Interaction of chloramphenicol tripeptide analogs with ribosomes

Abstract

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.

Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome

“…Consistent with previously published results, removal of the dichloroacetyl moiety from CLM results in nearly complete loss of its biological activity (Figure 2B, CLM vs. CAM) [23], likely due to the loss of interactions of the dichloroacetic moiety with the ribosome [6]. However, similar to the previous studies employing peptide derivatives of CLM [5, 20, 31], the inhibitory activity of CAM can be partially restored by incorporation of specific amino acids (Figure 2B), indicating that the contacts of the aminoacyl side chain of AA-CAMs with the ribosome may compensate for the interactions lost upon the removal of the dichloroacetic group. However, most of the tested AA-CAM-derivatives were less potent inhibitors of translation than CLM (Figure 2B).…”

“…Although the overall inhibitory activity of AA-CAM derivatives improved in the PURE system compared to their effect upon translation in the cell extract, it still did not reach the level of inhibition elicited by the CLM, suggesting that stability of our compounds is unlikely to be the key issue and indicating that the equilibrium binding of the inhibitors to a vacant ribosome often does not directly correlate with their inhibitory properties. Several peptide derivatives of CAM have been prepared and tested previously in the binding and inhibition assays [20, 31]. Similar to our findings, those derivatives were found to be poor inhibitors of translation.…”

Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents

“…All new semi-synthetic CAM-Cn-TPP compounds were expected to bind and act on bacterial ribosomes by inhibiting protein synthesis, which is similar to CAM-C4-TPP and the PTC-targeting parent antibiotic CHL. To assess the affinity of CAM-C10-TPP and CAM-C14-TPP for the bacterial 70S ribosome ( Figure 2 A), a competition-binding assay exploiting BODIPY-labeled erythromycin (BODIPY-ERY) was used [ 47 , 52 , 53 ]. CAM-C10-TPP was found to have significantly greater (~7-fold) affinity to the ribosome, compared to the parent CHL, and a slightly greater affinity (~1.5-fold), compared to CAM-C4-TPP (K Dapp = 0.4 ± 0.04 µM for CAM-C10-TPP vs. 2.8 ± 0.4 µM for CHL; and K Dapp = 0.61 ± 0.07 µM for CAM-C4-TPP [ 51 ]).…”

“…The scheme for the synthesis of chloramphenicol triphenyphosphonium analogs (CAM-C10-TPP and CAM-C14-TPP) is represented in Figure 1 . (1R,2R)-2-amino-1-(4-nitrophenyl)propane-1,3-diol (chloramphenicol amine, CAM, 2) was obtained via the acid hydrolysis of chloramphenicol (CHL, 1), according to a procedure [ 55 ] described in [ 47 , 53 ]. (10-carboxydecyl)(triphenyl)phosphonium bromide (5) was obtained by the condensation of 11-bromoundecanoic acid ( 3 ) and triphenylphosphin (4) for 12 h at 85 °C.…”

“…The binding affinity of CAM-Cn-TPP to E. coli ribosomes was analyzed by a competitionbinding assay using the fluorescently-labeled BODIPY-ERY, as described before [47,52,53,69]. BODIPY-Ery (16 nM) was incubated with the ribosomes (50 nM) in the buffer containing 20 mM HEPES-KOH (pH 7.5), 50 mM NH 4 Cl, 10 mM Mg(CH 3 COO) 2 , 4 mM mercaptoethanol, and 0.05% Tween-20 for 30 min at 25 • C. Solutions of CHL, CAM-Cn-TPP, and CnTPP in different concentration ranges were added to the formed complex.…”