Novel antimicrobials targeting type IV pilus and type 2 secretion systems

针对 IV 型菌毛和 2 型分泌系统的新型抗菌药物

基本信息

批准号：
9386954
负责人：
MICHAEL S DONNENBERG
金额：
$ 16.44万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9386954
关键词：
Acinetobacter Acinetobacter baumannii Active Sites Adherence Animal Model Antibiotic Resistance Antibiotics Antimicrobial Resistance Aspartic Acid Bacteria Bacterial Infections Binding Biogenesis Biological Assay Characteristics Chemicals Chimeric Proteins Complex Disease Drug Targeting DsRed Energy Transfer Enzymes Equipment Escherichia coli Fimbriae Proteins Fluorescence Fluorescence Anisotropy Fluorescence Polarization Formulation Fostering Goals Growth Health Human Immunoblotting In Vitro Infection Lead Libraries Maryland Medicine Membrane Modeling Molecular Machines Molecular Models Mutation N-terminal Neisseria gonorrhoeae Organism Pathogenesis Peptide Hydrolases Peptide Signal Sequences Performance Pharmaceutical Chemistry Phase Physicians Pilum Pneumonia Preclinical Drug Evaluation Preclinical Testing Prevention Probability Property Protease Inhibitor Proteins Pseudomonas aeruginosa Pseudomonas aeruginosa pneumonia Quality Control Recombinant Proteins Resistance Resources Series Specificity Structure Surface System Testing Therapeutic Toxin Universities Validation Virulence analog antimicrobial antimicrobial drug appendage drug development drug discovery efficacy testing enteropathogenic Escherichia coli experience improved in vitro activity in vivo inhibitor/antagonist innovation killings molecular modeling mouse model mutant novel novel therapeutics pathogen prevent protein S precursor screening small molecule success therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Bacterial pathogens have conserved complex molecular machines that are required to cause disease. Among these machines are those that make up a type IV pilus (T4P) biogenesis apparatus and the related type II secretion (T2S) machine. T4Ps are surface appendages essential for adherence and host colonization by many important bacterial pathogens; T2S systems export toxins that are critical for many diseases. Acinetobacter baumanii, Pseudomonas aeruginosa, and Neisseria gonorrhoeae all produce T4Ps. Among the components common to both machines is a pre-pilin peptidase enzyme that specifically removes the characteristic N-terminal signal sequence from pre-pilin proteins and N-methylates the nascent amino terminus. These enzymes are highly conserved, absolutely required for T4P and T2S function, and represent a novel class of neutral pH aspartic acid peptidases that do not resemble mammalian enzymes. As such, they are ideal targets for new drug discovery. We have devised an in vivo Föster resonance energy transfer (FRET) system comprised of three recombinant proteins expressed in E. coli: the pre-pilin peptidase, the pre-pilin protein fused to enhanced yellow fluorescent protein (eYFP), and an essential assembly protein to which the pre-pilin binds fused to dsRed. Cleavage of the pre-pilin fusion protein by the pre-pilin peptidase releases eYFP and prevents FRET with dsRed. If the pre-pilin peptidase is inactivated by mutation, then FRET occurs. We will adapt this extremely sensitive system and a related even simpler system that relies on fluorescence anisotropy to develop in vitro high-throughput small molecule screens to identify pre-pilin peptidase inhibitor candidates. Secondary assays will establish specificity and spectrum of activity. The initial effort will provie candidates that will subsequently subjected to further chemical refinement to enhance potency, specificity and activity against whole bacteria. Finally, (a) lead compound(s) will be advanced to preclinical testing using an established murine model of P. aeruginosa pneumonia in which T4Ps are required for disease. Ultimately, the goal is to discover an entirely novel therapeutic class of anti-virulence agents to improve human health.

描述（由申请人提供）：细菌病原体具有引起疾病所需的复杂分子机器，其中包括构成 IV 型菌毛（T4P）生物发生装置和相关的 II 型分泌（T4P）机器的分子机器。是许多重要细菌病原体粘附和宿主定殖所必需的表面附属物；T2S系统输出对许多疾病至关重要的毒素。铜绿假单胞菌和淋病奈瑟菌均产生 T4P，这两种机器的共同成分是前菌毛蛋白肽酶，该酶可特异性去除前菌毛蛋白的特征性 N 末端信号序列，并对新生的氨基末端进行 N 甲基化。高度保守，是 T4P 和 T2S 功能所必需的，代表了一类新型的中性 pH 值天冬氨酸肽酶，不因此，它们是新药发现的理想靶点，我们设计了一种体内 Föster 共振能量转移 (FRET) 系统，由大肠杆菌中表达的三种重组蛋白组成：前菌毛蛋白肽酶、前菌毛蛋白肽酶。与增强型黄色荧光蛋白 (eYFP) 融合的菌毛蛋白，以及与 dsRed 融合的前菌毛蛋白结合的必需组装蛋白。前菌毛蛋白肽酶释放 eYFP 并阻止 dsRed 的 FRET 如果前菌毛蛋白肽酶因突变而失活，则我们将采用这种极其敏感的系统和依赖于荧光各向异性的相关更简单的系统来开发体外高荧光。 -用于鉴定前菌毛蛋白肽酶抑制剂候选物的通量小分子筛选将确定特异性和活性谱，初步努力将提供随后进行进一步研究的候选物。最后，（a）先导化合物将使用已建立的铜绿假单胞菌肺炎小鼠模型进行临床前测试，其中 T4P 是疾病所必需的。目标是发现一种全新的治疗类抗毒剂来改善人类健康。