药学学报

Information	Species
Structure
PDB ID	4Q35	4N4R	4RHB	5IVA	5IV9	7OMM	5IXM
Sequence	LptD	25-784 aa	226-786 aa	230-784 aa	317-898 aa	25-783 aa	88-801 aa	3-556 aa
LptE	20-169 aa	19-169 aa	20-169 aa	21-169 aa	20-169 aa	31-158 aa	2-146 aa

Information	Species
Structure
PDB ID	4Q35	4N4R	4RHB	5IVA	5IV9	7OMM	5IXM
Sequence	LptD	25-784 aa	226-786 aa	230-784 aa	317-898 aa	25-783 aa	88-801 aa	3-556 aa
LptE	20-169 aa	19-169 aa	20-169 aa	21-169 aa	20-169 aa	31-158 aa	2-146 aa

NCT number	Title	Primary outcome measure	Phase	Date	Oragnization	Status
NCT02165293	A single center study to evaluate the penetration of RO7033877 into the lung in healthy volunteers	Multiple dose PK of RO7033877: C_max, AUC, C_max/AUC	Phase I	2014.6-2014.9	Roche	Completed
NCT02156323	A single-center, open-label, randomized, two sequence, 3-way crossover study to investigate the interaction between multiple doses of colistin methanesulfonate sodium and multiple doses of RO7033877 in healthy subjects	Multiple dose PK of RO7033877 and colistin: C_max, AUC	Phase I	2014.7-2015.3	Roche	Completed
NCT02165332	A two-part, single-dose, randomized study to evaluate the safety of supra-therapeutic doses of RO7033877 and to investigate the effect of RO7033877 on the QTc interval	Part 1: Incidence of ADEs after single supratherapeutic dose of RO7033877; plasma and urine concentrations, AUC Part 2: ECG recordings	Phase I	2014.7-2014.11	Roche	Completed
NCT02110459	An open-label, non-randomized, monocenter, single-dose, phase I study to evaluate PK and safety of POL7080 administered as single intravenous infusion to subjects with renal impairment	Plasma concentrations of POL7080	Phase I	2013.4-2015.7	Polyphor	Completed
NCT02897869	A single-center, open-label, two sequence, crossover study to investigate the interaction between amikacin and POL7080 in healthy subjects	Multiple dose PK of POL7080 and amikacin: plasma C_max, AUC	Phase I	2016.4-2016.12	Polyphor	Completed
NCT02096328	A phase II, open-label, multi-center study to assess PK, safety and efficacy of POL7080 co-administered with standard of care treatment in patients with ventilator-associated pneumonia due to suspected or documented P. aeruginosa infection	Plasma concentrations of POL7080	Phase II	2013.10-2016.12	Polyphor	Completed
NCT02096315	A phase II, open-label, multicenter study to assess the tolerance, safety, efficacy and PK/PD of POL7080 in the treatment of patients with acute exacerbation of non-cystic fibrosis bronchiectasis due to P. aeruginosa infection requiring intravenous treatment	Sputum bacterial clearance	Phase II	2013.12-2015.11	Polyphor	Terminated
NCT03409679	A multicenter, open-label, randomized, active-controlled, parallel group, pivotal study to investigate the efficacy, safety and tolerability, and pharmacokinetics of murepavadin combined with one anti-pseudomonal antibiotic versus two anti-pseudomonal antibiotics in adult subjects with ventilator-associated bacterial pneumonia suspected or confirmed to be due to P. aeruginosa	Clinical cure rate	Phase III	2016.4-2016.12	Polyphor	Terminated
NCT03582007	A multicenter, open label, sponsor blinded, randomized, active controlled, parallel group, pivotal study to evaluate the efficacy, safety, and tolerability of murepavadin given with ertapenem versus an anti-pseudomonal-β-lactam-based antibiotic in adult subjects with nosocomial pneumonia suspected or confirmed to be due to P. aeruginosa	All cause mortality rates	Phase III	2018.10-2019.7	Polyphor	Terminated

NCT number	Title	Primary outcome measure	Phase	Date	Oragnization	Status
NCT02165293	A single center study to evaluate the penetration of RO7033877 into the lung in healthy volunteers	Multiple dose PK of RO7033877: C_max, AUC, C_max/AUC	Phase I	2014.6-2014.9	Roche	Completed
NCT02156323	A single-center, open-label, randomized, two sequence, 3-way crossover study to investigate the interaction between multiple doses of colistin methanesulfonate sodium and multiple doses of RO7033877 in healthy subjects	Multiple dose PK of RO7033877 and colistin: C_max, AUC	Phase I	2014.7-2015.3	Roche	Completed
NCT02165332	A two-part, single-dose, randomized study to evaluate the safety of supra-therapeutic doses of RO7033877 and to investigate the effect of RO7033877 on the QTc interval	Part 1: Incidence of ADEs after single supratherapeutic dose of RO7033877; plasma and urine concentrations, AUC Part 2: ECG recordings	Phase I	2014.7-2014.11	Roche	Completed
NCT02110459	An open-label, non-randomized, monocenter, single-dose, phase I study to evaluate PK and safety of POL7080 administered as single intravenous infusion to subjects with renal impairment	Plasma concentrations of POL7080	Phase I	2013.4-2015.7	Polyphor	Completed
NCT02897869	A single-center, open-label, two sequence, crossover study to investigate the interaction between amikacin and POL7080 in healthy subjects	Multiple dose PK of POL7080 and amikacin: plasma C_max, AUC	Phase I	2016.4-2016.12	Polyphor	Completed
NCT02096328	A phase II, open-label, multi-center study to assess PK, safety and efficacy of POL7080 co-administered with standard of care treatment in patients with ventilator-associated pneumonia due to suspected or documented P. aeruginosa infection	Plasma concentrations of POL7080	Phase II	2013.10-2016.12	Polyphor	Completed
NCT02096315	A phase II, open-label, multicenter study to assess the tolerance, safety, efficacy and PK/PD of POL7080 in the treatment of patients with acute exacerbation of non-cystic fibrosis bronchiectasis due to P. aeruginosa infection requiring intravenous treatment	Sputum bacterial clearance	Phase II	2013.12-2015.11	Polyphor	Terminated
NCT03409679	A multicenter, open-label, randomized, active-controlled, parallel group, pivotal study to investigate the efficacy, safety and tolerability, and pharmacokinetics of murepavadin combined with one anti-pseudomonal antibiotic versus two anti-pseudomonal antibiotics in adult subjects with ventilator-associated bacterial pneumonia suspected or confirmed to be due to P. aeruginosa	Clinical cure rate	Phase III	2016.4-2016.12	Polyphor	Terminated
NCT03582007	A multicenter, open label, sponsor blinded, randomized, active controlled, parallel group, pivotal study to evaluate the efficacy, safety, and tolerability of murepavadin given with ertapenem versus an anti-pseudomonal-β-lactam-based antibiotic in adult subjects with nosocomial pneumonia suspected or confirmed to be due to P. aeruginosa	All cause mortality rates	Phase III	2018.10-2019.7	Polyphor	Terminated

抗革兰阴性菌新靶标: 脂多糖转运蛋白LptDE及其抑制剂研究进展

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李玥 ¹ , 李国庆 ² , 田元元 ³ , 李聪然 ² , 杨信怡 ²^,^* , 姚开虎 ¹^,^* , 游雪甫 ²^,^*

药学学报 | 综述 2024,59(2): 279-288

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药学学报 | 综述 2024, 59(2): 279-288

抗革兰阴性菌新靶标: 脂多糖转运蛋白LptDE及其抑制剂研究进展

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李玥¹, 李国庆², 田元元³, 李聪然², 杨信怡²^,^*, 姚开虎¹^,^*, 游雪甫²^,^*

作者信息

1.国家儿童医学中心/首都医科大学附属北京儿童医院/北京市儿科研究所/国家呼吸系统疾病临床医学研究中心/教育部儿科重大疾病研究重点实验室/儿童呼吸道感染性疾病研究北京市重点实验室, 北京 100045

2.中国医学科学院、北京协和医学院医药生物技术研究所, 抗感染药物研究北京市重点实验室, 北京 100050

3.国家儿童医学中心/首都医科大学附属北京儿童医院皮肤科, 北京 100045

通讯作者:

^*杨信怡, E-mail: xinyiyang@imb.cams.cn;

姚开虎, E-mail: jiuhu2655@sina.com;

游雪甫, E-mail: xuefuyou@imb.pumc.edu.cn

Novel antibacterial drug target against Gram-negative bacteria: lipopolysaccharide transport protein LptDE and its inhibitors

Yue LI¹, Guo-qing LI², Yuan-yuan TIAN³, Cong-ran LI², Xin-yi YANG²^,^*, Kai-hu YAO¹^,^*, Xue-fu YOU²^,^*

Affiliations

1. National Center for Children's Health/Beijing Children's Hospital, Capital Medical University/Beijing Pediatric Research Institute/National Clinical Research Center for Respiratory Diseases/Key Laboratory of Major Diseases in Children, Ministry of Education/Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing 100045, China

2. Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

3. Department of Dermatology, National Center for Children's Health/Beijing Children's Hospital, Capital Medical University, Beijing 100045, China

出版时间: 2024-02-12 doi: 10.16438/j.0513-4870.2023-0903

文章导航

摘要

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富含脂多糖(lipopolysaccharide, LPS) 的外膜是绝大多数革兰阴性(Gram-negative, G^-) 菌生存必需的生物学屏障。LPS转运蛋白(lipopolysaccharide transport protein, Lpt) 复合体LptDE负责LPS转运和外膜组装的最后关键一步, 在G^-菌中普遍存在且结构-功能较为保守, 哺乳动物细胞中缺乏同源蛋白, 是颇具开发前景的抗菌新靶标。近10年, LptDE蛋白三维结构的破译为基于此类“非酶”蛋白靶标的药物发现奠定了基础, 首个作用于LptD的拟肽类化合物murepavadin开启了一类新作用机制的抗菌药物研究。本文将总结LptDE的分子特征、结构-功能, 梳理murepavadin等新型拟肽类抗菌药物的研究进展, 以期为相关研究提供参考。

关键词

革兰阴性菌 / 脂多糖外膜 / 脂多糖转运蛋白复合体DE / 抗菌新靶标 / 新型拟肽类抗菌药物

Abstract

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The outer membrane composed predominantly of lipopolysaccharide (LPS) is an essential biological barrier for most Gram-negative (G^-) bacteria. Lipopolysaccharide transport protein (Lpt) complex LptDE is responsible for the critical final stage of LPS transport and outer membrane assembly. The structure and function of LptDE are highly conserved in most G^- bacteria but absent in mammalian cells, and thus LptDE complex is regarded as an attractive antibacterial target. In recent 10 years, the deciphering of the three-dimensional structure of LptDE protein facilities the drug discovery based on such "non-enzyme" proteins. Murepavadin, a peptidomimetic compound, was reported to be the first compound able to target LptD, enlightening a new class of antibacterial molecules with novel mechanisms of action. This article is devoted to summarize the molecular characteristics, structure-function of LptDE protein complex and review the development of murepavadin and related peptidomimetic compounds, in order to provide references for relevant researches.

Key words

Gram-negative bacteria / lipopolysaccharide outer membrane / lipopolysaccharide transport protein complex DE / new antibacterial drug target / novel peptidomimetic compound

引用本文

李玥, 李国庆, 田元元, 李聪然, 杨信怡, 姚开虎, 游雪甫. 抗革兰阴性菌新靶标: 脂多糖转运蛋白LptDE及其抑制剂研究进展. 药学学报, 2024 , 59 (2) : 279 -288 . DOI: 10.16438/j.0513-4870.2023-0903

Yue LI, Guo-qing LI, Yuan-yuan TIAN, Cong-ran LI, Xin-yi YANG, Kai-hu YAO, Xue-fu YOU. Novel antibacterial drug target against Gram-negative bacteria: lipopolysaccharide transport protein LptDE and its inhibitors[J]. Acta Pharmaceutica Sinica, 2024 , 59 (2) : 279 -288 . DOI: 10.16438/j.0513-4870.2023-0903

正文

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革兰阴性(Gram-negative, G^-) 菌耐药日趋严重, 有效药物匮乏, 临床治疗困难。脂多糖(lipopolysaccharide, LPS) 是G^-菌外膜的关键结构单元, 不存在于革兰阳性(Gram-positive, G⁺) 菌和哺乳动物的细胞中, 因此构成G^-菌所特有的生物学屏障, 并是一种重要的毒力因子, 为绝大多数G^-菌生存所必需。LPS是携带负电荷的脂水两亲性生物大分子(图 1), 由脂质A、核心多糖和O-特异性寡糖三部分以共价结合方式相连。脂质A是LPS结构-功能的核心, 通常以二聚N-乙酰葡萄糖胺为骨架, 连接着6条长链脂肪酸, 具有两个磷酸化位点。脂肪酰基链在疏水作用的驱动下致密堆积, 携带负电荷的磷酸基团与插入LPS分子之间的二价阳离子(如Mg²⁺) 相互作用, 形成疏水-电荷双重渗透屏障。LPS外膜生物学屏障不仅导致G^-菌对已有的疏水性抗菌药物敏感性差, 也是抗G^-菌新药研发难度大、周期长的重要原因。

LPS生物合成和转运对保证外膜结构-功能完整性至关重要。早在1980s~1990s, LPS保守核心结构Kdo2-脂质A在胞浆中的生物合成通路已被Anderson等^[1]解析得较为清楚, 其中以限速酶UDP-3-O-(R-羟基十四酰)-N-乙酰氨基葡糖脱乙酰氨基酶(LpxC) 为靶标开发的抗菌药物候选物ACHN-975曾一度进入I期临床试验^[2]。近10年, 蛋白三维结构的解析和功能学研究不断深入与精细化, LPS跨膜运输和外膜组装机制逐渐清晰。其中, LPS转运蛋白(lipopolysaccharide transport protein, Lpt) LptD和LptE组成“桶-塞”结构蛋白复合体, 定位于G^-菌外膜, 负责LPS运输及插膜的最后一步, 对外膜合成和组装至关重要。2010年瑞士苏黎世大学的Robinson课题组基于天然抗菌肽protegrin I (PG-I) 研发了强效、特异性杀灭铜绿假单胞菌的环状拟肽, 机制研究推测其首要靶标为LptD。Polyphor公司开发的同类型拟肽murepavadin (POL7080或RO7033877) 是首个作用于G^-菌外膜蛋白的抗菌药物候选物。迄今, murepavadin注射剂治疗铜绿假单胞菌肺炎的最高研究阶段为III期临床试验(已终止)。

尽管murepavadin掀起了LptDE作为新型抗G^-菌分子靶标的热度, 但是目前还没有真正通过“定靶筛选”发现的LptDE抑制剂, 导致基于LptDE的抗菌药物研究无法突破以拟肽为主的单一结构类型。当前面临的研究瓶颈与LptDE这类“非酶”膜蛋白结构-功能的复杂性直接相关。本文将从LPS转运的全过程谈起, 着重总结LptDE的分子生物学和生物化学性质、三维结构特征、重要功能结构域, 梳理新型拟肽类抗菌药物的研究进展, 期为针对该靶标的相关药物研究提供参考。

1 LPS转运蛋白及其跨膜运输机制

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1989年, Sampson^[3]发现了一个与细菌膜通透性相关的基因, 命名为imp (increased membrane permeability)。进一步研究发现imp与大肠埃希菌对有机溶剂正己烷的耐受水平相关, 故又将其称作ostA (organic solvent tolerance)^[4]。2002年, 普林斯顿大学发表研究揭示了Imp/OstA是大肠埃希菌外膜形成和细菌存活的必需蛋白, 属于β-桶状蛋白, 定位于外膜^[5]。2006年普林斯顿大学和哈佛大学医学院联合发现Imp/OstA与脂蛋白RlpB以复合体形式存在^[6]。2007~2008年间, 陆续鉴定了其他的LPS转运蛋白, 包括位于周质空间的LptA (YhbN)、LptC (YrbK) 与位于内膜的LptB (YhbG)、LptF (YjgP) 和LptG (YjgQ), 并将Imp/OstA和RlpB重新命名为LptD和LptE^{[7, 8]}。

2008年, Sperandeo等^[9]基于MsbA和7个转运蛋白(LptA~G) 的细胞定位、生物化学特点和生物学功能提出LPS跨越内膜-周质空间-外膜转运的分子模型(图 1): LPS在内膜内侧合成, 由内膜上的ATP结合盒转运体MsbA将其从内膜内侧翻转至内膜外侧, ABC转运复合体LptB₂FG将LPS从内膜外页(inner leaflet) 移出并引导至LptC, LptC继而将LPS传递给周质空间转运蛋白LptA, 多聚化的LptA蛋白纤丝搭建起LPS跨越周质空间的桥梁, LptD周质空间结构域接收由LptA呈递的LPS, 通过LptDE复合体跨膜区将LPS插入外膜外页(outer leaflet) 中。2011年至今, 7个LPS转运蛋白的三维结构均已得到解析, 从结构生物学角度佐证了Sperandeo提出的LPS跨膜转运模型。

既往研究显示, LPS转运蛋白功能如果存在缺陷, 通常引发外膜结构-功能异常、细胞形态异常、细胞内容物外泄直至死亡等一系列生物学改变^{[5, 6]}, 主要包括: ①细菌对疏水性化合物(抗菌药物、染料等) 通透性增加; ②细菌胞内蛋白渗出; ③电镜下细菌黏连, 呈现长丝样改变(细胞分裂异常); ④膜样物质在周质空间异常堆积; ⑤ LPS/磷脂比例异常, 外膜密度改变; ⑥外膜蛋白PagP/PagL被激活并对LPS进行化学修饰; ⑦活细菌数随细菌繁殖逐代降低。

2 LptDE生物化学特征

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据粗略估计, 单个G^-菌外膜上大约分布着60个LptDE分子, 属于极低丰度蛋白, 无法通过免疫印迹(Western blot, WB) 的方法在全细胞裂解液中检测到, 需分离提取外膜蛋白后方可检测^{[5, 10]}。LptDE复合体为1∶1异源二聚体。LptD大致由780~800个氨基酸(amino acid, aa) 组成, 分子量~90 kD, 芳香族氨基酸占比超过13%; LptE为~20 kD的脂蛋白, 常由169个aa组成, 通过N-末端半胱氨酸残基翻译后脂化锚定在外膜内侧^[11]。

LptD的两对二硫键Cys173-Cys725和Cys31-Cys724对维系β桶高级结构至关重要, 导致LptD变性前后在凝胶电泳中的迁移速率不同。天然状态的LptD或与其他LPS转运蛋白形成高分子复合体, 可能由于分子量过大不能进入SDS-聚丙烯酰胺凝胶(SDS-PAGE) 的孔隙中, 无法通过凝胶电泳检测到。LptDE复合体对1.5%的SDS稳定, LptDE核心复合物(包括LptD 521~542 aa和LptE 146~157 aa) 可以抵抗胰蛋白酶降解^[11]。360 mmol·L^-1 β-巯基乙醇使大肠埃希菌LptDE复合物彻底解离和变性, LptD、LptE分别在~83 kD和~20 kD处呈现单一条带; 非还原态下从30 ℃加热至100 ℃, 复合体逐渐变性, LptD的迁移位置在83~110 kD之间变化, 可能同时出现2~3个条带^[12]。

LptD至少需要和LptE的C-末端共表达才能形成具有天然折叠状态的β桶状高级结构。2014~2022年间, 多个研究团队尝试实现不同种属G^-菌LptDE的体外重组表达, 普遍发现全长的LptDE可溶性差, 去掉LptD的N-末端周质空间结构域后, LptDE的可溶性表达大大提高^[13-15]。

3 LptDE三维结构特征

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2014年7月3日, 中国科学院生物物理所的Qiao等^[16]和东英吉利大学的Dong等^[17]于同一天在《Science》上发表两篇文章, 首次解析了福氏志贺菌和鼠伤寒沙门菌LptDE的三维结构。目前, 共7个种属G^-菌的LptDE三维结构得到解析(表 1), 其中福氏志贺菌、肺炎克雷伯菌和淋病奈瑟菌的LptDE三维结构是包含LptD N-末端周质空间结构域的全长蛋白。不同种属G^-菌LptDE三维结构高度相似, 下文多以福氏志贺菌LptDE三维结构(PDB ID: 4Q35) 为例进行结构-功能阐述。

LptD是迄今发现的最大的β桶状蛋白, 也是唯一以一个独立的蛋白(LptE) 作为“塞子”的β桶状蛋白^[17]。LptD三维结构可进一步分为C-末端β桶跨膜区(图 2A, 蓝色) 和N-末端周质空间区(图 2A, 绿色和红色)。如图 2B所示, LptD的C-末端β桶跨膜区由26个β片层(β1~β26) 正-反向折叠环绕成肾型的桶样结构, 在β1和β26两条β片层之间闭合, 各β片层由环状结构(loop, L) 连接, 胞外侧的环明显比周质空间侧的环长, 其中L4和L8两条环伸向β桶中部孔隙, 其他环伸向β桶表面。β桶孔隙内遍布携带电荷的氨基酸, 形成亲水环境, 有利于LPS分子中的亲水性糖链进入而不利于脂质部分进入。N-末端周质空间区可以进一步分为N-末端环(图 2A, 红色) 和β-jellyroll (图 2A, 绿色), β-jellyroll由11个正-反向折叠的β片层构成蛋糕卷样结构, 中部凹槽形成V型疏水腔(图 2C)。

LptE (图 2A, 紫色) 大体位于LptD的β桶中部孔隙内, 约有1/3的体积暴露于周质空间中, LptE并未填满整个β桶, 在周质空间侧留有约45 Å × 35 Å的空隙, 足够LPS进入β桶中^[15]。LptE是一个罕见的脂蛋白, 如图 2D所示由2个α螺旋(α1、α2) 和4个β折叠(β1~β4) 组成, 4个β折叠包括3条长链(β2~β4) 和一条短链(β1), LptE的三维结构与节肢动物鲎所产生的抗内毒素蛋白LALF较为相似(图 2D)^[18]。LptE通过N-末端半胱氨酸残基脂质化锚定在外膜内侧。

4 LptDE功能结构域预测

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当前的研究推测, LptDE至少应当具备以下5种分子功能: ① LPS单体插入外膜的门控功能; ② LptD-LptE相互作用的功能; ③结合转运底物LPS的功能; ④辅助自身β桶折叠的功能; ⑤与其他LPS转运蛋白相互作用的功能。目前, LptDE功能结构域的研究多基于结构预测和分子模拟, 分子机制不完全清楚, 下文将对其进行总结。

4.1 LPS插膜门控区

LPS插膜门控区大致位于LptD的β桶上。如图 3A显示β折叠β1、β2、β3上分布着3个高度保守的脯氨酸(Pro231、Pro246、Pro261), 导致这3条β折叠之间所形成的氢键很少, 堆叠疏松、柔性强^{[13, 15-17]}。Qiao等^[16]推测β1-β2发挥门的作用, 在β3和β26之间形成一个16 Å的横向裂隙, 这个裂隙很可能是LPS插入外膜的出口。Lundquist等^[19]采用分子动力学模拟LptD转运LPS的动态过程, 推测LPS在微秒级别上激活了LptD。

4.2 LptD-LptE“桶-塞”互作区

LptD-LptE广泛的互作界面维系了“桶-塞”高级结构。采用交联法发现大肠埃希菌LptD上有27个氨基酸直接与LptE相互作用^[20], 其遍布LptD的β桶内部。基于鼠伤寒沙门菌LptDE三维结构全面预测了两者的相互作用(图 3B), 发现以LptDE核心复合物中的氨基酸残基相互作用较为强烈, 组成核心复合物的LptD胞外环L8上的Ser533、Asp531、Leu535与LptE的Glu139、Ala125、Met142相互作用, 另外LptD胞外环L4 (335~354 aa) 与LptE也可以形成多位点互作^[16]。互作氨基酸突变菌株lptE14相较野生型菌株对红霉素和利福平的药物敏感性明显提高, 但不影响LPS转运和LptD折叠, 推测“桶-塞”互作位点改变后可能导致LptE“塞子”松动, 抗菌药物或直接通过β-桶中部孔隙进入胞内^[21]。

4.3 底物结合区

4.3.1 LptD底物结合区

研究普遍推测N-末端β-jellyroll是LptD结合LPS的结构域。如图 3C所示, β-jellyroll由11个正-反向折叠的β片层组成蛋糕卷样结构, 其内表面形成V-型疏水腔, 这一结构特征与另外两个LPS转运蛋白LptC、LptA高度相似, 有利于结合LPS的疏水区。根据LptA与LPS的结合位点推测β-jellyroll上的Val51、Tyr112和Leu128 (图 3C) 是其与LPS互作的关键氨基酸^[16], 但尚缺乏体内、体外实验证据。

4.3.2 LptE底物结合区

体外分子互作实验显示, LptE可以特异性结合LPS, 当LptE达到一定浓度时可以使LPS多聚体解聚。基于LptE和抗LPS因子LALF的结构相似性, 推测连接β2-β3和连接β4-α2的两个环状结构上的阳离子氨基酸R91、K136或与lipid A携带负电的磷酸基团相互吸引(图 3D), 发挥结合底物LPS的作用。R91、K136位点突变后显著影响LPS多聚体的解聚, 双位点突变菌株对万古霉素和利福平的药物敏感性显著提高^[18]。加入LPS前后铜绿假单胞菌LptE连接β折叠和α螺旋的环状结构上有部分氨基酸残基发生了光谱学改变^[13]。

4.4 LptE辅助β桶折叠的功能区

研究发现, LptD前体蛋白只有先结合LptE, 才能被周质空间氧化酶DsbA氧化形成二硫键, 继而形成具有β桶结构的成熟蛋白^[11], 推测LptE结合LptD前体蛋白后促进了远距离半胱氨酸在空间结构上相互靠近。后续研究进一步定位了辅助β桶折叠的关键氨基酸, 发现LptE上116~120 aa区段由YPISA变成YRA后(图 3E), 氧化态的LptD明显减少, LptD蛋白稳定性显著降低, 突变菌株对杆菌肽和利福平的敏感性提高^[22]。

4.5 与其他LPS转运蛋白相互区

在7个LPS转运蛋白中, LptA和LptD的β-jellyroll具有高度的结构-功能相似性, 分子模拟推测它们通过相似的V型疏水腔结合和传递LPS, 在时间和空间上相互承接。LptD的β-jellyroll三维结构分辨率明显低于β-桶, 相较于福氏志贺菌, 肺炎克雷伯菌LptD的β-jellyroll发生了21 Å的扭转^[15] (图 3F)。以上研究均显示, β-jellyroll为柔性较强的结构域, 或通过适度扭转协调LPS转运桥的稳定性。

5 新型阳离子拟肽类抗菌药物murepavadin

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5.1 研发历程

PG-I是猪白细胞产生的天然阳离子抗菌肽, 由18个氨基酸组成, 富含半胱苷酸, Cys6-Cys15和Cys8-Cys13在两个反向折叠的β片层之间形成两对二硫键, 使得PG-I呈现β-发夹样三维结构(图 4A)。PG-I主要通过裂解生物膜发挥广谱抗菌活性, 具有很强的溶血性, 100 μg·mL^-1 PG-I溶血率为37%。2002年瑞士苏黎世大学的Robinson课题组^[12]将类似PG-I的环状多肽连接在D-脯氨酸-L-脯氨酸刚性模板上合成了β-发夹样拟肽分子库, 初步筛选发现100 μg·mL^-1 14元环拟肽L8-1溶血率降低至1%。

其后, Robinson课题组以L8-1为先导化合物进行结构优化(图 4A), 其中L27-11对铜绿假单胞菌PAO1的最低抑菌浓度(minimal inhibitory concentration, MIC) 为0.004 μg·mL^-1, 对其他受试G^-菌和G⁺菌的MIC值均≥ 64 μg·mL^-1 (图 4B), 体现出特异、强效杀灭铜绿假单胞菌的活性^[23]。L27-11浓度提高至100 mg·mL^-1时没有观察到明显的溶血作用。但L27-11容易被人血清中的胰蛋白酶降解。瑞士的Polyphor公司进一步优化了此类拟肽的成药性, 采用α, γ-二氨基丁酸(Dab) 替代L27-11中的赖氨酸或精氨酸, 开发了POL7080 (也称RO7033877), 药品名称为murepavadin。Murepavadin是首个作用于G^-菌外膜蛋白的“first-in-class”抗菌药物, 主要用于治疗铜绿假单胞菌引起的医院获得性细菌性肺炎和呼吸机相关细菌性肺炎。Murepavadin静脉注射剂最高研究阶段为III期临床试验, 目前2项III期临床试验均已终止(表 2)。终止原因或与患者发生急性肾损伤的频率高于预期有关。Polyphor公司(2022年更名为SPEXIS公司) 官网最新消息显示, 他们目前正积极推动murepavadin吸入给药剂型治疗肺纤维化感染的临床研究。

Murepavadin III期临床试验终止至今尚无新的LptDE抑制剂问世。由于LptDE是具有复杂结构和多种功能的大分子膜蛋白, 笔者推测缺乏酶活性、难以可溶性表达、柔性强、构象变化、功能结构域未十分明确等问题会给药物筛选模型的构建带来很大挑战, 故而从“源头”上获得新的先导化合物困难大、周期长。此外, murepavadin和LptD的分子间相互作用还不十分清楚, 因此基于murepavadin的化学改造、构-效关系研究等方面也缺乏可靠的分子基础。

5.2 药理学研究进展

5.2.1 体外药理学研究

Murepavadin对全球范围内分离的1 219株铜绿假单胞菌的50%和90%最低抑菌浓度值(MIC_50/90值) 为0.12 μg·mL^-1/0.12 μg·mL^-1[24]; 对785株泛耐药铜绿假单胞菌的MIC_50/90值为0.12 μg·mL^-1/0.25 μg·mL^-1[25]; 抑制铜绿假单胞菌生物被膜形成的90%最低杀膜浓度值(MBBC₉₀值) 为64 μg·mL^-1[26]。

Murepavadin的最低杀菌浓度(minimum bactericidal concentration, MBC) 与MIC比值为1~2。其发挥杀菌作用具有一定的浓度、时间依赖性, 在1~4× MIC值范围内使活菌数降低至初始接种量的1/1 000所需时间为3~7 h。

观察体外培养条件对murepavadin抗菌活性的影响, 发现50%的人类血清可以使murepavadin抗铜绿假单胞菌的MIC值升高8倍, 初始接种量(5×10⁵~1×10⁸ CFU·mL^-1)、pH值(5.5~8.5) 和50%的肺表面活性物质对murepavadin体外抗菌活性影响不大^[27]。

Murepavadin的抗生素后效应为1.2~4.2 h^[28]。1× MIC值浓度下铜绿假单胞菌对murepavadin的随机耐药突变率为10^-8~10^{-9 [29]}。Murepavadin联合阿奇霉素、氨曲南、头孢他啶、环丙沙星、黏菌素、亚胺培南抗铜绿假单胞菌均未体现出明显的协同或拮抗作用^[27]。

5.2.2 体内药理学研究

皮下注射murepavadin治疗铜绿假单胞菌感染所致小鼠败血症的半数有效剂量范围为0.25~0.55 mg·kg^{-1 [23]}; 治疗敏感(MIC = 0.125 μg·mL^-1) 和耐药(MIC = 16 μg·mL^-1) 铜绿假单胞菌引起的小鼠败血症的生存率达到100%时所需的药物剂量分别为1和3 mg·kg^{-1 [27]}。

采用小鼠腿部感染模型和肺炎模型研究murepavadin的药效指数和药效靶值, 发现主要疗效参数为游离血药浓度-时间曲线下面积(free-drug area under the concentration-time curve, fAUC) 和MIC值的比值即fAUC/MIC。以小鼠肺内活菌数降低至初始接种量的1/10为药效靶值, fAUC/MIC需要达到39.85^[30]。

小鼠、大鼠、家兔和猴4种实验动物的murepavadin单剂量药代动力学研究显示, 峰浓度(maximum plasma concentration, C_max)、AUC与给药剂量呈线性关系, 符合二房室模型, 半衰期(t_1/2) 为5~8 h, 血浆清除率(plasma clearance, CL) 与肾小球过滤率相似。皮下注射生物利用度为67%~79%, 口服生物利用度 < 0.01%^[31]。Murepavadin主要分布于肾脏、心脏、肝脏和肺中, 肾脏药物浓度最高, 脑组织药物浓度最低。Murepavadin经蛋白降解后主要由尿液排出^[31]。

5.2.3 临床研究

Murepavadin在美国FDA注册的临床试验共9项, 包括I期临床试验5项, II期临床试验2项, III期临床试验2项(表 2)。健康受试者静脉输注murepavadin单次剂量达4.5 mg·kg^-1、多次剂量达5 mg·kg^-1, 持续6天, 未出现严重不良反应或死亡^[32]。相较于健康受试者, 肾脏功能损伤者静脉输注murepavadin单次剂量为2.2 mg·kg^-1时, AUC_0–∞增加2~2.5倍, C_max增加1.5倍, CL由7 L·h^-1降低至2.4~3.8 L·h^-1, t_1/2由7.7 h延长至24 h^[33]。常见的不良事件包括输液部位反应、导管部位相关反应、头晕、感觉异常和口周感觉异常, 大多是轻微、一过性现象^{[32, 33]}。PK/PD模型预测, murepavadin治疗敏感铜绿假单胞菌(MIC = 0.25 μg·mL^-1) 医院获得性肺炎, 药效达标率为100%^[34]。目前, murepavadin联合其他抗菌药物治疗铜绿假单胞菌肺炎的两项III期临床试验均已提前终止。美国临床试验数据库(ClinicalTrials.gov) 中murepavadin所登记的两项III期临床试验终止的原因均简要记录为“安全数据审查”, Polyphor公司曾发表声明称III期临床试验数据显示, 患者血清中肌酸酐浓度升高, 急性肾损伤的频率高于预期^[35]。

5.2.4 作用机制研究

Murepavadin等新型阳离子拟肽具有独特的抗菌谱, 与已知抗菌药物无明显交叉耐药性, 也体现出新机制、新靶标抗菌活性物的优势所在。系列研究显示, L27-11等抑制LPS向外膜转运, 导致LPS不能被外膜蛋白PagL催化脱去3-O-脂肪酰基链^[36]。此类药物显著提高铜绿假单胞菌对去污剂(SDS和TritonX-100)、抗菌药物(替加环素、利福平等) 和荧光染料diSC3的通透性, 能够引起细胞内膜样物质堆积、细胞表面出泡、多细胞黏连等形态学改变^{[23, 36, 37]}。

Murepavadin诱导耐药研究显示, 高水平耐药突变菌株(MIC > 32 μg·mL^-1) 相较于野生型菌株在lptD的β-jellyroll编码区628~645 nt出现了18个碱基的重复串联^[23]。体外分子互作实验显示, L27-11与LptDE具有纳摩尔级别的亲和力[K_D = (32 ± 10)×10^-9 mol·L^-1]。以L27-11结构类似物PAL-6作为光标记探针分子, 在铜绿假单胞菌全细胞水平进行靶标垂钓发现, LptD与PAL-6存在特异性相互作用。通过胰蛋白酶降解使LptD片段化, 发现PAL-6结合在LptD的周质空间区段, 质谱鉴定与PAL-6结合的肽段为位于LptD周质空间β-jellyroll上的199~201 aa和插入序列上的85~87 aa^[37]。相较于其他常见的G^-菌, 周质空间插入序列是铜绿假单胞菌LptD所特有的结构^[13], PAL-6靶向此结构也一定程度解释了murepavadin窄谱、特异性杀灭铜绿假单胞菌的独特药理学特性。

另有一些研究揭示了此类拟肽可能的多机制、多靶标效应。如murepavadin可以通过MRGPRX2激活人体肥大细胞的免疫调节功能, 有助于细菌清除和伤口愈合^[38]; PmrB变异后的LPS化学修饰也可以引发铜绿假单胞菌对murepavadin耐药^[39]; 同类拟肽JB-95不仅作用于LptD, 而是作用于更多的β-桶状外膜蛋白^[40]。

以往研究显示, 抗菌肽不仅可以直接作用于细菌发挥抗/杀菌活性, 且常常具有免疫调节功能。但是目前的研究显示murepavadin不能中和LPS诱导的巨噬细胞活化, 没有明显的免疫调节功能和抗炎作用^[41]。

6 展望

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LptDE蛋白复合体因其特殊的细胞定位、重要的生物学功能、高度的G^-菌特异性被视为理想的抗菌新靶标, murepavadin所体现出来的窄谱、强效杀铜绿假单胞菌活性印证了LptDE的可靶性, 开创了以抑制LPS转运蛋白为作用机制的新型抗菌药物研究, 为临床最为棘手的耐药铜绿假单胞菌抗感染治疗带来了希望。

从murepavadin等拟肽类抗菌药物的研究历程可以看出, 此类药物并不是基于靶标发现的, 而是通过诱导耐药、靶标垂钓等反向找靶的方法定位到其作用靶标。Murepavadin首次报道的时间甚至要早于LptDE三维结构破译4年。目前为止, 还没有一个真正基于靶标发现的LptDE抑制剂。

LptDE是具有复杂结构和功能的膜蛋白, 可能通过瞬时的动态变构完成多种分子功能。基于此类“非酶”靶标的药物发现具有一定挑战性, 一方面需要尽可能精细化靶标蛋白的功能-结构域、分子机制, 另一方面需要借助高效、准确的实体或虚拟筛选策略获得先导化合物。本课题组尝试基于分子间相互作用构建药物高通量筛选模型并初步得到一些与LptDE结合能力较强的活性分子, 其靶标特异性和分子机制有待进一步验证。当前人工智能(artificial intelligence, AI) 技术逐渐应用于先导化合物发现和优化、药物分子从头生成、分子设计等新药研究的各个环节。相较于传统计算机辅助的苗头化合物识别, 基于AI的药物发现更为准确、快速、有效, 未来将有望助力基于此类“非酶”靶标的新药发现。另外, 蛋白水解靶向嵌合体(PROTACs) 等新技术的发展, 特别是最近报道的BacPROTACs技术^[42], 也扩展了细菌中可靶蛋白的范围, 或为干预复杂结构-功能的蛋白靶标(诸如LptDE在细胞质中的前体蛋白) 提供全新的解决方案。预计在不久的将来, 基于LptDE的药物研究定会有所发现, 突破当前单一的结构类型, 为临床抗G^-菌感染提供更为丰富的候选药物。

作者贡献: 李玥负责文献检索和综述撰写; 李国庆、田元元、李聪然负责文章修改; 杨信怡、姚开虎、游雪甫负责文章的思路指导、审阅及完善工作。

利益冲突: 所有作者均声明不存在利益冲突。

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国家自然科学基金资助项目(82204465)

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2024年第59卷第2期

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doi: 10.16438/j.0513-4870.2023-0903

接收时间：2023-07-25
首发时间：2025-11-28
出版时间：2024-02-12

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出版历史

收稿日期：2023-07-25
修回日期：2023-09-14

基金

国家自然科学基金资助项目(82204465)

作者信息

2.中国医学科学院、北京协和医学院医药生物技术研究所, 抗感染药物研究北京市重点实验室, 北京 100050

3.国家儿童医学中心/首都医科大学附属北京儿童医院皮肤科, 北京 100045

通讯作者:

^*杨信怡, E-mail: xinyiyang@imb.cams.cn;

姚开虎, E-mail: jiuhu2655@sina.com;

游雪甫, E-mail: xuefuyou@imb.pumc.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Information	Species
Structure
PDB ID	4Q35	4N4R	4RHB	5IVA	5IV9	7OMM	5IXM
Sequence	LptD	25-784 aa	226-786 aa	230-784 aa	317-898 aa	25-783 aa	88-801 aa	3-556 aa
LptE	20-169 aa	19-169 aa	20-169 aa	21-169 aa	20-169 aa	31-158 aa	2-146 aa

Information

Species

S. flexneri

S. typhimurium

E. coli

P. aeruginosa

K. pneumoniae

N. gonorrhoeae

Y. pestis

Structure

PDB ID

4Q35

4N4R

4RHB

5IVA

5IV9

7OMM

5IXM

Sequence

LptD

25-784 aa

226-786 aa

230-784 aa

317-898 aa

25-783 aa

88-801 aa

3-556 aa

LptE

20-169 aa

19-169 aa

20-169 aa

21-169 aa

20-169 aa

31-158 aa

2-146 aa

NCT number	Title	Primary outcome measure	Phase	Date	Oragnization	Status
NCT02165293	A single center study to evaluate the penetration of RO7033877 into the lung in healthy volunteers	Multiple dose PK of RO7033877: C_max, AUC, C_max/AUC	Phase I	2014.6-2014.9	Roche	Completed
NCT02156323	A single-center, open-label, randomized, two sequence, 3-way crossover study to investigate the interaction between multiple doses of colistin methanesulfonate sodium and multiple doses of RO7033877 in healthy subjects	Multiple dose PK of RO7033877 and colistin: C_max, AUC	Phase I	2014.7-2015.3	Roche	Completed
NCT02165332	A two-part, single-dose, randomized study to evaluate the safety of supra-therapeutic doses of RO7033877 and to investigate the effect of RO7033877 on the QTc interval	Part 1: Incidence of ADEs after single supratherapeutic dose of RO7033877; plasma and urine concentrations, AUC Part 2: ECG recordings	Phase I	2014.7-2014.11	Roche	Completed
NCT02110459	An open-label, non-randomized, monocenter, single-dose, phase I study to evaluate PK and safety of POL7080 administered as single intravenous infusion to subjects with renal impairment	Plasma concentrations of POL7080	Phase I	2013.4-2015.7	Polyphor	Completed
NCT02897869	A single-center, open-label, two sequence, crossover study to investigate the interaction between amikacin and POL7080 in healthy subjects	Multiple dose PK of POL7080 and amikacin: plasma C_max, AUC	Phase I	2016.4-2016.12	Polyphor	Completed
NCT02096328	A phase II, open-label, multi-center study to assess PK, safety and efficacy of POL7080 co-administered with standard of care treatment in patients with ventilator-associated pneumonia due to suspected or documented P. aeruginosa infection	Plasma concentrations of POL7080	Phase II	2013.10-2016.12	Polyphor	Completed
NCT02096315	A phase II, open-label, multicenter study to assess the tolerance, safety, efficacy and PK/PD of POL7080 in the treatment of patients with acute exacerbation of non-cystic fibrosis bronchiectasis due to P. aeruginosa infection requiring intravenous treatment	Sputum bacterial clearance	Phase II	2013.12-2015.11	Polyphor	Terminated
NCT03409679	A multicenter, open-label, randomized, active-controlled, parallel group, pivotal study to investigate the efficacy, safety and tolerability, and pharmacokinetics of murepavadin combined with one anti-pseudomonal antibiotic versus two anti-pseudomonal antibiotics in adult subjects with ventilator-associated bacterial pneumonia suspected or confirmed to be due to P. aeruginosa	Clinical cure rate	Phase III	2016.4-2016.12	Polyphor	Terminated
NCT03582007	A multicenter, open label, sponsor blinded, randomized, active controlled, parallel group, pivotal study to evaluate the efficacy, safety, and tolerability of murepavadin given with ertapenem versus an anti-pseudomonal-β-lactam-based antibiotic in adult subjects with nosocomial pneumonia suspected or confirmed to be due to P. aeruginosa	All cause mortality rates	Phase III	2018.10-2019.7	Polyphor	Terminated

NCT number

Title

Primary outcome measure

Phase

Date

Oragnization

Status

NCT02165293

A single center study to evaluate the penetration of RO7033877 into the lung in healthy volunteers

Multiple dose PK of RO7033877: C_max, AUC, C_max/AUC

Phase I

2014.6-2014.9

Roche

Completed

NCT02156323

A single-center, open-label, randomized, two sequence, 3-way crossover study to investigate the interaction between multiple doses of colistin methanesulfonate sodium and multiple doses of RO7033877 in healthy subjects

Multiple dose PK of RO7033877 and colistin: C_max, AUC

Phase I

2014.7-2015.3

Roche

Completed

NCT02165332

A two-part, single-dose, randomized study to evaluate the safety of supra-therapeutic doses of RO7033877 and to investigate the effect of RO7033877 on the QTc interval

Part 1: Incidence of ADEs after single supratherapeutic dose of RO7033877; plasma and urine concentrations, AUC
Part 2: ECG recordings

Phase I

2014.7-2014.11

Roche

Completed

NCT02110459

An open-label, non-randomized, monocenter, single-dose, phase I study to evaluate PK and safety of POL7080 administered as single intravenous infusion to subjects with renal impairment

Plasma concentrations of POL7080

Phase I

2013.4-2015.7

Polyphor

Completed

NCT02897869

A single-center, open-label, two sequence, crossover study to investigate the interaction between amikacin and POL7080 in healthy subjects

Multiple dose PK of POL7080 and amikacin: plasma C_max, AUC

Phase I

2016.4-2016.12

Polyphor

Completed

NCT02096328

A phase II, open-label, multi-center study to assess PK, safety and efficacy of POL7080 co-administered with standard of care treatment in patients with ventilator-associated pneumonia due to suspected or documented P. aeruginosa infection

Plasma concentrations of POL7080

Phase II

2013.10-2016.12

Polyphor

Completed

NCT02096315

A phase II, open-label, multicenter study to assess the tolerance, safety, efficacy and PK/PD of POL7080 in the treatment of patients with acute exacerbation of non-cystic fibrosis bronchiectasis due to P. aeruginosa infection requiring intravenous treatment

Sputum bacterial clearance

Phase II

2013.12-2015.11

Polyphor

Terminated

NCT03409679

A multicenter, open-label, randomized, active-controlled, parallel group, pivotal study to investigate the efficacy, safety and tolerability, and pharmacokinetics of murepavadin combined with one anti-pseudomonal antibiotic versus two anti-pseudomonal antibiotics in adult subjects with ventilator-associated bacterial pneumonia suspected or confirmed to be due to P. aeruginosa

Clinical cure rate

Phase III

2016.4-2016.12

Polyphor

Terminated

NCT03582007

A multicenter, open label, sponsor blinded, randomized, active controlled, parallel group, pivotal study to evaluate the efficacy, safety, and tolerability of murepavadin given with ertapenem versus an anti-pseudomonal-β-lactam-based antibiotic in adult subjects with nosocomial pneumonia suspected or confirmed to be due to P. aeruginosa

All cause mortality rates

Phase III

2018.10-2019.7

Polyphor

Terminated