Gram-negative bacteria such as Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. comprise the “critical” tier of the World Health Organization’s priority list of drug-resistant pathogens.(1) Rising resistance to carbapenems and colistin (polymyxin E), traditionally the drugs of last resort, has led to global concern. New drugs are urgently needed, but the pace of discovery has been hampered by a dearth of novel and practical targets. A prime barrier to developing agents against Gram-negative bacteria is their outer membrane, composed primarily of phospholipids, proteins, and lipopolysaccharide (LPS). The outer membrane prevents entry of many types of compounds and houses several key multidrug efflux pumps that remove molecules that do enter the cell. However, the unique environment and components of the outer membrane and the periplasmic space also offer potential targets for selective anti-Gram-negative agents. Recently, several macrocyclic peptides that do just that have been reported. A team composed of scientists in academia and industry revealed a new antibiotic target as well as a potential strategy for overcoming Gram-negative colistin resistance. The group initially identified the potent Pseudomonas-specific antibiotic murepavadin (Figure 1) from a library of cyclic β-hairpin-containing peptidomimetics.(2) Murepavadin (POL7080), currently in late stage clinical development, targets the Pseudomonas homologue of the β-barrel outer membrane protein (OMP) LptD, which is widely present in Gram-negative bacteria and is essential for LPS transport to the outer membrane in Escherichia coli. In a more recent report,(3) the team optimized murepavadin for broad spectrum activity against Gram-negative bacteria. Figure 1. Chemical structures of murepavadin and colistin, with cartoon format of the same structures illustrated under each structure. Arrows point in the C-terminal direction of the amide chain. Also shown are cartoon representations of two chimeric peptides containing linked murepavadin-like and colistin-like macrocycles. Residues derived from murepavadin and colistin are colored red and yellow, respectively. Residues mutated for conjugation or artificial macrocyclization are colored blue, and residues mutated during lead optimization are colored green. Undisclosed residues (X₁–X₅) on peptide 2 are colored gray. The structure of the natural product darobactin is shown for comparison. Abbreviations: Dab, 2,4-diaminobutyric acid; Hse, homoserine; ^tBuGly, tert-leucine. The authors hypothesized that murepavadin’s mode of action could be synergistic with colistin (Figure 1), which targets the lipid A component of LPS. They synthesized a series of chimeric peptides that connect murepavadin-like β-hairpins to colistin-like macrocycles (Figure 1). A subset of these peptides [e.g., peptides 1 and 2 (Figure 1)] were inactive against Gram-positive bacteria but had potent activity against a wide range of Gram-negative bacteria, including carbapenem-resistant and colistin-resistant strains of A. baumannii, K. pneumoniae, P. aeruginosa, Enterobacter cloacae, and E. coli. Compound 1 was also active against an LPS-deficient A. baumannii mutant, suggesting that LPS cannot be the sole target. Mechanism of action studies showed that treated cells released β-lactamase and β-galactosidase from the periplasm and cytoplasm, respectively, indicating permeabilization of both the inner and the outer membrane. However, enantiomers of the active structures had very low activity, suggesting a target with a chiral structure. Fluorescent peptide derivatives concentrated in foci on cell membranes, consistent with binding to outer membrane protein complexes, and photo-cross-linking experiments in E. coli and sequencing of resistant mutants of K. pneumoniae pointed to a protein called BamA. This outer membrane β-barrel protein is integral to the assembly complex that catalyzes the folding of β-barrel OMPs and their insertion into the outer membrane.(4) Fluorescence polarization experiments confirmed BamA binding with submicromolar affinity. Subsequent characterization of the BamA–peptide interaction was performed by two-dimensional nuclear magnetic resonance spectroscopy using BamA embedded in lipid nanodiscs. Binding-dependent chemical shift and intensity perturbations were concentrated in loops on the extracellular face of BamA, suggesting a possible peptide binding site, with the peptide causing a shift in the equilibrium between the two primary conformers of BamA to the closed conformation. In addition to BamA, which was photolabeled by all chimeric peptides carrying photoaffinity probes, other β-barrel OMPs such as LamB, LptD, and LptE were labeled by individual peptides, which may be related to the dependence of these proteins on the β-barrel assembly machinery (BAM) for folding and membrane insertion. The exact mechanism of membrane disruption and cell death by the chimeric peptides and how these effects are related to BamA binding are currently still unresolved. In vivo experiments were performed to measure efficacy against Gram-negative infections in several mouse models. Administration in moderate doses was effective against septicemia, thigh infection, and peritonitis, including infections with extensively drug-resistant and colistin-resistant isolates. The peptides were also superior to colistin in measures of nephrotoxicity, which normally limits the clinical use of colistin to life-threatening infections. Resistance frequencies of the pathogens tested were low as was cytotoxicity against mammalian cells. Thus, these compounds are very promising leads as antimicrobial agents that are selective for a broad range of Gram-negative organisms. One of the most intriguing aspects of this study is the combination of two individual pharmacophores (murepavadin and colistin) to form a chimeric molecule with an entirely different target. As noted above, BamA, LptD, and LPS are not completely unrelated as the BAM complex catalyzes the folding and membrane insertion of LptD, and LptD is involved in the transport of LPS to the outer membrane leaflet. However, determining the molecular mechanism underlying the dramatic shift in binding between the chimeric peptides and their two individual components will require more detailed characterization of both LPS transport and OMP maturation. Time will tell whether targeting the BAM complex yields clinically useful antibiotics, but it appears that Nature has already exploited this strategy. For instance, darobactin, a recently reported antibacterial peptide from Photorhabdus, also appears to bind BamA and has a spectrum of activity similar to that of the chimeric peptides.(5) Darobactin also has two macrocycles formed by cross-linking of the side chains of Trp and Lys (Figure 1), although the rings are much smaller than those in chimeric peptides 1 and 2. The compound is a member of the ribosomally synthesized and post-translationally modified peptides (RiPPs) and was discovered by screening Photorhabdus symbionts present in the gut microbiome of nematodes. E. coli resistance mutants all had mutations in the gene encoding BamA, and these mutations were sufficient to confer darobactin resistance on previously sensitive strains. Furthermore, darobactin was shown to inhibit BamA-mediated in vitro protein refolding. The discoveries in these two recent reports suggest that BAM inhibition, and outer membrane biogenesis in general, may be a fruitful area for future Gram-negative antibiotic development. W.A.v.d.D. is a Howard Hughes Medical Investigator. The authors declare no competing financial interest. β-barrel assembly machinery lipopolysaccharide ribosomally synthesized and post-translationally modified peptide outer membrane protein 2,4-diaminobutyric acid homoserine tert-leucine. This article references 5 other publications.

中文翻译：

---

指环王：大环抗生素肽揭示了抗革兰氏阴性靶标。

肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌等革兰氏阴性菌属 构成世界卫生组织优先耐药病原体清单的“关键”层级。(1) 对碳青霉烯类和粘菌素（多粘菌素 E）的耐药性不断上升，传统上是不得已的药物，已引起全球关注。迫切需要新药，但由于缺乏新颖实用的靶标，发现的步伐受到了阻碍。开发针对革兰氏阴性菌的药物的主要障碍是它们的外膜，主要由磷脂、蛋白质和脂多糖 (LPS) 组成。外膜可防止多种化合物进入，并装有几个关键的多药外排泵，可去除进入细胞的分子。然而，外膜和周质空间的独特环境和成分也为选择性抗革兰氏阴性药物提供了潜在靶点。最近，已经报道了几种具有这种作用的大环肽。一个由学术界和工业界的科学家组成的团队揭示了一个新的抗生素目标以及克服革兰氏阴性粘菌素耐药性的潜在策略。该小组最初确定了强效假单胞菌特异性抗生素 murepavadin（图 1）来自含有环状 β-发夹的拟肽库。(2) Murepavadin (POL7080) 目前处于后期临床开发阶段，靶向 β-桶外膜蛋白 (OMP) 的假单胞菌同系物) LptD，广泛存在于革兰氏阴性菌中，对大肠杆菌中 LPS 转运至外膜至关重要. 在最近的一份报告中，(3) 该团队针对革兰氏阴性菌的广谱活性优化了 murepavadin。图 1. murepavadin 和粘菌素的化学结构，每个结构下都有相同结构的卡通形式。箭头指向酰胺链的 C 端方向。还显示了两个嵌合肽的卡通表示，其中包含连接的 murepavadin 样和粘菌素样大环化合物。来自 murepavadin 和粘菌素的残基分别呈红色和黄色。为共轭或人工大环化而突变的残基为蓝色，在前导优化过程中突变的残基为绿色。肽2上未公开的残基 (X ₁ –X ₅ )是灰色的。显示天然产物 darobactin 的结构以供比较。缩写：Dab，2,4-二氨基丁酸；Hse，高丝氨酸；^t BuGly，叔-亮氨酸。作者假设 murepavadin 的作用方式可能与粘菌素具有协同作用（图 1），粘菌素靶向 LPS 的脂质 A 成分。他们合成了一系列嵌合肽，将 murepavadin 样 β 发夹连接到粘菌素样大环化合物（图 1）。这些肽的一个子集 [例如，肽1和2（图 1）] 对革兰氏阳性菌无活性，但对多种革兰氏阴性菌具有有效活性，包括耐碳青霉烯和耐粘菌素的曲霉菌菌株。鲍曼不动杆菌,肺炎克雷伯菌、铜绿假单胞菌、阴沟肠杆菌和大肠杆菌。化合物1对缺乏 LPS 的鲍曼不动杆菌突变体也有活性，表明 LPS 不是唯一的靶标。作用机制研究表明，经处理的细胞分别从周质和细胞质中释放 β-内酰胺酶和 β-半乳糖苷酶，表明内膜和外膜均发生透化作用。然而，活性结构的对映异构体具有非常低的活性，表明具有手性结构的目标。荧光肽衍生物集中在细胞膜上，与外膜蛋白复合物的结合以及大肠杆菌中的光交联实验一致肺炎克雷伯菌耐药突变体的测序指向一种叫做 BamA 的蛋白质。这种外膜 β 桶蛋白是组装复合物的组成部分，该复合物催化 β 桶 OMP 的折叠及其插入外膜。(4) 荧光偏振实验证实 BamA 以亚微摩尔亲和力结合。使用嵌入脂质纳米圆盘中的 BamA，通过二维核磁共振波谱法对 BamA-肽相互作用进行了后续表征。依赖于结合的化学位移和强度扰动集中在 BamA 细胞外表面的环中，表明可能存在肽结合位点，肽导致 BamA 的两个主要构象异构体之间的平衡向闭合构象转变。除了 BamA，它被所有携带光亲和探针的嵌合肽光标记，其他 β 桶 OMP，如 LamB、LptD 和 LptE 被单独的肽标记，这可能与这些蛋白质对折叠和膜插入的 β 桶组装机制 (BAM) 的依赖性有关。嵌合肽引起的膜破坏和细胞死亡的确切机制以及这些效应如何与 BamA 结合相关目前仍未解决。体内进行了实验以测量在几种小鼠模型中对抗革兰氏阴性菌感染的功效。中等剂量给药可有效对抗败血症、大腿感染和腹膜炎，包括广泛耐药和粘菌素耐药分离株的感染。这些肽在肾毒性方面也优于粘菌素，这通常将粘菌素的临床使用限制在危及生命的感染中。测试的病原体的耐药频率低，对哺乳动物细胞的细胞毒性也低。因此，这些化合物是非常有前途的线索，可以作为对广泛的革兰氏阴性生物体具有选择性的抗菌剂。这项研究最有趣的方面之一是结合两个单独的药效团（murepavadin 和粘菌素）形成具有完全不同靶标的嵌合分子。如上所述，BamA、LptD 和 LPS 并非完全无关，因为 BAM 复合物催化 LptD 的折叠和膜插入，而 LptD 参与 LPS 向外膜小叶的转运。然而，要确定嵌合肽与其两个单独成分之间结合发生显着变化的分子机制，将需要对 LPS 转运和 OMP 成熟进行更详细的表征。时间会证明靶向 BAM 复合物是否会产生临床上有用的抗生素，但大自然似乎已经利用了这一策略。例如，达罗巴汀，Photorhabdus似乎也与 BamA 结合，并且具有与嵌合肽相似的一系列活性。(5) Darobactin 还具有两个通过 Trp 和 Lys 侧链交联形成的大环（图 1），尽管环远小于嵌合肽1和2中的那些。该化合物是核糖体合成和翻译后修饰肽 (RiPP) 的成员，是通过筛选存在于线虫肠道微生物组中的Photorhabdus共生体发现的。大肠杆菌抗性突变体都在编码 BamA 的基因中发生突变，这些突变足以使以前敏感的菌株产生 darobactin 抗性。此外，darobactin 显示可抑制 BamA 介导的体外蛋白质再折叠。最近这两份报告中的发现表明，BAM 抑制和一般的外膜生物发生可能是未来革兰氏阴性抗生素开发的一个富有成效的领域。WAvdD 是霍华德休斯医学调查员。作者声明没有相互竞争的经济利益。β-桶组装机械脂多糖核糖体合成和翻译后修饰肽外膜蛋白2,4-二氨基丁酸高丝氨酸叔亮氨酸。本文引用了其他 5 篇出版物。