Discovery of an antivirulence compound that reverses β-lactam resistance in MRSA

Abstract

Staphylococcus aureus is the leading cause of infections worldwide, and methicillin-resistant strains (MRSA) are emerging. New strategies are urgently needed to overcome this threat. Using a cell-based screen of ~45,000 diverse synthetic compounds, we discovered a potent bioactive, MAC-545496, that reverses β-lactam resistance in the community-acquired MRSA USA300 strain. MAC-545496 could also serve as an antivirulence agent alone; it attenuates MRSA virulence in Galleria mellonella larvae. MAC-545496 inhibits biofilm formation and abrogates intracellular survival in macrophages. Mechanistic characterization revealed MAC-545496 to be a nanomolar inhibitor of GraR, a regulator that responds to cell-envelope stress and is an important virulence factor and determinant of antibiotic resistance. The small molecule discovered herein is an inhibitor of GraR function. MAC-545496 has value as a research tool to probe the GraXRS regulatory system and as an antibacterial lead series of a mechanism to combat drug-resistant Staphylococcal infections.

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Fig. 1: High-throughput screening identifies potent bioactives that reverse β-lactam resistance in MRSA.
Fig. 2: MAC-545496 targets GraR, a regulator of the cell-envelope stress response.
Fig. 3: MAC-545496 inhibits S. aureus USA300 biofilm formation.
Fig. 4: MAC-545496 abrogates intracellular survival of S. aureus USA300.
Fig. 5: MAC-545496 attenuates virulence in vivo.
Fig. 6: MAC-545496 potentiates components of the innate immune response (oxidative stress and lysozyme).

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). Source data for Figs. 1–6 are presented with the paper.

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Acknowledgements

We thank A. Keddie from the University of Alberta for providing valuable advice on breeding Galleria and supplying the first batch of larvae, and B. Weber and A. Khaled for their help in breeding Galleria. We thank G. Wright from McMaster University for providing S. aureus clinical isolates. We also thank S. French for preparing the graphical abstract. This work was supported by grants from the Canadian Institutes of Health Research (foundation grant FRN-143215), the Canadian glycomics network (GlycoNet, https://doi.org/10.13039/501100009056, a National Centre of Excellence) and a Tier I Canada Research Chair award to E.D.B. D.E.H. acknowledges operating grant support from Cystic Fibrosis Canada. Studies performed in the laboratory of M.G.O. were funded by the Ontario Research Foundation. O.M.E.-H. was supported by a Michael G. DeGroote Fellowship Award in Basic Biomedical Science.

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O.M.E.-H., T.L.C. and E.D.B. conceived and designed the research. O.M.E.-H. performed all experiments and analyzed all data unless otherwise stated. T.L.C. performed the primary screen. R.S.F. performed the macrophage intracellular assays, supervised by D.E.H. R.C.K. constructed promoter–reporter transcriptional fusions, supervised by M.J.M. J.D., A.S. and P.E. synthesized and characterized MAC-545496 analogs, supervised by M.G.O. J.C.B. helped O.M.E.-H. perform and analyze the ITC assays and performed the circular dichroism assays, supervised by R.M.E. O.M.E.-H and E.D.B. wrote the paper. All authors approved the final version.

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Correspondence to Eric D. Brown.

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E.D.B., O.M.E.-H., T.L.C., J.D., M.G.O., R.S.F. and D.E.H. are inventors on a patent application on the use of MAC-545496 and analogs thereof, alone and in combination with other antibiotics, for the treatment of MRSA infections.

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El-Halfawy, O.M., Czarny, T.L., Flannagan, R.S. et al. Discovery of an antivirulence compound that reverses β-lactam resistance in MRSA. Nat Chem Biol 16, 143–149 (2020). https://doi.org/10.1038/s41589-019-0401-8

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