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

阳离子肽抗生素对粘菌素耐药菌活性的结构决定因素和机制

• 批准号: 10733264
• 负责人: Berthony Deslouches
• 金额: $ 65.39万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10733264
• 关键词: Address; Affinity; Amino Acids; Animal Model; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Antimicrobial Effect; Antimicrobial Resistance; Bacteremia; Bacteria; Bacterial Drug Resistance; Biophysics; Charge; Circular Dichroism; Circulation; Clinical; Clinical Research; Colistin; Cytolysis; Data; Development; Dose; Endotoxins; Engineering; Erythrocytes; Genetic; Goals; Gram-Negative Bacteria; Hydrophobicity; In Vitro; Infection; Intravenous; Investigation; Lead; Length; Leukocytes; Libraries; Lipid A; Lipopolysaccharides; Mammalian Cell; Maximum Tolerated Dose; Measures; Medical; Membrane; Minimum Inhibitory Concentration measurement; Modeling; Molecular; Multi-Drug Resistance; Multiple Bacterial Drug Resistance; Mus; Outcome; Patients; Peptide Antibiotics; Peptide Library; Peptides; Periodicity; Pharmaceutical Preparations; Phase II Clinical Trials; Predisposition; Prevalence; Property; Pseudomonas aeruginosa infection; Public Health; Research Personnel; Resistance; Resistance development; Safety; Serial Passage; Series; Structure; Surface Plasmon Resonance; Testing; Therapeutic; Therapeutic Index; Therapeutic Uses; Thinking; Time; Toxic effect; Toxicology; United States; Variant; Work; antibiotic design; antimicrobial; antimicrobial drug; antimicrobial peptide; bacterial resistance; bactericide; cell type; clinical development; clinically significant; colistin resistance; cytotoxic; defined contribution; design; emerging antibiotic resistance; experimental study; immunogenicity; in vitro activity; in vivo; indexing; infectious disease treatment; intraperitoneal; iterative design; lead candidate; multi-drug resistant pathogen; multidrug-resistant Pseudomonas aeruginosa; multiple drug use; novel; peptide drug; phosphoethanolamine; preclinical study; public health relevance; rational design; rational function; resistance frequency; resistance mechanism; resistant Klebsiella pneumoniae; resistant strain; response; structural determinants; success; therapeutically effective

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.

抽象。抗生素耐药性（AR）的出现强调了开发新型抗生素的迫切需要。 因此，可以使用能够克服当前AR机制的抗微生物剂。抗菌肽（AMP）往往 破坏细菌膜，而不管对传统抗生素的耐药性如何。然而，细菌也 进化为通过减少（例如，掺入磷酸乙醇胺），而不是消除， 脂质A的净电负性电荷，脂质A是AMP靶向的LPS的重要组分。因为 最小的电负性电荷对于细菌膜完整性是必不可少的，AMP可以被优化以 克服LPS的有限变化，LPS是针对内源性AMP的最常见的抗性机制， 粘菌素我们已经开发了一个迭代框架，用于合理设计工程阳离子肽抗生素 (PAX)优化了针对多药耐药（MDR）细菌的广泛活性。我们合理地使用图书馆 设计了一系列新颖的富含Trp的肽，每次相差2个残基，1个瓦尔和1个Arg，以建立 抗微生物效力的结构决定因素与宿主毒性的结构决定因素之间的区别。正如预测的那样， 对MDR临床分离株达到最低的最低抑菌浓度（MIC）， 在最大试验浓度下，红细胞溶解或对白色细胞的毒性增加。值得注意的是，一 选择的PAX（E35）中的一种在以4- 5 mg/kg或单次给药时表现出有效性。 剂量（MDR铜绿假单胞菌感染）或多次剂量（MDR肺炎克雷伯菌感染）。Pax E35还显示出30 mg/kg的最大耐受剂量，相比之下，我们的初始先导肽为12-15 mg/kg WLBU 2目前处于2期临床试验中。我们的迭代框架最大限度地减少了设计PAX的尝试和错误， 增强对MDR细菌的全身功效。鉴于对粘菌素耐药（Col-R）细菌的成功， 这种倾向是声称我们的工作是在没有考虑到当前PAX的潜在缺点的情况下完成的。 然而，还有太多的未知数。(1)MDR细菌尚未在临床上暴露于肽 就像他们对粘菌素一样。一旦他们这样做，几乎可以肯定的是，阻力频率将稳步上升。 (2)目前的结构优化只是最小的，因为我们还没有确定如何每个 阳离子（C）和疏水（H）基序有助于细菌识别和杀灭。因此，关键是 继续这项重要的工作，而一些PAX可能会进展到先进的临床前和临床研究。 我们建议使用Col-R菌株来定义这2个基序对靶标识别和消除的贡献 可以通过传代进化出对当前PAX的抗性。本提案的总体目标是阐明 通过解偶联PAX的C和H基序， C/H与LPS结构变化的相关性和使用col-R和 PAX-R MDR临床分离株。影响：我们将从最小的单一药物发展到全面的药物- 具有根据临床挑战设计或重新设计PAX的能力。