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Non-classical transpeptidases yield insight into new antibacterials

Abstract

Bacterial survival requires an intact peptidoglycan layer, a three-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by D,D-transpeptidases, enzymes that are inhibited by the β-lactams, which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of β-lactams are distinctly effective not only because they inhibit D,D-transpeptidases and are poor substrates for β-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the L,D-transpeptidases, which generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of L,D-transpeptidases of Mycobacterium tuberculosis and a range of bacteria including ESKAPE pathogens, and used this information to design, synthesize and test simplified carbapenems with potent antibacterial activity.

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Figure 1: Interactions of β-lactams with mycobacterial L,D-transpeptidases and activity of biapenem and faropenem in the mouse model of TB.
Figure 2: Molecular details of interactions of M. tuberculosis L,D-transpeptidases with (carba)penems.
Figure 3: Design and activity of evolved carbapenems.
Figure 4: Crystal structures of LdtMt2 with evolved carbapenems.
Figure 5: Proposed mechanism of acylation of L,D-transpeptidases by (carba)penems.

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Acknowledgements

Assistance of V. Chauhan for protein expression is appreciated. We thank L. Basta for critical discussions. P. aeruginosa PA14 was a kind gift from S. Lory, Harvard University. Discovery Studio was kindly provided to S.E. and J.S.F. by Biovia. This study was supported by NIH awards R21AI111739 and DP2OD008459 to G.L. Structural results shown in this report are derived in part from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357.

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Contributions

P.K., protein/ligand, crystallization and structure studies; A.K., antibacterial potency, animal studies; E.P.L., antibacterial synthesis and mass-spectrometry, mechanism; S.-G.L., antibacterial synthesis (contributed equally with E.P.L.); R.M., D.T.B., enzyme kinetics; T.A.Z. and A.L.P., structure data analysis; S.E., antibacterial cheminformatics; S.L.G., crystal data collection/analysis; C.A.T., antibacterial design/synthesis, mass-spectrometry, data analysis, mechanism; J.S.F., antibacterial design/synthesis and data analysis; G.L., overall study conception, cloning, animal studies, data analysis. P.K., N.C.A. and G.L. prepared the manuscript and all authors contributed to the final draft.

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Correspondence to Gyanu Lamichhane.

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Supplementary Results, Supplementary Tables 1–12 and Supplementary Figures 1–9. (PDF 2555 kb)

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Synthetic procedures. (PDF 828 kb)

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Kumar, P., Kaushik, A., Lloyd, E. et al. Non-classical transpeptidases yield insight into new antibacterials. Nat Chem Biol 13, 54–61 (2017). https://doi.org/10.1038/nchembio.2237

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