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Expanding the antibacterial selectivity of polyether ionophore antibiotics through diversity-focused semisynthesis

Abstract

Polyether ionophores are complex natural products capable of transporting cations across biological membranes. Many polyether ionophores possess potent antimicrobial activity and a few selected compounds have the ability to target aggressive cancer cells. Nevertheless, ionophore function is believed to be associated with idiosyncratic cellular toxicity and, consequently, human clinical development has not been pursued. Here, we demonstrate that structurally novel polyether ionophores can be efficiently constructed by recycling components of highly abundant polyethers to afford analogues with enhanced antibacterial selectivity compared to a panel of natural polyether ionophores. We used classic degradation reactions of the natural polyethers lasalocid and monensin and combined the resulting fragments with building blocks provided by total synthesis, including halogen-functionalized tetronic acids as cation-binding groups. Our results suggest that structural optimization of polyether ionophores is possible and that this area represents a potential opportunity for future methodological innovation.

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Fig. 1: Accessing structural diversity within the polyether ionophores.
Fig. 2: Building block synthesis.
Fig. 3: Fragment coupling via boron trifluoride-mediated Mukaiyama aldol reaction.
Fig. 4: Final coupling of tetronic acid derivatives.
Fig. 5: Oxidative deconstruction of monensin for hybrid polyether synthesis.

Data availability

MIC and IC50 values for cell viability for each biological replicate are available in Supplementary Table 1. The morphological profiling data (aggregated profiles and correlation matrix), included in Supplementary Figs. 39, have been deposited at Mendeley Data (https://doi.org/10.17632/jv3sjk8wy4.2). The images from morphological profiling are too large to be deposited at Mendeley Data (which has a limit of 10 GB per data set), but can be obtained upon request. The X-ray crystallography data have been deposited in the Cambridge Crystallographic Data Centre (CCDC) using the following identifiers (www.ccdc.cam.ac.uk/structures/): 1920656 (6-Na), 1920657 (14), 1920658 (24), 1920659 (29-Na) and 1920660 (31-Na).

Code availability

Code/scripts for analysis of morphological profiling data and Cell Profiler pipelines have been deposited at Mendeley Data (https://doi.org/10.17632/jv3sjk8wy4.2).

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Acknowledgements

This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement number 865738). Financial support from the Carlsberg Foundation (grant CF17-0800), Novo Nordisk Foundation (grant NNF19OC0054782) and Independent Research Fund Denmark (grants 9040-00117B and 6110-00600B) is acknowledged. We thank E. Jung and A. Johnbeck for technical assistance with the organic syntheses, A. Bodholt Nielsen for technical assistance with NMR spectroscopy, and I.C. Stensgaard Jensen for technical assistance with microbiology.

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Contributions

T.B.P. conceived and supervised the study. T.B.P., S.L., H.L. and E.B.S. designed the experiments. S.L., H.L., J.M.V. and C.N.P. performed the organic syntheses. E.B.S. and K.M.J. conducted the cell biology experiments and analysed data. M.W. and F.D.A. conducted the microbiology experiments. T.T. supervised the microbiology experiments and analysed data. P.N. carried out the X-ray crystallographic analyses. T.B.P., S.L., H.L. and E.B.S. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Thomas B. Poulsen.

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Extended data

Extended Data Fig. 1 Strategies for the synthesis of protected 5-(halo)methylidene tetronic acid building blocks and subsequent coupling.

a, Chemical structure of the targeted 3-acyltetronic acid-derivatives found in nonthmicin/ecteinamycin and 6. No previous syntheses of the halogenated variants have been reported. b, Examples of known methods used to prepare non-halogenated variants. c, Failed attempts and model studies toward 5-chloromethylidene tetronate. I) Unsuccessful synthesis of 5-tributylstannylmethylidene tetronate. II) Unsuccessful chlorination of 5-dimethylaminomethylidene tetronate. III) Direct and indirect chlorination of 5-methylidene tetronate methyl ether. In practice, the removal of the methyl group from the chlorinated tetronate product using LiCl/DMSO or BBr3 was difficult. Thus, we moved to synthesize the tetronate derivatives bearing a TMSE group, which can be removed under much milder condition (TBAF/DMF). DMSO = dimethylsulfoxide DCC = N,N’-dicyclohexylcarbodiimide, DMAP = 4-dimethylaminopyridine, Ms = methanesulfonyl, MOM = methoxymethyl, DCE = 1,2-dichloroethane, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, TMS = trimethylsilyl, NCS = N-chlorosuccinimide, AIBN = azobisisobutyronitrile.

Extended Data Fig. 2 Synthesis of hybrid polyether HL324 (32).

Preparation of analogue HL324 (32) was performed through anti-selective (Evans-Saksena) reduction of the ketone-functionality of 26 followed by DCC-mediated coupling with 5-chloromethylidene tetronate. rt = room temperature, TBAF = tetrabutylammonium fluoride, DMF = N,N-dimethylformamide, DCC = N,N’-dicyclohexylcarbodiimide, DMAP = 4-dimethylaminopyridine, HPLC = high pressure liquid chromatography.

Extended Data Fig. 3 Droplet screen for antibacterial activity in B. cereus.

a, Representative images of inhibition zones of all compounds from B. cereus. b, Heatmap representing no (red), slight (yellow) or large (green) inhibition zones. All compounds were tested at 10 mM in DMSO except monensin (1 mM) and HL160 (7 mM). c) Chemical structures of acid-containing synthetic fragments. SL382 = 27; SL415 = 26 – see Fig. 3.

Extended Data Fig. 4 Use of morphological profiling via cell painting to determine bioactivity threshold in U-2OS cells.

a, Plots of cell count (grey) and Mahalanobis distance (‘Bioactivity’; red and green) against concentration of compound. Green points indicate significant activity (mp-value < 0.01) while red points indicate no significant activity (mp-value > 0.01). Many ionophores induce an active profile without loss of cell viability while bioactivity of the hybrid ionophores is correlated with a loss of cells, indicating a profile representing toxicity. The bioactivity threshold is determined as the first concentration to reach significance (mp-value < 0.01). b, Dose-dependent morphological profiles from which the bioactivity threshold is determined. Note that some ionophores change profile at higher concentrations, for example calcimycin (Cal) and nanchangmycin (Nan), typically correlated with a loss of cells. See Supplementary Fig. 39 for representative images, all dose-response plots, profiles and correlation matrices in both U-2OS and Vero cells.

Supplementary information

Supplementary Information

Supplementary Figs. 1–9, X-ray crystallography methods and comments, morphological profiling imaging settings, synthesis protocols and characterization data, references and NMR spectra.

Reporting Summary

Supplementary Table 1

Complete data from MIC determinations and cell viability experiments.

Supplementary Data 1

Cif file for compound 6-Na.

Supplementary Data 2

Structure factors file for compound 6-Na.

Supplementary Data 3

Cif file for compound 14.

Supplementary Data 4

Structure factors file for compound 14.

Supplementary Data 5

Cif file for compound 24.

Supplementary Data 6

Structure factors file for compound 24.

Supplementary Data 7

Cif file for compound 29-Na.

Supplementary Data 8

Structure factors file for compound 29-Na.

Supplementary Data 9

Cif file for compound 31-Na.

Supplementary Data 10

Structure factors file for compound 31-Na.

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Lin, S., Liu, H., Svenningsen, E.B. et al. Expanding the antibacterial selectivity of polyether ionophore antibiotics through diversity-focused semisynthesis. Nat. Chem. 13, 47–55 (2021). https://doi.org/10.1038/s41557-020-00601-1

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