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Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition

Nature Chemistry volume 6, pages 1084–1090 (2014)Cite this article

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Abstract

The use of β-lactam antibiotics is compromised by resistance, which is provided by β-lactamases belonging to both metallo (MBL)- and serine (SBL)-β-lactamase subfamilies. The rhodanines are one of very few compound classes that inhibit penicillin-binding proteins (PBPs), SBLs and, as recently reported, MBLs. Here, we describe crystallographic analyses of the mechanism of inhibition of the clinically relevant VIM-2 MBL by a rhodanine, which reveal that the rhodanine ring undergoes hydrolysis to give a thioenolate. The thioenolate is found to bind via di-zinc chelation, mimicking the binding of intermediates in β-lactam hydrolysis. Crystallization of VIM-2 in the presence of the intact rhodanine led to observation of a ternary complex of MBL, a thioenolate fragment and rhodanine. The crystallographic observations are supported by kinetic and biophysical studies, including 19F NMR analyses, which reveal the rhodanine-derived thioenolate to be a potent broad-spectrum MBL inhibitor and a lead structure for the development of new types of clinically useful MBL inhibitors.

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Figure 1: Mode of action of β-lactams and their inactivation by β-lactamases.
Figure 2: Crystallographic analysis unexpectedly reveals that ML302 undergoes fragmentation to yield a thioenolate.
Figure 3: Crystallographic analysis reveals that the binding modes of ML302M and ML302F are similar to those of the MBL-product complexes resulting from β-lactam antibiotic hydrolysis.
Figure 4: Inhibition of VIM-2 by ML302, ML302F and ML302M reveals >20-fold difference in IC50 values for ML302M compared to ML302 or ML302F alone.
Figure 5: Crystallographic analysis of ML302F in complex with Bacillus cereus MBL (BcII) reveals that the observed binding mode of ML302 also applies to other MBLs.

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Acknowledgements

The authors acknowledge support from a Medical Research Council (MRC)/Canadian grant G1100135 and the Biotechnology and Biological Sciences Research Council (BBSRC; J.B. and C.J.S). Cancer Research UK (CRUK) is acknowledged for supporting S.S.v.B. and C.J.S. The authors acknowledge support from the Dulverton Trust (A.M.R.), the Royal Society Dorothy Hodgkin Fellowship (A.K.) and a German Academic Exchange Service (DAAD) Postdoctoral Fellowship (K-D.U.). The authors thank T. Walsh for supplying the clinically isolated strains used in this study.

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  1. Jürgen Brem, Sander S. van Berkel, WeiShen Aik, Anna M. Rydzik, Matthew B. Avison, Ilaria Pettinati, Klaus-Daniel Umland, Akane Kawamura, James Spencer, Timothy D. W. Claridge, Michael A. McDonough and Christopher J. Schofield: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK

    Jürgen Brem, Sander S. van Berkel, WeiShen Aik, Anna M. Rydzik, Ilaria Pettinati, Klaus-Daniel Umland, Akane Kawamura, Timothy D. W. Claridge, Michael A. McDonough & Christopher J. Schofield

  2. School of Cellular and Molecular Medicine, University of Bristol, Medical Sciences Building, Bristol, BS8 1TD, UK

    Matthew B. Avison & James Spencer

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Contributions

J.B., S.S.v.B. and C.J.S. conceived the research and designed the experiments. S.S.v.B. and K-D.U. synthesized and characterized the compounds used for the study. J.B. and I.P carried out the kinetic studies, purified the enzymes used in the biochemical studies, and crystallized the inhibitors with VIM-2 and BcII MBLs. W.S.A., J.B. and M.A.M. collected X-ray data and solved the crystal structures. A.M.R. designed and performed the NMR spectroscopy experiments with the help of J.B. and T.D.W.C. M.B.A. designed and performed the MIC experiments with the help of J.S. A.K. performed the cytotoxicity testing and helped with the design of the selectivity assays. J.B and C.J.S. wrote the first draft of the manuscript. All authors discussed the results and contributed to writing the manuscript.

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Correspondence to Christopher J. Schofield.

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Brem, J., van Berkel, S., Aik, W. et al. Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition. Nature Chem 6, 1084–1090 (2014). https://doi.org/10.1038/nchem.2110

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