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.
Subscribe to Journal
Get full journal access for 1 year
only $14.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Cassini, A. et al. Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis. Lancet Infect. Dis. 19, 56–66 (2019).
Tacconelli, E. et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 18, 318–327 (2018).
Tong, S. Y. C., Davis, J. S., Eichenberger, E., Holland, T. L. & Fowler, V. G. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin. Microbiol. Rev. 28, 603–661 (2015).
Harms, A., Maisonneuve, E. & Gerdes, K. Mechanisms of bacterial persistence during stress and antibiotic exposure. Science 354, aaf4268 (2016).
Ling, L. L. et al. A new antibiotic kills pathogens without detectable resistance. Nature 517, 455–459 (2015).
Sass, P. et al. Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ. Proc. Natl Acad. Sci. USA 108, 17474–17479 (2011).
Smith, P. A. et al. Optimized arylomycins are a new class of Gram-negative antibiotics. Nature 561, 189–194 (2018).
Kurosu, M. & Begari, E. Bacterial protein kinase inhibitors. Drug Dev. Res 71, 168–187 (2010).
Miller, J. R. et al. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore. Proc. Natl Acad. Sci. USA 106, 1737–1742 (2009).
Chang, H.-C. et al. In vitro and in vivo activity of a novel sorafenib derivative SC5005 against MRSA. J. Antimicrob. Chemother. 71, 449–459 (2016).
Roberts, J. L. et al. GRP78/DNA K is a target for Nexavar/Stivarga/Votrient in the treatment of human malignancies, viral infections and bacterial diseases. J. Cell. Physiol. 230, 2552–2578 (2015).
Pujol, E. et al. Pentafluorosulfanyl-containing triclocarban analogues with potent antimicrobial activity. Molecules 23, 2853 (2018).
Walsh, S. E. et al. Activity and mechanisms of action of selected biocidal agents on Gram-positive and -negative bacteria. J. Appl. Microbiol. 94, 240–247 (2003).
Conlon, B. P. et al. Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503, 365–370 (2013).
Springer, M. T., Singh, V. K., Cheung, A. L., Donegan, N. P. & Chamberlain, N. R. Effect of clpP and clpC deletion on persister cell number in Staphylococcus aureus. J. Med. Microbiol. 65, 848–857 (2016).
Waters, E. M., Rowe, S. E., O’Gara, J. P. & Conlon, B. P. Convergence of Staphylococcus aureus persister and biofilm research: can biofilms be defined as communities of adherent persister cells? PLoS Pathog. 12, e1006012 (2016).
Hamamoto, H. et al. Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane. Nat. Chem. Biol. 11, 127–133 (2015).
Evans, M. J. & Cravatt, B. F. Mechanism-based profiling of enzyme families. Chem. Rev. 106, 3279–3301 (2006).
Fonović, M. & Bogyo, M. Activity-based probes as a tool for functional proteomic analysis of proteases. Expert Rev. Proteomics 5, 721–730 (2008).
Kleiner, P., Heydenreuter, W., Stahl, M., Korotkov, V. S. & Sieber, S. A. A whole proteome inventory of background photocrosslinker binding. Angew. Chem. Int. Ed. 56, 1396–1401 (2017).
Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A. J. R. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494 (2009).
Rao, C. V. S., Waelheyns, E. D., Economou, A. & Anné, J. Antibiotic targeting of the bacterial secretory pathway. Biochim. Biophys. Acta 1843, 1762–1783 (2014).
Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513–2526 (2014).
Boersch, M., Rudrawar, S., Grant, G. & Zunk, M. Menaquinone biosynthesis inhibition: a review of advancements toward a new antibiotic mechanism. RSC Adv. 8, 5099–5105 (2018).
Kurosu, M. & Begari, E. Vitamin K2 in electron transport system: are enzymes involved in vitamin K2 biosynthesis promising drug targets? Molecules 15, 1531–1553 (2010).
Fey, P. D. et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. mBio 4, e00537 (2013).
Craney, A., Dix, M. M., Adhikary, R., Cravatt, B. F. & Romesberg, F. E. An alternative terminal step of the general secretory pathway in Staphylococcus aureus. mBio 6, e01178 (2015).
Benkovic, S. J. et al. Identification of borinic esters as inhibitors of bacterial cell growth and bacterial methyltransferases, CcrM and MenH. J. Med. Chem. 48, 7468–7476 (2005).
Rao, C. V. S. et al. Enzymatic investigation of the Staphylococcus aureus type I signal peptidase SpsB—implications for the search for novel antibiotics. FEBS J. 276, 3222–3234 (2009).
Therien, A. G. et al. Broadening the spectrum of β-lactam antibiotics through inhibition of signal peptidase type I. Antimicrob. Agents Chemother. 56, 4662–4670 (2012).
Antes, I. DynaDock: a new molecular dynamics-based algorithm for protein–peptide docking including receptor flexibility. Proteins 78, 1084–1104 (2010).
Craney, A. & Romesberg, F. E. The inhibition of type I bacterial signal peptidase: biological consequences and therapeutic potential. Bioorg. Med. Chem. Lett. 25, 4761–4766 (2015).
Smith, P. A. & Romesberg, F. E. Mechanism of action of the arylomycin antibiotics and effects of signal peptidase I inhibition. Antimicrob. Agents Chemother. 56, 5054–5060 (2012).
Walsh, S. I., Craney, A. & Romesberg, F. E. Not just an antibiotic target: exploring the role of type I signal peptidase in bacterial virulence. Bioorg. Med. Chem. 24, 6370–6378 (2016).
Schallenberger, M. A., Niessen, S., Shao, C., Fowler, B. J. & Romesberg, F. E. Type I signal peptidase and protein secretion in Staphylococcus aureus. J. Bacteriol. 194, 2677–2686 (2012).
Chao, M. C. et al. Protein complexes and proteolytic activation of the cell wall hydrolase RipA regulate septal resolution in mycobacteria. PLoS Pathog. 9, e1003197 (2013).
Frankel, M. B., Hendrickx, A. P. A., Missiakas, D. M. & Schneewind, O. LytN, a murein hydrolase in the cross-wall compartment of Staphylococcus aureus, is involved in proper bacterial growth and envelope assembly. J. Biol. Chem. 286, 32593–32605 (2011).
Pinho, M. G., Kjos, M. & Veening, J.-W. How to get (a)round: mechanisms controlling growth and division of coccoid bacteria. Nat. Rev. Microbiol. 11, 601–614 (2013).
Makhlin, J. et al. Staphylococcus aureus ArcR controls expression of the arginine deiminase operon. J. Bacteriol. 189, 5976–5986 (2007).
Ernst, C. M. & Peschel, A. Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids. Mol. Microbiol 80, 290–299 (2011).
Jones, T. et al. Failures in clinical treatment of Staphylococcus aureus infection with daptomycin are associated with alterations in surface charge, membrane phospholipid asymmetry, and drug binding. Antimicrob. Agents Chemother. 52, 269–278 (2008).
Roy, H. Tuning the properties of the bacterial membrane with aminoacylated phosphatidylglycerol. IUBMB Life 61, 940–953 (2009).
Médard, G. et al. Optimized chemical proteomics assay for kinase inhibitor profiling. J. Proteome Res. 14, 1574–1586 (2015).
Fish, D. N. & Chow, A. T. The clinical pharmacokinetics of levofloxacin. Clin. Pharmacokinet. 32, 101–119 (1997).
Scaglione, F., Mouton, J. W., Mattina, R. & Fraschini, F. Pharmacodynamics of levofloxacin and ciprofloxacin in a murine pneumonia model: peak concentration/MIC versus area under the curve/MIC ratios. Antimicrob. Agents Chemother. 47, 2749–2755 (2003).
Nosengo, N. Can you teach old drugs new tricks? Nature 534, 314–316 (2016).
Xu, H. H. et al. Staphylococcus aureus TargetArray: comprehensive differential essential gene expression as a mechanistic tool to profile antibacterials. Antimicrob. Agents Chemother. 54, 3659–3670 (2010).
Wan, P. T. C. et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855–867 (2004).
Wu, P., Nielsen, T. E. & Clausen, M. H. FDA-approved small-molecule kinase inhibitors. Trends Pharmacol. Sci. 36, 422–439 (2015).
Sukheja, P. et al. A novel small-molecule inhibitor of the Mycobacterium tuberculosis demethylmenaquinone methyltransferase MenG is bactericidal to both growing and nutritionally deprived persister cells. mBio 8, e02022 (2017).
Paetzel, M., Dalbey, R. E. & Strynadka, N. C. J. Crystal structure of a bacterial signal peptidase in complex with a β-lactam inhibitor. Nature 396, 186–190 (1998).
Dreisbach, A., van Dijl, J. M. & Buist, G. The cell surface proteome of Staphylococcus aureus. Proteomics 11, 3154–3168 (2011).
Gatlin, C. L. et al. Proteomic profiling of cell envelope-associated proteins from Staphylococcus aureus. Proteomics 6, 1530–1549 (2006).
Hempel, K. et al. Quantitative cell surface proteome profiling for SigB-dependent protein expression in the human pathogen Staphylococcus aureus via biotinylation approach. J. Proteome Res. 9, 1579–1590 (2010).
Eirich, J. et al. Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy. Mol. BioSyst. 8, 2067–2075 (2012).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
Vizcaíno, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, 11033–11033 (2016).
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).
Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279–D285 (2016).
Nielsen, H. Predicting secretory proteins with SignalP. Methods Mol. Biol. 1611, 59–73 (2017).
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.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Kinase inhibitor and SFN analogues libraries.
Target identification related proteomic data.
Proteome, secretome, surfaceome related proteomic data.
Genome sequencing of SFN-resistant isolates.
Kinobead pull-down related proteomic data.
About this article
Cite this article
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