生素耐药性(AR)的出现突显了开发新型抗生素的迫切需要 能够克服当前AR机制的抗菌剂。抗菌肽(AMP)倾向于 破坏细菌膜，而不考虑对传统抗生素的耐药性。然而，细菌也已经 进化为通过减少(例如，加入磷乙醇胺)而不是消除，来抵抗AMPS的影响， 脂类A的净电负性电荷，脂类A是AMPS靶向的脂多糖的重要组成部分。因为一个 最小的电负电荷对细菌膜的完整性是必不可少的，AMPS可以优化为 克服内毒素的有限变化，内源性AMP最常见的耐药机制 粘菌素。我们开发了一个合理设计工程阳离子多肽抗生素的迭代框架。 (Pax)针对多药耐药(MDR)细菌的广泛活性进行了优化。我们理性地使用库 设计了一系列新型的富含Trp的多肽，每次有两个残基，一个Val和一个Arg，以建立 抗菌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。正如预测的那样， 对多药耐药临床分离株的最低最低抑菌浓度(MIC)是在没有实质性 在最大检测浓度下，增加红细胞裂解或对白细胞的毒性。值得注意的是，其中一个 选定的PAX(E35)中的一种在系统地以4-5 mg/kg的速度给药时显示出疗效 剂量(耐多药铜绿假单胞菌感染)或多剂量(耐多药肺炎克雷伯菌感染)。和平 E35还显示出最大耐受量为30毫克/公斤，而我们最初的铅多肽为12-15毫克/公斤 WLBU2目前处于第二阶段临床试验。我们的迭代框架将设计PAX时的试错降至最低 增强了对耐多药细菌的全身药效。鉴于对粘菌素耐药(COL-R)细菌的成功， 人们倾向于声称，我们的工作是在没有考虑当前PAX的潜在缺点的情况下完成的。 然而，仍然有太多的未知数。(1)临床上未见多药耐药细菌暴露于这些多肽 就像他们对粘菌素所做的那样。一旦他们这样做了，几乎可以肯定的是，抵抗频率将稳步增加。 (2)目前的结构优化只是最小的，因为我们还没有确定每个 阳离子(C)和疏水(H)基序有助于识别和杀死细菌。因此，至关重要的是 继续这项重要的工作，同时一些PAX可能会进展到高级临床前和临床研究。 我们建议使用COL-R菌株来定义这两个基序对靶识别和消除的贡献 可以通过传代来进化出对当前PAX的抵抗力。这项提议的首要目标是澄清 解偶联PAX的C和H基序以允许细菌对PAX产生耐药性的分子决定因素 用COL-R和Col-R研究脂多糖结构变化和相应杀菌活性的C/H相关性 Pax-R多药耐药临床分离株。影响：我们将从最小的单一药物进步到全面的药物- 治疗类方法，具有根据临床挑战设计或重新设计PAX的能力。

Abstract. The emergence of antibiotic resistance (AR) underscores the urgent need to develop novel antimicrobial agents that are able to overcome current AR mechanisms. Antimicrobial peptides (AMPs) tend to disrupt bacterial membranes regardless of resistance to traditional antibiotics. However, bacteria have also evolved to resist the effects of AMPs by reducing (e.g., incorporation of phosphoethanolamine), not eliminating, the net electronegative charge of lipid A, an important component of LPS targeted by AMPs. Because a minimum electronegative charge is essential to bacterial membrane integrity, AMPs can be optimized to overcome limited changes in LPS, the most common resistance mechanism against endogenous AMPs and colistin. We have developed an iterative framework for rational design of engineered cationic peptide antibiotics (PAX) optimized for broad activity against multidrug-resistant (MDR) bacteria. We used libraries of rationally designed series of novel Trp-rich peptides differing by 2 residues at a time, 1 Val and 1 Arg to establish the distinction between structural determinants of antimicrobial potency and those of host toxicity. As predicted, lowest minimum inhibitory concentrations (MIC) are achieved against MDR clinical isolates without substantial increase in red blood cell lysis or toxicity to white blood cells at maximum test concentrations. Remarkably, one of the selected PAX (E35) demonstrates efficacy when systemically administered at 4-5mg/kg either a single dose (MDR P. aeruginosa infection) or multiple doses (MDR Klebsiella pneumonia infection) in mice. PAX E35 also displayed a maximum tolerated dose of 30 mg/kg compared to 12-15 mg/kg of our initial lead peptide WLBU2 now in phase 2 clinical trial. Our iterative framework minimizes trial and error for designing PAX with enhanced systemic efficacy against MDR bacteria. Given the success against colistin-resistant (Col-R) bacteria, the tendency is to claim that our work is done without thinking of potential shortcomings of the current PAX. However, there are still too many unknowns. (1) MDR bacteria have not been exposed clinically to the peptides as they have been to colistin. Once they do, it is almost certain that resistance frequency will steadily increase. (2) The current structural optimization is only minimal because we have not yet determined how each of the cationic (C) and hydrophobic (H) motifs contributes to bacterial recognition and killing. Thus, it is critical to continue this important work while some of the PAX may progress to advanced pre-clinical and clinical studies. We propose to define the contribution of the 2 motifs to target recognition and elimination using Col-R strains that can be passaged to evolve resistance to the current PAX. The overarching goal of this proposal is to elucidate the molecular determinants for bacterial resistance to PAX by uncoupling the C and H motifs of PAX to allow C/H correlation with changes in LPS structures and corresponding bactericidal activity using both col-R and PAX-R MDR clinical isolates. Impact: we will progress from a minimal one-drug to a comprehensive one- therapeutic class approach with the ability to design or re-design PAX based on clinical challenges.