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基本代谢过程，也是开发新抗生素的目标。在我们最初资助的SCORE基金中，我们基于polyU mRNA指导的蛋白质合成开发了来自铜绿假单胞菌的氨基酰化/翻译（A/T）系统。该系统被优化并开发成一个平台，以针对系统的活性进行化合物的高通量筛选。在此期间，一些抑制性化合物（6）被确定和表征，并开发成先导化合物。围绕六种先导化合物建立了一个小的（5-6个）同系物组。目前提案的目标是围绕这些先导化合物中的每一种开发一个先导系列，并继续发现抑制致病菌生长的其他化合物。为了开发先导系列，我们将进行结构活性关系（SAR）研究，以增加抑制效力、溶解度、ADME和抑制生物膜形成的能力。同时，我们将尝试降低药物-药物诱导的酶抑制和对人细胞系的毒性的可能性。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，对我们的系统进行测试，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。最初将确定每种新发现的抑制剂对一组病原菌的IC 50和MIC。这些数据将 确定新的命中化合物是否进入如上所述的SAR研究。 接下来，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。在最初的赠款期间，我们能够完成的比提议的要多得多。我们已经克隆、表达并分离了11个参与蛋白质合成的额外蛋白质：7个额外的阿尔斯蛋白质、3个翻译起始因子（IF-1、IF-2和IF 3)和甲硫氨酰-tRNA甲酰基转移酶。将这些蛋白质掺入A/T测定中，沿着设计的天然样mRNA，允许短肽的翻译，将允许我们在一个测定中筛选核糖体和15种辅助蛋白的抑制剂。我们已经为系统的每个组件开发了功能检测，并将能够快速确定抑制剂的分子靶点。将根据最初供资期间取得的经验开发和优化这一系统。化合物文库的筛选将继续使用闪烁邻近测定（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。为了确定这些抑制性化合物的作用机制，我们将评估有希望的先导化合物在铜绿假单胞菌的超敏感菌株的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的结构已经解决，我们将与UTPA的Kotsikorou博士合作，使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。我们还将模拟化合物在PheRS活性位点的结合，以更好地了解抑制活性的机制。