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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

基本信息

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
项目类别：
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

项目摘要

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.