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（通过应用程序提供）：蛋白质合成是所有细菌中必不可少的代谢过程，也是开发新抗生素的靶标。在我们最初的资助得分赠款中，我们根据Polyu mRNA定向蛋白质合成开发了铜绿假单胞菌的氨基化/翻译（A/T）系统。该系统被优化并发展为一个平台，以对系统活性进行高吞吐量筛选。在此期间，许多抑制性化合物（六）被鉴定出来并表征并发展为铅化合物。一个小的（5-6）同类小组建造了六种铅大约二种铅大约。当前建议的目的是围绕每种铅化合物开发铅系列，并继续发现抑制致病细菌生长的其他化合物。为了开发铅系列，我们将进行结构活动关系（SAR）研究，以提高抑制性，溶解度，ADME和抑制生物膜形成的能力。同时，我们将尝试减少药物诱导酶抑制和对人细胞系的毒性的潜力。在持续的发现过程中，我们将积极招募合作者进行隔离自然化合物的工作，以测试我们的系统，以识别新的抑制性化合物，并获得小型集中的合成化合物库。最初，针对发现的每个新抑制剂，将确定IC50和针对致病细菌小组的MIC。这些数据将 确定新命中化合物是否如上所述输入SAR研究。接下来，我们建议将A/T最小蛋白质合成系统扩展到更自然的蛋白质合成系统。在最初的赠款期间，我们能够完成比提出的要多得多。我们已经克隆，表达和隔离了11种参与蛋白质合成的其他蛋白质：另外7种AARS蛋白，三个翻译起始因子（IF-1，IF-2以及IF） 3）和甲基-TRNA甲基转移酶。将这些蛋白质掺入A/T分析中，以及像MRNA这样的设计器的天然，使短肽的翻译将使我们能够在一个测定中筛选核糖体抑制剂和15种辅助蛋白的抑制剂。我们为系统的每个组件开发了功能测定，并且能够快速确定抑制剂的分子靶标。该系统将根据初始资金期间获得的经验开发和优化。复合库的筛选将继续使用闪烁接近测定（SPA）以高通量格式进行。四种铅化合物通过抑制phers的活性来抑制蛋白质合成。为了确定这些抑制性化合物的作用机理，我们将评估有希望的铅化合物，以产生赞助者自发突变体或在铜绿假单胞菌过敏性菌株的序列亚培养后产生抗性。我们将筛选在编码词体基因中的突变。已解决了铜绿假单胞菌的结构，我们将与UTPA的Kotsikorou博士合作，并使用晶体结构和分子建模方法来解释突变对抗菌化合物抗性的影响。我们还将建模化合物在pHOR的活性位点的结合，以更好地了解抑制活性的机制。