ИСТИНА |
Войти в систему Регистрация |
|
ИПМех РАН |
||
The coronavirus crisis in 2020 demonstrated that humanity is still susceptible to infections. Nevertheless, in the last decade, more and more bacteria with outstanding resistance have been discovered. As a result, It caused a gradual decrease in the efficiency of the known antibiotics. Therefore, searching for novel compounds with antibiotic activity is extremely important, as well as an understanding of its mechanism of action. The method for cost-effective and convenient for high throughput screening for antibiotics based on the expression of two fluorescent proteins was designed in our laboratory. Using this method novel antibiotic was discovered. This compound was purified by the HPLC method. The structure was determined by the tandem of HRMS and NMR methods. It has the empirical formula C23H25NO4, and according to the solved structure, it is a novel molecule. This compound - Auroplanin, was extracted from a cultural broth of Actinoplanes sp. VKM Ac-2862. Auroplanin induces a translational part of the reporter system, so we supposed it could be a translational inhibitor. This hypothesis was supported by in vitro translation inhibition in E.coli S30 extract and by inhibition of vivo incorporation of C14-valine in proteins on JW5503 E.coli cells. Furthermore, the pattern of translation arrest on the primer extension inhibition assay revealed that Auroplanin acts as an inhibitor of translation elongation. Cryo-EM study of Auroplanin complex with E.coli ribosome shows that it binds close to 560 loop of 30S ribosomal subunit. Resistant clones for this compound had substitutions of C564G, G558U, and G566A nucleotides in 16S rRNA, relative to the binding site according to the determined structure. All mutations were not previously described in the literature. Thus, Auroplanin has a novel binding site, and study its mechanism of action can clarify some details in ribosome work. The reported study was funded by RFBR according to the research project № 20-34-90048