Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms

Abstract

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, and shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, which included interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltransferase and the stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associated antibiotic effects, especially the lack of resistance development, probably stem from the compound’s polypharmacology.

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Fig. 1: Antibacterial properties of SFN and PK150.
Fig. 2: Target identification by chemical proteomic profiling in S. aureus.
Fig. 3: Validation of cellular targets with putative roles in the antibiotic mechanism.
Fig. 4: SAR study of SFN and mode of action analysis by chemical proteomics.
Fig. 5: FESEM and TEM of S. aureus NCTC 8325.
Fig. 6: In-depth analysis of SFN-resistant S. aureus isolates and accompanying consequences for compound-induced SpsB stimulation.
Fig. 7: Pharmacokinetic and pharmacodynamic parameters of PK150 and in vivo efficacy.

Data availability

The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE59 partner repository with the dataset identifier PXD012946. Whole-genome sequencing data and metadata are available on the SRA repository under the Bioproject number PRJNA525411. Bacterial strains and plasmids used in this work are readily available from the authors, or can be purchased commercially as stated in the Supplementary Information.

Code availability

All computer code used is either publicly available software, described in prior publications31 or available from the authors upon request. For details on the versions and parameters used, please refer to the respective sections in the Supplementary Information.

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Acknowledgements

We thank the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for the supply of the Nebraska Transposon Mutant Library (NTML). Furthermore, we thank S. Grond for providing arylomycin and F. Romesberg for providing S. aureus N315 ARC0001ΔSpsB. We also thank S. Miami and E. Rubin for determining the antimicrobial activities against M. tuberculosis. We thank A. Klaschwitz, F. Kortmann, S. Hifinger and C. Lierse von Gostomski for the scintillation measurement of radioactively labelled menaquinone. S.A.S. was funded by the Center for Integrated Protein Science Munich (CIPSM), Deutsche Forschungsgemeinschaft SFB1035 and European Research Council (ERC) and the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 725085, CHEMMINE, ERC consolidator grant). E.K. was supported by a doctoral fellowship of the Fonds der Chemischen Industrie. R.M. was supported by a doctoral fellowship of the Boehringer Ingelheim Fonds. K.R. was supported by the German Centre for Infection Research (DZIF) (TTU 09.710). I.A. acknowledges funding by Deutsche Forschungsgemeinschaft SFB1035. W.M.W. was funded by the National Science Foundation (CHE-1454116) and the National Institute of General Medical Sciences (R35 GM119426). M.C.J. acknowledges a National Science Foundation predoctoral grant (DGE-1144462). S.M.H. acknowledges financial support by a Liebig fellowship of the Fonds der Chemischen Industrie. M.W.H., C.F. and F.A.M.M. were funded by the Federal Ministry for Education and Research (BMBF) under the framework programme ‘VIP+’—project ‘aBacter’. We thank D. Mostert for excellent experimental support, M. Wolff, K. Bäuml, K. Gliesche, L. Nguyen and J. Schreiber for excellent technical support and M. Stahl for critical comments on the manuscript.

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Contributions

P.L., E.K., R.M. and S.A.S. designed the experiments, interpreted the results and wrote the manuscript with input from all the authors. P.L. synthesized the library compounds and probes and performed SAR studies. V.S.K. assisted in the chemical synthesis of the AfBPP probes. E.K. and P.L. performed gel- and MS-based labelling and analysis of the MS data, as well as SpsB target deconvolution and validation experiments. E.K. analysed mass spectrometry-based data and conducted bioinformatics analyses. R.M. performed target identification, MS data analysis and validation experiments in the context of the menaquinone biosynthesis pathway and assisted in further validation experiments. E.K., P.L. and S.M.H. performed the bacterial resistance development studies. E.K. carried out persister assays and time-kill assays. M.W.H., C.F. and F.A.M.M. performed time-kill assays as well as biofilm and persister studies. M.C.J. and W.M.W. helped in the biofilm studies. J.L. performed microbiological studies in mycobacteria. D.C.-M. and D.H.P. conducted the whole-genome sequencing of resistant bacterial isolates and analysed the related data. I.U. and I.A. performed molecular docking and dynamic studies and interpreted the related data. K.R. and M.Rohde performed electron microscopy studies and analysed the related data. M.Reinecke and B.K. performed kinobead pull-down experiments and analysed the related data. K.R. and E.M. performed animal studies and analysed the related data.

Corresponding author

Correspondence to Stephan A. Sieber.

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Competing interests

P.L., E.K. and S.A.S. are co-inventors on a European patent (EP 16 171 906.7) that covers the structure of PK150. All the other authors declare no competing interests.

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Supplementary information

Supplementary Information

Reporting Summary

Supplementary Data 1

Kinase inhibitor and SFN analogues libraries.

Supplementary Data 2

Antibacterial activities.

Supplementary Data 3

Target identification related proteomic data.

Supplementary Data 4

Proteome, secretome, surfaceome related proteomic data.

Supplementary Data 5

Genome sequencing of SFN-resistant isolates.

Supplementary Data 6

Kinobead pull-down related proteomic data.

Supplementary Data 7

NMR data.

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Le, P., Kunold, E., Macsics, R. et al. Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms. Nat. Chem. 12, 145–158 (2020). https://doi.org/10.1038/s41557-019-0378-7

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