Construction of a Protein Synthesis System from Pseudomonas aeruginosa for Screening for Antibacterial Candidates

铜绿假单胞菌蛋白质合成系统的构建用于筛选抗菌候选物

• 批准号: 9267492
• 负责人: James M Bullard
• 金额: $ 10.9万
• 依托单位: UNIVERSITY OF TEXAS RIO GRANDE VALLEY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-19 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9267492
• 关键词: Active Sites; Acylation; Adverse effects; Aminoacylation; Anti-Bacterial Agents; Antibiotics; Area; Bacteria; Bacterial Drug Resistance; Bacterial Infections; Benign; Binding; Biochemical; Biological Assay; Cell Culture Techniques; Chemicals; Chronic; Codon Nucleotides; Collaborations; Crystallization; Cystic Fibrosis; Cytochrome P450; Data; Development; Drug resistance; Enzyme Inhibition; Evolution; Funding; Genes; Glycine decarboxylase; Goals; Grant; Growth; Human; Human Cell Line; Hypersensitivity; In Vitro; Infection; Lead; Libraries; Lung; Medical; Messenger RNA; Metabolism; Methionyl-tRNA formyltransferase; Methods; Microbial Biofilms; Modeling; Molecular Models; Molecular Target; Mutation; Natural Products; Pathway interactions; Patient-Focused Outcomes; Peptide Elongation Factor G; Peptide Initiation Factors; Peptides; Pharmaceutical Preparations; Poly U; Process; Production; Protein Biosynthesis; Protein Engineering; Proteins; Pseudomonas aeruginosa; Public Health; Recruitment Activity; Research; Resistance; Resistance development; Respiratory Tract Infections; Ribosomes; Series; Solubility; Specificity; Structure; Structure-Activity Relationship; Symbiosis; System; Testing; Time; Toxic effect; Toxicity Tests; Toxicology; Translations; Vendor; Work; bacterial resistance; base; cystic fibrosis patients; design; drug candidate; experience; high throughput screening; improved; inhibitor/antagonist; lead series; molecular modeling; mortality; mutant; mutation screening; novel; pathogen; pathogenic bacteria; pressure; public health relevance; resistance mechanism; screening; translation assay

 DESCRIPTION (provided by applicant): Protein synthesis is an essential metabolic process in all bacteria and a target for the development of new antibiotics. In our initially funded SCORE grant we developed an amino acylation/translation (A/T) system from P. aeruginosa based on polyU mRNA directed protein synthesis. This system was optimized and developed into a platform to perform high throughput screening of chemical compounds against the activity of the system. During this period a number of inhibitory compounds (six) were identified and characterized and developed into lead compounds. A small (5-6) congener group was built around six of the lead compounds. The goal of the current proposal is to develop a lead series around each of these lead compounds as well as to continue discovery of additional compounds that inhibit growth of pathogenic bacteria. To develop a lead series we will carry out structure activity relationship (SAR) studies to increase the inhibitory potency, solubility, ADME and abilit to inhibit biofilm formation. At the same time we will attempt to decrease the potential for drug-drug induced enzyme inhibition and toxicity to human cell lines. In the continued discovery process, we will actively recruit collaborators doing work in isolation of natural compounds to test against our system for identification of new inhibitory compounds as well as obtain small focused synthetic compound libraries. The IC50 as well as the MIC against a panel of pathogenic bacteria will initially be determined for each new inhibitor discovered. These data will determine if new hit compounds enter SAR studies as described above. Next, we propose to expand the A/T minimal protein synthesis system into a more natural like protein synthesis system. During the initial grant period, we were able to accomplish much more than was proposed. We have cloned, expressed, and isolated 11 additional proteins involved in protein synthesis: seven additional aaRS proteins, three translation initiation factors (IF-1, IF-2, and IF 3) and the methionyl-tRNA formyltransferase. Incorporation of these proteins into the A/T assay, along with a designer natural like mRNA, allowing translation of a short peptide will allow us to screen for inhibitors of the ribosome and 15 accessory proteins in one assay. We have functional assays developed for each component of the system and will be able to quickly determine the molecular target of an inhibitor. This system will be developed and optimized based on the experience gained in the initial funding period. Screening of compound libraries will continue to be carried out in a high throughput format using scintillation proximity assays (SPA). Four of the lead compounds inhibit protein synthesis by inhibiting the activity of PheRS. To determine mechanism of action of these inhibitory compounds we will evaluate promising lead compounds for their ability to generate spontaneous mutants or develop resistant after serial sub-culturing of hypersensitive strains of P. aeruginosa. We will screen for mutations in the gene encoding PheRS. The structure for P. aeruginosa PheRS has been solved and we will collaborate with Dr. Kotsikorou at UTPA and use the crystal structure and molecular modeling methods to explain the effects of the mutations on resistance to the antibacterial compounds. We will also model the binding of the compounds in the active site of PheRS to better understand the mechanism of inhibitory activity.

 描述（由申请人提供）：蛋白质合成是所有细菌的重要代谢过程，也是开发新抗生素的目标。在我们最初资助的 SCORE 资助中，我们开发了铜绿假单胞菌的氨酰化/翻译 (A/T) 系统。基于 PolyU mRNA 指导的蛋白质合成，该系统被优化并开发成一个平台，用于针对该系统的活性对化合物进行高通量筛选。在此期间，鉴定、表征和开发了多种抑制化合物（六种）。围绕六种先导化合物建立了一个小型（5-6）同系物组，当前提案的目标是围绕每种先导化合物开发一个先导系列，并继续发现其他化合物。为了开发先导系列，我们将进行结构活性关系 (SAR) 研究，以提高抑制效力、溶解度、ADME 和抑制生物膜形成的能力。药物-药物诱导酶在持续的发现过程中，我们将积极招募从事天然化合物分离工作的合作者，以测试我们的系统以鉴定新的抑制化合物，并获得小型的集中合成化合物库。以及针对一组致病菌的 MIC，将首先确定所发现的每种新抑制剂的 MIC。 确定新的热门化合物是否进入 SAR 研究，如上所述，我们建议将 A/T 最小蛋白质合成系统扩展为更自然的蛋白质合成系统。我们已经克隆、表达和分离了 11 种参与蛋白质合成的其他蛋白质：另外 7 种 aaRS 蛋白质、三种翻译起始因子（IF-1、IF-2 和 IF）。 3) 和甲硫氨酰-tRNA 甲酰基转移酶 将这些蛋白质与设计的天然类似 mRNA 结合在一起，允许翻译短肽，这将使我们能够筛选核糖体抑制剂和 15 种辅助蛋白质。我们为该系统的每个组件开发了功能分析，并将能够根据初始资助期间获得的经验来快速确定抑制剂的分子靶标。将继续使用闪烁邻近分析 (SPA) 以高通量形式进行。四种先导化合物通过抑制 PheRS 的活性来抑制蛋白质合成。为了确定这些抑制化合物的作用机制，我们将评估有前景的先导化合物。铜绿假单胞菌过敏菌株连续传代培养后产生自发突变体或产生抗性的能力 我们将筛选编码 PheRS 的基因中的突变。 铜绿假单胞菌 PheRS 的结构具有。我们将与 UTPA 的 Kotsikorou 博士合作，利用晶体结构和分子建模方法来解释突变对抗菌化合物耐药性的影响，我们还将对 PheRS 活性位点中化合物的结合进行建模。更好地了解抑制活性的机制。