Abstract
Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.
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Data availability
All data supporting the findings of this study, including statistical analyses, are available within the article and its Supplementary Information or Source Data files. The cryo-EM structure and supporting data have been deposited to the PDB under the accession code 8GK7. Source data are provided with this paper.
Code availability
No proprietary code was used in this work.
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Acknowledgements
We thank D. Libardo and S. Dong for their careful review of the paper and helpful suggestions, as well as K. Smith, S. Zhou and I. Etim for their help with the plasma protein binding determinations. We thank the scientists at Evotec for their contributions to protein production and the scientists at HD Biosciences for testing the expanded strain panel including the CRAB isolates.
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S.S.W., C.J.B., P.S., H.W., J.S., I.R., R.T., T.A.B., A.I., Y.-T.C., D.J.K. and M.B. designed the experiments and wrote the paper. H.W. and R.E.P. designed and conducted microbial target identification and biological experiments. K.B., Z.W., J.S., A.W.S., C.W., L.T., M.L., H.J.M., D.S., J.M., T.M., W.L., J.M., A.C., A.B., L.-K.Z., M.X. and J.L. designed, constructed or analysed chemical matter. R.T., R.R.M., A.L. and T.D.C. designed and conducted formulation and pharmacokinetics studies, and analysed data. P.S., D.M. and H.L. designed, conducted and analysed in vivo efficacy studies. A.I., Y.-T.C., D.J.K. and G.S. designed and conducted cryo-EM structure experiments. C.B.-T., L.D. and M.B. designed and conducted biochemical experiments. Y.L., J.C.X., Q.S., P.A.M. and R.E.P. conducted antimicrobial and cell-based assays.
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All authors are or were, at the time this work was conducted, employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA and may be shareholders in Merck & Co., Inc., Rahway, NJ, USA. Cerastecin A–D are the subjects of two patent applications (PCT/US2023/022198 and PCT/US2023/022200) by Merck Sharp & Dohme LLC, Rahway, NJ, USA. I.R., C.J.B., J.S., M.L., H.J.M., L.T., H.W., C.W., A.B., L.-K.Z., J.L., K.B., Z.W., A.W.S. and A.C. are inventors on either or both applications.
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Extended data
Extended Data Fig. 1 Bactericidal activity of cerastecin B.
The time-dependent effects of cerastecin B exposure at 8 X MIC on A. baumannii ATCC19606 and CLB 21655 cell viability. Results from independent single experiments using two A. baumannii strains, ATCC19606 and CLB21655 are shown.
Extended Data Fig. 2 Chemical genetic confirmation of cerastecin mechanism of action.
a, Sensitivity of wild-type (upper panel), cerastecin B-resistant (B5, middle panel), and lpxA deleted (lower panel) A. baumannii ATCC19606 to the indicated compounds. Dilutions of compounds from high (top) to low (bottom), indicated by black wedge, were placed directly on the agar surface. Col, colistin; Rif, rifampicin; Bac, bacitracin. Photographs are unprocessed and uncropped. b, Detection of LOS in wild-type and cerastecin B-resistant mutant B5 A. baumannii ATCC19606 by the HEK-Blue™ cell assay. Test done in duplicate, both data sets shown. c, RT-qPCR to measure relative expression of MsbA in wild-type, mutant B1 (V39F) and B3 (K6K) A. baumannii ATCC19606 (n = 4). Data plotted with mean +/- SEM. Statistical significance relative to the wild-type strain is denoted as * (p = 0.003) and *** (p = 0.00038) was analyzed in MS Excel (v. 2302) using a two-tailed Student t-test. d, Sensitivity of wild-type (upper panel), cerastecin B-resistant mutants B1 (V39F) and B3 (K6K) to the indicated compounds. Dilutions of compounds were placed directly on the agar surface from high to low concentration as in a. Cer. B, cerastecin B; other abbreviations as above. The LpxC inhibitor CHIR-9025 was used at 4 mg mL−1 in the agar. Photographs are unprocessed and cropped only to fit space constraints and allow easier comparisons between images.
Extended Data Fig. 3 MsbA-cerastecin B binding, kinetics, and activity in vitro.
a, Kinetic binding parameters and affinity of cerastecin B binding to wild-type MsbA in nanodiscs. b, Activation of the ATPase activity of MsbA in proteoliposome (lipo) or in amphipols (amphi). Tested in duplicate, each data point shown for comparison. c, Michaelis-Menten parameters for cerastecin B stimulation of wild-type MsbA in nanodiscs (mean ± std. dev.). Supporting data. d, Image of instrument output of single cycle binding kinetic data. Red line, experimental sensogram trace; black line, single cycle kinetic fit. Axis details enlarged for readability. e, Image of binding affinity fit data from panel d (red diamonds, experimental data; black line, fit of data). Axis details enlarged for readability.
Extended Data Fig. 4 Pharmacokinetics of cerastecin D in mice.
Three fasted C57BL/6 mice were dosed subcutaneously at 300 mg kg−1 and tail vein samples were taken at the indicated times for quantitation by LC-MS as described. Data for all three mice are shown with the mean values at each time point in grey. Dashed lavender line indicates the serum-shifted MIC for cerastecin D (Table 1).
Extended Data Fig. 5 Molecular interactions of inhibitors with MsbA.
a, Cryo-EM structure depicting important amino acid residues in both MsbA monomers involved in cerastecin binding. Predicted hydrogen bonds are shown as yellow dashed lines. b, Overlay of the cerastecin C binding pocket with the existing MsbA binding structures. LPS is shown in stick form, Genentech inhibitor (G907, Fig. 1b) is shown as cyan spheres (both from PDB ID 6BPL), cerastecin C and AMP-PNP are shown as purple and green spheres respectively. c, Overlay of cerastecin C binding with TBT1 (Fig. 1b) (PDB ID 7MET). Ordered (shown in red) and disordered (shown in yellow) protomers of the TBT1-bound structure are overlayed with cerastecin C bound MsbA. TBT1 and cerastecin C molecules are shown as sticks in green and magenta, respectively.
Supplementary information
Supplementary Information
Cryo-EM image capture information, RT-qPCR primer sequences and synthesis of cerastecin A–D.
Source data
Source Data Fig. 2b,c
MsbA ATPase activation and MsbA (V39F) characterization.
Source Data Fig. 4
In vivo efficacy data.
Source Data Extended Data Fig. 1
Bactericidal activity of cerastecin B.
Source Data Extended Data Fig. 2b,c
Chemical genetic confirmation of cerastecin mechanism of action.
Source Data Extended Data Fig. 3b
MsbA ATPase activation in lipid environments.
Source Data Extended Data Fig. 4
Pharmacokinetics of cerastecin D.
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Wang, H., Ishchenko, A., Skudlarek, J. et al. Cerastecins inhibit membrane lipooligosaccharide transport in drug-resistant Acinetobacter baumannii. Nat Microbiol (2024). https://doi.org/10.1038/s41564-024-01667-0
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DOI: https://doi.org/10.1038/s41564-024-01667-0
