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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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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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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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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DOI: https://doi.org/10.1038/nchem.2110
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