In the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL—CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome—PTC and peptide exit tunnel.

In order to obtain antimicrobial compounds with improved properties, new conjugates comprising two different biologically active agents within a single chimeric molecule based on chloramphenicol (CHL) and a hydrophobic cation were synthesized and studied. Chloramphenicol amine (CAM), derived from the ribosome-targeting antibiotic CHL, and the plant isoquinoline alkaloid berberine (BER) are connected by alkyl linkers of different lengths in structures of these conjugates. Using competition binding, double reporter system, and toeprinting assays, we showed that synthesized CAM-Cn-BER compounds bound to the bacterial ribosome and inhibited protein synthesis like the parent CHL. The mechanism of action of CAM-C5-BER and CAM-C8-BER on the process of bacterial translations was similar to CHL. Experiments with bacteria demonstrated that CAM-Cn-BERs suppressed the growth of laboratory strains of CHL and macrolides-resistant bacteria. CAM-C8-BER acted against mycobacteria and more selectively inhibited the growth of Gram-positive bacteria than the parent CHL and the berberine derivative lacking the CAM moiety (CH₃-C8-BER). Using a potential-sensitive fluorescent probe, we found that CAM-C8-BER significantly reduced the membrane potential in B. subtilis cells. Crystal violet assays were used to demonstrate the absence of induction of biofilm formation under the action of CAM-C8-BER on E. coli bacteria. Thus, we showed that CAM-C8-BER could act both on the ribosome and on the cell membrane of bacteria, with the alkylated berberine fragment of the compound making a significant contribution to the inhibitory effect on bacterial growth. Moreover, we showed that CAM-Cn-BERs did not inhibit eukaryotic translation in vitro and were non-toxic for eukaryotic cells.

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.