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

 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。在我们最初资助的评分拨款中，我们开发了一个基于PolyU mRNA指导的蛋白质合成的铜绿假单胞菌氨基酰化/翻译(A/T)系统。该系统被优化并开发成一个针对系统活性进行高通量化合物筛选的平台。在此期间，一些抑制化合物(6个)被鉴定和表征，并发展成为先导化合物。围绕六种先导化合物建立了一个很小的(5-6)同系基。目前提议的目标是围绕每一种先导化合物开发一系列铅，并继续发现更多抑制病原菌生长的化合物。为了开发一系列的铅，我们将进行结构活性关系(SAR)研究，以增加抑制效力，溶解性，ADME和能力，以抑制生物被膜的形成。同时，我们将努力降低药物引起的酶抑制和对人类细胞株的毒性。在持续的发现过程中，我们将积极招募在分离天然化合物方面进行工作的合作者，以针对我们的系统进行测试，以识别新的抑制性化合物，并获得小型专注的合成化合物文库。对于发现的每一种新的抑制剂，最初将确定其对一组致病菌的IC50以及MIC。这些数据将 确定是否有新的Hit化合物进入如上所述的SAR研究。下一步，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，我们能够完成的工作比计划的多得多。我们已经克隆、表达和分离了另外11个与蛋白质合成有关的蛋白质：7个额外的AARS蛋白、3个翻译起始因子(IF-1、IF-2和IF 3)和甲硫酰-tRNA甲酰转移酶。将这些蛋白质结合到A/T分析中，再加上一种设计的类似天然的mRNA，允许翻译短肽，将使我们能够在一次检测中筛选出核糖体和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分析，并将能够快速确定抑制剂的分子靶标。这一系统将根据最初筹资期间取得的经验进行开发和优化。化合物文库的筛选将继续以高通量格式进行，使用闪烁邻近分析(SPA)。其中四种先导化合物通过抑制phers的活性来抑制蛋白质的合成。为了确定这些抑制化合物的作用机制，我们将评估有希望的先导化合物在铜绿假单胞菌超敏菌株连续传代后产生自发突变或产生抗药性的能力。我们将筛查phers编码基因的突变。铜绿假单胞菌的结构已经解决，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活性部位的结合，以更好地了解抑制活性的机制。

 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.