The rapid increase in antimicrobial resistance underscores the urgent need for new antibacterial agents. One promising strategy involves designing novel compounds through targeted chemical modifications of existing antibiotics. Azithromycin (AZI), a widely used macrolide, has served as a versatile scaffold for developing numerous antibacterial candidates. However, the mechanistic consequences of such modifications remain largely unexplored. Here, we characterize the activity and mechanism of action of three AZI-benzoxaborole (AZI-BB) conjugates. We show that these compounds inhibit bacterial translation in vitro and remain active against a model Escherichia coli strain carrying an inducible ermCL-ermC operon, which confers resistance to macrolide antibiotics. Unlike erythromycin, these derivatives, along with AZI itself, exhibit minimal induction of ErmC expression. Structural analysis reveals that the benzoxaborole moiety of AZI-BB2 forms additional interactions with nucleotides C2441 and C2586 of 23S rRNA, likely contributing to premature ribosome stalling at the ermCL regulatory sequence and thereby preventing ErmC expression. Furthermore, high-throughput toeprinting analysis combined with deep sequencing (Toe-seq) demonstrates that AZI-BB2 exhibits reduced sequence specificity for canonical macrolide-sensitive stalling motifs. Altogether, these findings demonstrate that targeted chemical modification of AZI can reshape its context-specific interaction with the ribosome and attenuate the induction of macrolide resistance mechanisms.

The dynamic equilibrium between synthesis and degradation of biomolecules is maintained by cells, however, with aging, this balance is disrupted, resulting in the onset of diseases, including diabetes and neurodegenerative diseases. A decrease in autophagy, a key cellular process that is involved in lysosome-mediated degradation of damaged or dysfunctional cellular components, may contribute to this imbalance. Autophagy is strictly regulated within the cell through multiple signaling pathways, e.g., through the AMPK-dependent pathway, which functions as a key sensor of cellular energy limitation. In this study, we assessed the autophagy/mitophagy activation ability of a small set of 1,3-diaza-2-oxophenoxazine derivatives and analogs using a fluorescent reporter assay and immunoblot analysis. The two lead compounds, AR493 and AR900, which exhibited the highest autophagy induction levels, were demonstrated to activate the AMPK-dependent pathway. The introduction of a 2'-hydroxyl group into AR493 had almost no influence on its activity, while subsequent attachment of a metabolizable masked phosphate group resulted in a notable increase in activity, although accompanied by substantial toxicity. When analyzing the specificity of the lead compounds to AMPK and its main upstream regulator SIRT1 on the corresponding knockout cell lines, AR493 demonstrated the greatest specificity of action to AMPK. Molecular docking revealed that AR493 binds to Site 2 of the AMPK γ-subunit, which may promote AMPK activation by two possible mechanisms: by preventing ATP binding to Site 3, thus favoring AMP binding; and by directly engaging the αRIM2 motif to stabilize its interaction with the γ-subunit.

Oxydifficidin is a natural polyketide antibiotic that is a long-known ribosome-targeting antibiotic that inhibits protein synthesis. In this paper, we describe Bacillus velezensis strain EV17 and compare its complete genome sequence with that of the previously characterized strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry, to elucidate the production of oxydifficidin by these strains. Isolated oxydifficidin was determined to increase generalized inhibition of translation at each step of protein biosynthesis using toeprinting and small fluorescent peptide assays. In previous studies, it has been demonstrated that oxydifficidin targets L7/L12 protein. Although spontaneous mutations in ribosomal protein L7/L12, located relatively close to the thiostrepton binding site on uL11, confer resistance to oxydifficidin, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We show that this compound does not affect eukaryotic translation and has no toxic effect on eukaryotic cells. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic activity.

Metastatic evolution of malignant tumors following standard anticancer therapies and the emergence of resistant cancer cell populations remain major challenges in oncology. One promising strategy is to develop compounds that selectively target mechanisms of therapeutic resistance. Unlike therapy-sensitive malignant cells, which rely primarily on glycolysis for energy, many chemoresistant cells and cancer stem cells (CSCs) preferentially utilize mitochondrial oxidative phosphorylation (OXPHOS). In this study, we employed a triple-negative breast cancer model to demonstrate that short antimicrobial peptides can significantly suppress the metastatic potential of resistant cancer cells and reduce the formation of CSC-like mammospheres by disrupting mitochondrial respiration. This effect was further enhanced by conjugating the peptides to the mitochondrial-targeting cation triphenylphosphonium (TPP). Mechanistic studies revealed that these compounds induce oxidative stress and mitophagy and suppress mitochondrial translation. Collectively, these findings suggest that TPP-conjugated peptides represent a promising therapeutic strategy for targeting OXPHOS-dependent resistance in aggressive solid tumors.

Oxydifficidin is a natural polyketide antibiotic that has long been recognized as a ribosome-targeting agent that inhibits protein synthesis. In this paper, we describe Bacillus velezensis strain EV17 and compare its complete genome sequence with that of the previously characterized B. velezensis strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry to elucidate the production of oxydifficidin by these strains. Toeprinting and small fluorescent peptide assays showed that isolated oxydifficidin induces a generalized inhibition of translation at every step of elongation in protein biosynthesis. In previous studies, it has been demonstrated that oxydifficidin targets bL12 protein. Although spontaneous mutations conferring resistance to oxydifficidin in ribosomal protein bL12 located relatively close to the thiostrepton binding site on uL11, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We are the first to show that this compound does not affect eukaryotic translation and has two orders of magnitude lower effect on eukaryotic cells compared to bacteria. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic agent.

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.

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.

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.

A fluorescence polarization immunoassay method has been developed for the determination of antibiotic tylosin in honey. For this purpose, rabbits were immunized with a conjugate of tylosin and bovine serum albumin, and antisera were obtained. The conditions for the interaction of antisera in a competitive format with tylosin in the sample and with tylosin labeled with fluorescein were optimized. The developed method is characterized by a limit of detection for tylosin of 34.7 ng/mL and an analytical range of 65.2 to 564.0 ng/mL. High reproducibility of measurements using a portable detector was demonstrated, with relative standard deviation (RSD) values of 1.5–2.5

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.

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.

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.

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 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.

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.

Volume 63, no. 6, e02266-18, 2019, https://doi.org/10.1128/AAC.02266-18. Acknowledgments: the 3rd paragraph of this section should read as follows. “This work was supported by Illinois State start-up funds (to Y.S.P.) and by the National Institutes of Health (grant R21-AI137584 to Y.S.P.), Russian Foundation for Basic Research (grant 17-00-00366 to P.V.S.), the Russian Science Foundation (grant 17-14-01416 to A.L.K. [production of functional ribosomal complexes] and grant 18-44-04005 to I.A.O. [analysis of antibiotic’s interaction with the ribosome]), and the Moscow University Development Program (PNR 5.13 to P.V.S.).”

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.