Molecular mechanisms of action of macrolide antibiotics

描述(申请人提供)：大环内酯类抗生素通过干扰蛋白质合成来抑制细胞生长。根据广泛接受的观点，这些药物结合在新生多肽出口隧道中，并在翻译的前几轮抑制所有细胞蛋白质的合成。与这种传统的大环内酯作用模型相反，我们的初步研究表明，用大环内酯治疗革兰氏阳性和革兰氏阴性细菌允许继续翻译确定的蛋白质子集。此外，蛋白质逃避抑制的能力由其N-末端序列决定，该序列可以绕过出口隧道中的抗生素，而不会使药物从其结合部位移位。在最初的转流后，一些蛋白质的翻译继续进行，直到它们完成，而一些多肽的合成在后来的阶段可能被阻止。这两种作用都取决于抗生素的结构。尽管有功能性的 这些现象的医学意义，蛋白质逃避抑制能力的分子机制，以及药物诱导的翻译抑制的要求是未知的，将在这个项目中解决。全细胞蛋白质组学将被用来全面表征在抗生素存在的情况下其翻译继续的蛋白质。这项高度创新的核糖体图谱技术将提供全基因组范围内药物依赖的“晚期”翻译停滞部位的信息。在整个细胞研究之后，将对在无细胞翻译系统中进行的旁路和停滞的分子机制进行生化表征。最后，将分析核糖体出口通道中结合的抗生素的结构与经抗生素处理的细胞中合成的蛋白质谱之间的相关性，并将检查耐药组分变化的生理后果。这些预期的发现将大大扩展对临床上重要的大环内酯类抗菌药的一般作用模式的理解，并为开发具有优异抗生素性能的蛋白质合成抑制剂开辟新的场所。

DESCRIPTION (provided by applicant): Macrolide antibiotics inhibit cell growth by interfering with protein synthesis. These drugs bind in the nascent peptide exit tunnel and, according to the widely accepted view, inhibit synthesis of all cellular proteins at the early rounds of translation In contrast to this conventional model of macrolide action, our preliminary studies showed that treatment of Gram-positive and Gram-negative bacteria with macrolides allows for continued translation of a defined subset of proteins. Further, the ability of the protein to evade inhibitio is determined by its N-terminal sequence which can bypass the antibiotic in the exit tunnel without displacing the drug from its binding site. After the initial bypass, translation of some proteins cn continue until their completion, whereas synthesis of some polypeptides can be arrested at later stages. Both of these effects depend on the structure of the antibiotic. In spite of the functional and medical significance of these phenomena, the molecular mechanisms underlying the ability of the protein to evade inhibition and the requirements for the drug-induced translation arrest are unknown and will be addressed in this project. Whole-cell proteomics will be used to comprehensively characterize proteins whose translation continues in the presence of the antibiotic. The highly-innovative technique of ribosome profiling will provide genome-wide information of the sites of drug-dependent 'late' translation arrest. The whole cell-studies will b followed by biochemical characterization of molecular mechanisms of bypass and arrest carried out in a cell-free translation system. Finally, the correlation between the structure of the antibiotic bound in the ribosomal exit tunnel and the spectrum of proteins synthesized in antibiotic-treated cells will be analyzed and physiological consequences of the variation in the composition of the resistome will be examined. The anticipated findings should significantly expand the understanding of the general mode of action of clinically-important macrolide antibacterials and open new venues for development of protein synthesis inhibitors with superior antibiotic properties.