ИСТИНА

During protein biosynthesis, the growing polypeptide chain, passing through the ribosomal tunnel, can interact with the nucleotides forming its walls, thereby regulating the translation process. The study of such phenomena at the molecular level is complicated by the high mobility of the system. However, the use of low-molecular probes, which are conjugates of peptides and strongly binding antibiotics, makes it possible to study specific interactions between the amino acid residues of the peptide chain and the walls of the ribosomal tunnel [1]. In the case of the chloramphenicol (CHL), replacement of the dichloroacetyl residue with amino acid or peptide allows it to be oriented towards the ribosomal tunnel, similar to the growing polypeptide chain, while the unchanged amphenicol moiety (CAM) will anchor the entire molecule in the A site of the peptidyl transferase center [2, 3, 4]. In view of the growing bacterial resistance, such antibiotic analogs, capable of interacting with various functional sites of the ribosome, may be of interest as potential antibacterial drugs. The goal of this work was to design and synthesize peptide analogs of chloramphenicol in order to study interactions of amino acid residues with the ribosomal tunnel. Molecular docking of the previously synthesized amino acid chloramphenicol analogs into E. coli ribosome performed by means of the AutoDock Vina software [4] showed a strong correlation between calculated and experimentally determined ligand efficiencies. To find the optimal amino acid sequences of the peptides, virtual screening of all possible N-acetyl tripeptide analogs of chloramphenicol (8000 Ac-XXX-CAMs) for their binding to the ribosome was carried out using molecular docking. Ac-FWH-CAM, Ac-VFR-CAM, and Ac-RAW-CAM were selected for further synthesis. Ac-AAA-CAM was chosen as a control compound. Peptide analogs with free α-amino groups H-FWH-CAM, H-VFR-CAM, H-RAW-CAM, and H-AAA-CAM were also synthesized. The ability of the synthesized compounds to bind to bacterial ribosomes was studied by displacement of the fluorescent analog of erythromycin (BODIPY-erythromycin) from its complex with E. coli ribosomes. KDs of the complexes were measured (Table). RAW-CAM was found to bind to the ribosome 30 times better than the original antibiotic. Experimentally measured ligand efficiency values are consistent with molecular docking results. Table. Dissociation constants of complexes of synthesized peptide analogs of chloramphenicol with ribosomes (± 95% confidence interval). Compound KD, μM X : H- Ac- X-AAA-CAM 10 ± 1 71 ± 17 X-FWH-CAM 0.13 ± 0.02 0.53 ± 0.07 X-VFR-CAM 0.45 ± 0.07 2.8 ± 0.4 X-RAW-CAM 0.09 ± 0.02 1.0 ± 0.2 CHL 2.8 ± 0.5 Chemical probing of 23S rRNA in the presence of synthesized compounds revealed protection of nucleotides A2058 and A2059, which indicates the interaction of peptide analogs of chloramphenicol with the ribosomal tunnel. Structures of H-FWH-CAM, H-VFR-CAM, H-RAW-CAM bound to E. coli ribosome obtained by molecular docking represent the possible interactions between the elements of the ribosomal tunnel and the peptide moieties and provide a model for interpretation of the results of chemical probing. The ability of some compounds to inhibit protein biosynthesis in vitro was comparable to that of chloramphenicol, making the synthesized analogues of chloramphenicol promising for further antibacterial activity studies. This work was supported by the grants RFBR (16-04-00709) and RSF (14-24-00061-P).