Interaction of Chloramphenicol Cationic Peptide Analogues with the Ribosome.

Virtual screening of all possible tripeptide analogues of chloramphenicol was performed using molecular docking to evaluate their affinity to bacterial ribosomes. Chloramphenicol analogues that demonstrated the lowest calculated energy of interaction with ribosomes were synthesized. Chloramphenicol amine (CAM) derivatives, which contained specific peptide fragments from the proline-rich antimicrobial peptides were produced. It was demonstrated using displacement of the fluorescent erythromycin analogue from its complex with ribosomes that the novel peptide analogues of chloramphenicol were able to bind bacterial ribosome; all the designed tripeptide analogues and one of the chloramphenicol amine derivatives containing fragment of the proline-rich antimicrobial peptides exhibited significantly greater affinity to Escherichia coli ribosome than chloramphenicol. Correlation between the calculated and experimentally evaluated levels of the ligand efficiencies was observed. In vitro protein biosynthesis inhibition assay revealed, that the RAW-CAM analogue shows activity at the level of chloramphenicol. These data were confirmed by the chemical probing assay, according to which binding pattern of this analogue in the nascent peptide exit tunnel was similar to chloramphenicol.

New Fluorescent Macrolide Derivatives for Studying Interactions of Antibiotics and Their Analogs with the Ribosomal Exit Tunnel.

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.

Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes.

Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory "stop peptides" - MRL, IRA, IWP - were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin analog from its complex with E. coli ribosomes. It was found that peptide chloramphenicol analogs are able to bind to bacterial ribosomes. The dissociation constants were 4.3-10 µM, which is 100-fold lower than the corresponding values for chloramphenicol amine-ribosome complex. Interaction of the chloramphenicol peptide analogs with ribosomes was simulated by molecular docking, and the most probable contacts of "stop peptide" motifs with the elements of nascent peptide exit tunnel were identified.

Modeling Interactions of Erythromycin Derivatives with Ribosomes.

Using a method of static simulation, a series of erythromycin A analogs was designed with aldehyde functions introduced instead of one of the methyl substituents in the 3'-N-position of the antibiotic that was potentially capable of forming a covalent bond with an amino group of one of the nucleotide residues of the 23S rRNA in the ribosomal exit tunnel. Similar interaction is observed for antibiotics of the tylosin series, which bind tightly to the large ribosomal subunit and demonstrate high antibacterial activity. Binding of novel erythromycin derivatives with the bacterial ribosome was investigated with the method of fluorescence polarization. It was found that the erythromycin analog containing a 1-methyl-3-oxopropyl group in the 3'-N-position demonstrates the best binding. Based on the ability to inhibit protein biosynthesis, it is on the same level as erythromycin, and it is significantly better than desmethyl-erythromycin. Molecular dynamic modeling of complexes of the derivatives with ribosomes was conducted to explain the observed effects.