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Macrolones target bacterial ribosomes and DNA gyrase and can evade resistance mechanisms

Nature Chemical Biology volume 20, pages 1680–1690 (2024)Cite this article

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Abstract

Growing resistance toward ribosome-targeting macrolide antibiotics has limited their clinical utility and urged the search for superior compounds. Macrolones are synthetic macrolide derivatives with a quinolone side chain, structurally similar to DNA topoisomerase-targeting fluoroquinolones. While macrolones show enhanced activity, their modes of action have remained unknown. Here, we present the first structures of ribosome-bound macrolones, showing that the macrolide part occupies the macrolide-binding site in the ribosomal exit tunnel, whereas the quinolone moiety establishes new interactions with the tunnel. Macrolones efficiently inhibit both the ribosome and DNA topoisomerase in vitro. However, in the cell, they target either the ribosome or DNA gyrase or concurrently both of them. In contrast to macrolide or fluoroquinolone antibiotics alone, dual-targeting macrolones are less prone to select resistant bacteria carrying target-site mutations or to activate inducible macrolide resistance genes. Furthermore, because some macrolones engage Erm-modified ribosomes, they retain activity even against strains with constitutive erm resistance genes.

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Fig. 1: Macrolones inhibit DNA gyrase and protein synthesis in vivo.
Fig. 2: Macrolones inhibit protein synthesis and DNA gyrase in vitro.
Fig. 3: Structures of MCX-66, MCX-91 and MCX-128 in complex with the wild-type T. thermophilus 70S ribosome.
Fig. 4: Identification of cellular targets of macrolones by selecting resistant mutants.
Fig. 5: Macrolones are poor inducers of the expression of inducible macrolide resistance genes.
Fig. 6: Structure of macrolone MCX-128 bound to the Erm-methylated 70S ribosome.

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Data availability

Coordinates and structure factors were deposited to the Research Collaboratory for Structural Bioinformatics (RCSB) PDB under the following accession codes: 8VTU for the wild-type T. thermophilus 70S ribosome in complex with macrolone MCX-66, mRNA, aminoacylated A-site Phe-tRNAPhe, aminoacylated P-site fMet-tRNAiMet and deacylated E-site tRNAPhe; 8VTV for the wild-type T. thermophilus 70S ribosome in complex with macrolone MCX-91, mRNA, aminoacylated A-site Phe-tRNAPhe, aminoacylated P-site fMet-tRNAiMet and deacylated E-site tRNAPhe; 8VTW for the wild-type T. thermophilus 70S ribosome in complex with macrolone MCX-128 and protein Y; 8VTX for the m26A2058 T. thermophilus 70S ribosome in complex with macrolone MCX-128, mRNA, aminoacylated A-site Phe-tRNAPhe, aminoacylated P-site fMet-tRNAiMet and deacylated E-site tRNAPhe; 8VTY for the wild-type T. thermophilus 70S ribosome in complex with CIP and protein Y. All previously published structures that were used in this work for structural comparisons were retrieved from the RCSB PDB under accession codes 6XHW, 6XHX and 7ZTA. No sequence data were generated in this study. Source data are provided with this paper.

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Acknowledgements

We thank M. Svetlov for valuable discussions and assistance with the data processing. We thank the Analysis and Testing Center at the Beijing Institute of Technology for collecting and analyzing the spectral data. We thank the staff at Northeastern Collaborative Access Team (NE-CAT) beamlines 24ID-C and 24ID-E for help with X-ray diffraction data collection, especially M. Capel, F. Murphy, S. Banerjee, I. Kourinov, D. Neau, J. Schuermann, N. Sukumar, A. Lynch, J. Withrow, K. Perry, A. Kaya and C. Salbego. This work is based upon research conducted at the NE-CAT beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (NIH; grant P30-GM124165 to NE-CAT). The Eiger 16M detector on the 24ID-E beamline is funded by an NIH-ORIP HEI grant (S10-OD021527 to NE-CAT). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. This work was supported by the National Institute of General Medical Sciences of the NIH (grant R35-GM127134 to A.S.M.), the National Institute of Allergy and Infectious Diseases of the NIH (grant R21-AI137584 to A.S.M. and Y.S.P.), the Illinois State startup funds (to Y.S.P.), the National Key Research and Development Program of China (grant 2018YFA0901800 to J.-H.L.) and the National Natural Science Foundation of China (grant 81673335 to J.-H.L.). The funders had no role in study design, data collection and analysis, decision to publish or manuscript preparation.

Author information

Author notes

  1. These authors contributed equally: Elena V. Aleksandrova, Cong-Xuan Ma, Dorota Klepacki.

Authors and Affiliations

  1. Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Elena V. Aleksandrova & Yury S. Polikanov

  2. Key Laboratory of Medicinal Molecule Science and Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, China

    Cong-Xuan Ma & Jian-Hua Liang

  3. Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Dorota Klepacki, Faezeh Alizadeh, Nora Vázquez-Laslop, Yury S. Polikanov & Alexander S. Mankin

  4. Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, USA

    Dorota Klepacki, Faezeh Alizadeh, Nora Vázquez-Laslop, Yury S. Polikanov & Alexander S. Mankin

Authors
  1. Elena V. Aleksandrova
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  2. Cong-Xuan Ma
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  3. Dorota Klepacki
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  4. Faezeh Alizadeh
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  5. Nora Vázquez-Laslop
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  6. Jian-Hua Liang
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  7. Yury S. Polikanov
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  8. Alexander S. Mankin
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Contributions

C.-X.M. performed the chemical synthesis, purification and microbiological characterization of MCX compounds. D.K. and F.A. performed the in vivo dual-reporter assay, mutant selection and microbiological characterization of the selected MCX-resistant mutant strains. D.K. also performed the in vitro translation inhibition, gyrase inhibition and toeprinting assays. E.V.A. and Y.S.P. designed and performed X-ray crystallography experiments. A.S.M., N.V.-L., Y.S.P. and J.-H.L. designed and supervised the experiments. All authors interpreted the results. A.S.M., N.V.-L., Y.S.P. and J.-H.L. wrote the manuscript.

Corresponding authors

Correspondence to Jian-Hua Liang, Yury S. Polikanov or Alexander S. Mankin.

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Competing interests

The authors declare no competing interests.

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Nature Chemical Biology thanks Graeme Conn, Suparna Sanyal and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Inhibition of translation and ribosome binding of macrolones.

(a) Inhibition of production of the green flourescent protein (GFP) in cell-free translation system by varying concentrations of macrolones relative to uninhibited reaction. Shown are the results of two independent experiments. (b) Competitive binding of [14C]-ERY and macrolones to the E. coli ribosome. Unlabeled ERY was used as a control (black circles). Experimental details are presented in the Online Methods section. Shown are the results of two independent experiments.

Extended Data Fig. 2 Effects of macrolones on in vitro translation.

Mapping the sites of macrolone-mediated ribosome arrests (blue arrows) at the early codons of the model ORF derived from the E. coli yrbA gene. The classic macrolide ERY is included for comparison. Due to the presence of the Thr-RS inhibitor borrelidin, the ribosomes that did not stall at the early codons are eventually trapped at the Gln12 codon when Thr13 needs to be incorporated into the growing protein (grey arrow). The AUG start codon is marked with a black arrow. The sample labeled as ‘NONE’ contained only borrelidin but no ERY or macrolones. Amino acid and nucleotide sequences of yrbA gene are shown on the left. Sequencing lanes are marked as C, U, A, G. This experiment was repeated independently twice and produced similar results.

Extended Data Fig. 3 Electron density maps of ribosome-bound MCX-66, MCX-91, and MCX-128.

(a-c) 2Fo-Fc Fourier electron density maps of MCX-66 (a, magenta), MCX-91 (b, green), and MCX-128 (c, yellow) in complex with the T. thermophilus 70S ribosome (blue mesh) shown in two mutually perpendicular views. The refined models of ribosome-bound MCX compounds are displayed in their respective electron density maps after the refinement contoured at 1.0σ. Carbon atoms are colored magenta (MCX-66), green (MCX-91), or yellow (MCX-128); nitrogen atoms are blue; oxygen atoms are red; fluorine atoms are dark green. Note that the locations of fluoroquinolone side chains can be unambiguously determined from the obtained electron density maps.

Extended Data Fig. 4 Comparison of the structures of ribosome-bound macrolones, macrolide erythromycin and tetracenomycin X.

(a, b) Superposition of the structure of ribosome-bound MCX-66 (magenta), MCX-91 (green), MCX-128 (yellow) with the previous structures of ERY (red, PDB entry 6XHX ref. 15), or TcmX (blue, PDB entry 7ZTA ref. 75). All structures were aligned based on domain V of the 23S rRNA.

Extended Data Fig. 5 Structure of ciprofloxacin (CIP) in complex with the 70S ribosome.

(a) 2Fo-Fc Fourier electron density map of ciprofloxacin (CIP, greencyan) in complex with the T. thermophilus 70S ribosome (blue mesh). The refined model of ribosome-bound CIP is displayed in its respective electron density map after the refinement contoured at 1.0σ. Carbon atoms are colored greencyan; nitrogen atoms are blue; oxygen atoms are red; fluorine atom is dark green. (b, c) Close-up views of CIP bound in the NPET of the 70S ribosome, highlighting its stacking (red arrows) interactions with the nucleotides of the 23S rRNA. (d) Superposition of the structures of ribosome-bound CIP and MCX-128 (yellow). The structures were aligned based on domain V of the 23S rRNA.

Extended Data Fig. 6 Macrolones inhibit DNA gyrase activity in vitro.

(a) Chemical structures of the macrolones used in the assay. (b) Effect of macrolones on the activity of DNA gyrase. Supercoiled or relaxed plasmid DNA bands are marked on the left. CIP and ERY were used as positive and negative controls, respectively. The sample labeled as ‘NONE’ contained no antibiotics. A control sample where gyrase was not added is marked as ‘-’.The experiment was repeated independently and produced similar results.

Extended Data Fig. 7 Electron density maps of A2058 nucleotide in Erm-modified and wild-type T. thermophilus 70S ribosome.

(a, c) Unbiased Fo-Fc (grey and green mesh) and (b, d) 2Fo-Fc (blue mesh) electron difference Fourier maps of nucleotide A2058 in the T. thermophilus 70S ribosome contoured at 3.0σ and 1.0σ, respectively. Grey mesh shows the Fo-Fc map after refinement with the entire modified nucleotide omitted. Green mesh, reflecting the presence of the two methyl groups at N6 position of the nucleobase, shows the Fo-Fc electron density map after refinement with the nucleotide A2058 built as a regular unmethylated adenine. The refined models of Erm-modified N6-dimethylated (a, b) or wild-type unmodified (c, d) nucleotide A2058 are displayed in the corresponding electron density maps. Both structures carry MCX-128 compound (not shown). Carbon atoms are colored dark blue for the Erm-modified A2058 and light blue for the unmodified A2058; nitrogens are dark blue; oxygens are red.

Extended Data Table 1 Minimal inhibitory concentrations (MICs) of macrolones against the parental E. coli strain SQ110DTC (WT) and selected resistant mutants
Full size table
Extended Data Table 2 Antibacterial activity of macrolones and reference antibiotics against macrolide-sensitive ATCC standard strains and macrolide-resistant clinical isolates
Full size table

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Note, Figs. 1–6 and References.

Reporting Summary

Source data

Source Data Fig. 2

Raw experimental data for Fig. 2a.

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Aleksandrova, E.V., Ma, CX., Klepacki, D. et al. Macrolones target bacterial ribosomes and DNA gyrase and can evade resistance mechanisms. Nat Chem Biol 20, 1680–1690 (2024). https://doi.org/10.1038/s41589-024-01685-3

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