Validation and development of trans-translation as a drug target for MRSA

描述(申请人提供)：金黄色葡萄球菌菌株对现有抗生素具有抗药性的持续传播给治疗金黄色葡萄球菌感染带来了严重的问题。开发有效对抗这些致命的MRSA菌株的药物的关键障碍之一是缺乏新的抗生素靶点。解决停滞核糖体的反式翻译途径是药物开发的潜在靶点，因为它存在于所有细菌中，是MRSA在培养中生长所必需的。该项目的长期目标是确定金黄色葡萄球菌中可用作药物开发先导化合物的特定反式翻译抑制物。这项建议的总体目标是验证反式翻译作为金黄色葡萄球菌可行的抗生素靶标，并开发一种与高通量筛选(HTS)和二次检测相兼容的一次检测方法，可用于鉴定金黄色葡萄球菌反式翻译的抑制剂。这项拟议研究的基本原理是，将MRSA中的反式翻译作为药物靶点的验证以及HTS检测方法的开发将导致快速识别新的抗生素，这将极大地改善MRSA的治疗。这项建议的具体目的是确认tmRNA和SmpB对金黄色葡萄球菌在培养和感染期间的生长是必不可少的，开发一次和二次筛选试验来鉴定金黄色葡萄球菌的反式翻译抑制物，进行tmRNA-SmpB抑制物的初步筛选，并使用已建立的体外和体内试验来表征筛选结果。枯竭菌株和小分子抑制剂将被用来在生长、培养和感染期间去除反式翻译活性。这些实验的结果将为理解金黄色葡萄球菌中反式翻译的重要性以及确定这一途径是否可以用于药物开发提供基础。已建立的HTS检测和tmRNA-SmpB活性检测将适用于金黄色葡萄球菌成分。这些研究的结果将使未来的HTS和候选药物的高级优化成为可能。通过瞄准一种尚未用于抗生素开发的途径，该项目将产生新的化合物，这些化合物可以单独使用，也可以与现有的MRSA疗法结合使用。

DESCRIPTION (provided by applicant): The continued spread of Staphylococcus aureus strains that are resistant to available antibiotics has created a severe problem for treating S. aureus infections. One of the critical impediments to developing drugs that are effective against these deadly MRSA strains is the lack of new antibiotic targets. The trans-translation pathway for resolving stalled ribosomes is a potential target for drug development because it is found in all bacteria and is required for growth of MRSA in culture. The long-term goal of this project is t identify specific inhibitors of trans-translation in S. aureus that can be used as lead compounds for drug development. The overall objective of this proposal is to validate trans-translation as a viable antibiotic target in S. aureus and to develop a primary assay compatible with high-throughput screening (HTS) and secondary assays that can be used to identify inhibitors of trans-translation in S. aureus. The rationale that underlies the proposed research is that validation of trans-translation in MRSA as a drug target and development of assays for HTS will lead to rapid identification of new antibiotics that will dramatically improve treatment for MRSA. The specific aims of this proposal are to confirm that tmRNA and SmpB are essential for S. aureus growth in culture and during infection, to develop primary and secondary screening assays to identify inhibitors of trans-translation in S. aureus, to conduct a pilot screen for tmRNA-SmpB inhibitors, and to use established ex vivo and in vivo assays to characterize hits from screening. Depletion strains and small molecule inhibitors will be used to remove trans-translation activity in during growth in culture and during infection. The results of these experiments will provide the basis for understanding the importance of trans-translation in S. aureus as well as determining if this pathway can be targeted for drug development. An established HTS assay and tmRNA-SmpB activity assays will be adapted for use with S. aureus components. Results from these studies will allow future HTS and high-level optimization of drug candidates. By targeting a pathway that has not been used for antibiotic development, this project will yield new compounds that can be used individually or in combination with existing MRSA therapies.