Berberine analog of chloramphenicol exhibits a distinct mode of action and unveils ribosome plasticity.

Chloramphenicol (CHL) is an antibiotic targeting the peptidyl transferase center in bacterial ribosomes. We synthesized a new analog, CAM-BER, by substituting the dichloroacetyl moiety of CHL with a positively charged aromatic berberine group. CAM-BER suppresses bacterial cell growth, inhibits protein synthesis in vitro, and binds tightly to the 70S ribosome. Crystal structure analysis reveals that the bulky berberine group folds into the P site of the peptidyl transferase center (PTC), where it competes with the formyl-methionine residue of the initiator tRNA. Our toe-printing data confirm that CAM-BER acts as a translation initiation inhibitor in stark contrast to CHL, a translation elongation inhibitor. Moreover, CAM-BER induces a distinct rearrangement of conformationally restrained nucleotide A2059, suggesting that the 23S rRNA plasticity is significantly higher than previously thought. CAM-BER shows potential in avoiding CHL resistance and presents opportunities for developing novel berberine derivatives of CHL through medicinal chemistry exploration.

Hybrid Molecules of Azithromycin with Chloramphenicol and Metronidazole: Synthesis and Study of Antibacterial Properties.

The sustained rise of antimicrobial resistance (AMR) causes a strong need to develop new antibacterial agents. One of the methods for addressing the problem of antibiotic resistance is through the design of hybrid antibiotics. In this work, we proposed a synthetic route for the conjugation of an azithromycin derivative with chloramphenicol and metronidazole hemisuccinates and synthesized two series of new hybrid molecules 4a-g and 5a-g. While a conjugation did not result in tangible synergy for wild-type bacterial strains, new compounds were able to overcome AMR associated with the inducible expression of the ermC gene on a model E. coli strain resistant to macrolide antibiotics. The newly developed hybrids demonstrated a tendency to induce premature ribosome stalling, which might be crucial since they will not induce a macrolide-resistant phenotype in a number of pathogenic bacterial strains. In summary, the designed structures are considered as a promising direction for the further development of hybrid molecules that can effectively circumvent AMR mechanisms to macrolide antibiotics.

Triphenylphosphonium Analogs of Short Peptide Related to Bactenecin 7 and Oncocin 112 as Antimicrobial Agents.

Antimicrobial peptides (AMPs) have recently attracted attention as promising antibacterial agents capable of acting against resistant bacterial strains. In this work, an approach was applied, consisting of the conjugation of a peptide related to the sequences of bactenecin 7 (Bac7) and oncocin (Onc112) with the alkyl(triphenyl)phosphonium (alkyl-TPP) fragment in order to improve the properties of the AMP and introduce new ones, expand the spectrum of antimicrobial activity, and reduce the inhibitory effect on the eukaryotic translation process. Triphenylphosphonium (TPP) derivatives of a decapeptide RRIRPRPPYL were synthesized. It was comprehensively studied how the modification of the AMP affected the properties of the new compounds. It was shown that while the reduction in the Bac7 length to 10 a.a. residues dramatically decreased the affinity to bacterial ribosomes, the modification of the peptide with alkyl-TPP moieties led to an increase in the affinity. New analogs with structures that combined a decapeptide related to Bac7 and Onc112-Bac(1-10, R/Y)-and TPP attached to the C-terminal amino acid residue via alkylamide linkers, inhibited translation in vitro and were found to be more selective inhibitors of bacterial translation compared with eukaryotic translation than Onc112 and Bac7. The TPP analogs of the decapeptide related to Bac7 and Onc112 suppressed the growth of both Gram-negative bacteria, similar to Onc112 and Bac7, and Gram-positive ones, similar to alkyl-TPP derivatives, and also acted against some resistant laboratory strains. Bac(1-10, R/Y)-C2-TPP, containing a short alkylamide linker between the decapeptide and TPP, was transferred into the E. coli cells via the SbmA transporter protein. TPP derivatives of the decapeptide Bac(1-10, R/Y) containing either a decylamide or ethylamide linker caused B. subtilis membrane depolarization, similar to alkyl-TPP. The Bac(1-10, R/Y)-C2-TPP analog was proven to be non-toxic for mammalian cells using the MTT test.

An easy tool to monitor the elemental steps of in vitro translation via gel electrophoresis of fluorescently labeled small peptides.

Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1-7 amino acids, BODIPY-labeled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.

Conjugates of Desmycosin with Fragments of Antimicrobial Peptide Oncocin: Synthesis, Antibacterial Activity, Interaction with Ribosome.

Design and synthesis of conjugates consisting of the macrolide antibiotic desmycosin and fragments of the antibacterial peptide oncocin were performed in attempt to develop new antimicrobial compounds. New compounds were shown to bind to the E. coli 70S ribosomes, to inhibit bacterial protein synthesis in vitro, as well as to suppress bacterial growth. The conjugates of N-terminal hexa- and tripeptide fragments of oncocin and 3,2',4''-triacetyldesmycosin were found to be active against some strains of macrolide-resistant bacteria. By simulating molecular dynamics of the complexes of these compounds with the wild-type bacterial ribosomes and with ribosomes, containing A2059G 23S RNA mutation, the specific structural features of their interactions were revealed.

Conjugates of Chloramphenicol Amine and Berberine as Antimicrobial Agents.

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.

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome.

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.

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

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.

Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

Structure of Dirithromycin Bound to the Bacterial Ribosome Suggests New Ways for Rational Improvement of Macrolides.

Although macrolides are known as excellent antibacterials, their medical use has been significantly limited due to the spread of bacterial drug resistance. Therefore, it is necessary to develop new potent macrolides to combat the emergence of drug-resistant pathogens. One of the key steps in rational drug design is the identification of chemical groups that mediate binding of the drug to its target and their subsequent derivatization to strengthen drug-target interactions. In the case of macrolides, a few groups are known to be important for drug binding to the ribosome, such as desosamine. Search for new chemical moieties that improve the interactions of a macrolide with the 70S ribosome might be of crucial importance for the invention of new macrolides. For this purpose, here we studied a classic macrolide, dirithromycin, which has an extended (2-methoxyethoxy)-methyl side chain attached to the C-9/C-11 atoms of the macrolactone ring that can account for strong binding of dirithromycin to the 70S ribosome. By solving the crystal structure of the 70S ribosome in complex with dirithromycin, we found that its side chain interacts with the wall of the nascent peptide exit tunnel in an idiosyncratic fashion: its side chain forms a lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4. To our knowledge, the ability of this side chain to form a contact in the macrolide binding pocket has not been reported previously and potentially can open new avenues for further exploration by medicinal chemists developing next-generation macrolide antibiotics active against resistant pathogens.

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

Antibiotic chloramphenicol (CHL) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino acid analogs of CHL. The L-histidyl analog binds to the ribosome with the affinity exceeding that of CHL by 10 fold. Several of the newly synthesized analogs were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CHL. However, the inhibitory properties of the semi-synthetic CHL analogs did not correlate with their affinity and in general, the amino acid analogs of CHL were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogs showed that CHL derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.

Conjugates of amino acids and peptides with 5-o-mycaminosyltylonolide and their interaction with the ribosomal exit tunnel.

During protein synthesis the nascent polypeptide chain (NC) extends through the ribosomal exit tunnel (NPET). Also, the large group of macrolide antibiotics binds in the nascent peptide exit tunnel. In some cases interaction of NC with NPET leads to the ribosome stalling, a significant event in regulation of translation. In other cases NC-ribosome interactions lead to pauses in translation that play an important role in cotranslational folding of polypeptides emerging from the ribosome. The precise mechanism of NC recognition in NPET as well as factors that determine NC conformation in the ribosomal tunnel are unknown. A number of derivatives of the macrolide antibiotic 5-O-mycaminosyltylonolide (OMT) containing N-acylated amino acid or peptide residues were synthesized in order to study potential sites of NC-NPET interactions. The target compounds were prepared by conjugation of protected amino acids and peptides with the C23 hydroxyl group of the macrolide. These OMT derivatives showed high although varying abilities to inhibit the firefly luciferase synthesis in vitro. Three glycil-containing derivatives appeared to be strong inhibitors of translation, more potent than parental OMT. Molecular dynamics (MD) simulation of complexes of tylosin, OMT, and some of OMT derivatives with the large ribosomal subunit of E. coli illuminated a plausible reason for the high inhibitory activity of Boc-Gly-OMT. In addition, the MD study detected a new putative site of interaction of the nascent polypeptide chain with the NPET walls.