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Thiouracil SecA inhibitors: bypassing the effects of efflux pumps and attenuating virulence factor secretion in MRSA and Bacillus anthracis

Abstract

We have previously reported a series of thiouracil analogs as bacterial SecA inhibitors. In this study, one potent thiouracil analog, SCA-15, was further evaluated for its inhibitory effects on SecA proteins and against strains of methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus anthracis Sterne. SCA-15 inhibited the ion-channel activities of SecA1 with IC50 of 2.0–2.8 μM and the ATPase activities of SecA2 with IC50 of 13–20 μM, respectively. Drug affinity responsive target stability (DARTS) and protein pull-down experiments further supported SaSecA1 and SaSecA2 as the targets of the inhibitor. SCA-15 showed promising bactericidal effects against MRSA and B. anthracis Sterne. In addition, SCA-15 could at least partially overcome the effects of efflux pumps responsible for multidrug resistance. Moreover, SCA-15 also inhibited the secretion of several important toxins of both S. aureus and B. anthracis. These results indicated that targeting SecA is a promising antimicrobial strategy against MRSA and B. anthracis.

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Fig. 1
Fig. 2: Altered trypsin sensitivity by SCA-15.
Fig. 3: Target pull-down from S. aureus lysate by SCA-105.
Fig. 4: Target pull-down from S. aureus by SCA-104.
Fig. 5: Bactericidal effects of SCA-15 against S. aureus and B. anthracis.
Fig. 6: Bactericidal effects of SCA-15 against S. aureus efflux strains.
Fig. 7: Inhibition of the secretion of S. aureus toxins.
Fig. 8: Inhibition of the secretion of B. anthracis toxins.

References

  1. Driessen AJ, Nouwen N. Protein translocation across the bacterial cytoplasmic membrane. Annu Rev Biochem. 2008;77:643–67.

    CAS  Article  Google Scholar 

  2. Wang HW, Chen Y, Yang H, Chen X, Duan MX, Tai PC, et al. Ring-like pore structures of SecA: implication for bacterial protein-conducting channels. Proc Natl Acad Sci USA. 2003;100:4221–6.

    CAS  Article  Google Scholar 

  3. Hsieh YH, Zhang H, Lin BR, Cui N, Na B, Yang H, et al. SecA alone can promote protein translocation and ion channel activity: SecYEG increases efficiency and signal peptide specificity. J Biol Chem. 2011;286:44702–9.

    CAS  Article  Google Scholar 

  4. Siboo IR, Chaffin DO, Rubens CE, Sullam PM. Characterization of the accessory Sec system of Staphylococcus aureus. J Bacteriol. 2008;190:6188–96.

    CAS  Article  Google Scholar 

  5. Lenz LL, Mohammadi S, Geissler A, Portnoy DA. SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc Natl Acad Sci USA 2003;100:12432–7.

    CAS  Article  Google Scholar 

  6. Jin J, Hsieh YH, Chaudhary AS, Cui J, Houghton JE, Sui SF. et al. SecA inhibitors as potential antimicrobial agents: differential actions on SecA-only and SecA-SecYEG protein-conducting channels. FEMS Microbiol Lett. 2018;365:fny145

    CAS  Article  Google Scholar 

  7. Chen W, Huang YJ, Gundala SR, Yang H, Li M, Tai PC, et al. The first low microM SecA inhibitors. Bioorg Med Chem. 2010;18:1617–25.

    CAS  Article  Google Scholar 

  8. Li M, Huang YJ, Tai PC, Wang B. Discovery of the first SecA inhibitors using structure-based virtual screening. Biochemical biophysical Res Commun. 2008;368:839–45.

    CAS  Article  Google Scholar 

  9. Chaudhary AS, Jin J, Chen W, Tai PC, Wang B. Design, syntheses and evaluation of 4-oxo-5-cyano thiouracils as SecA inhibitors. Bioorg Med Chem. 2015;23:105–17.

    CAS  Article  Google Scholar 

  10. Nguyen-Mau SM, Oh SY, Kern VJ, Missiakas DM, Schneewind O. Secretion genes as determinants of Bacillus anthracis chain length. J Bacteriol. 2012;194:3841–50.

    CAS  Article  Google Scholar 

  11. Lin BR, Gierasch LM, Jiang C, Tai PC. Electrophysiological studies in Xenopus oocytes for the opening of Escherichia coli SecA-dependent protein-conducting channels. J Membr Biol. 2006;214:103–13.

    CAS  Article  Google Scholar 

  12. Hsieh YH, Zou J, Jin JS, Yang H, Chen Y, Jiang C, et al. Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes. Anal Biochem. 2015;480:58–66.

    CAS  Article  Google Scholar 

  13. Jin J, Hsieh YH, Cui J, Damera K, Dai C, Chaudhary AS, et al. Using chemical probes to assess the feasibility of targeting SecA for developing antimicrobial agents against gram-negative bacteria. ChemMedChem. 2016;11:2511–21.

    CAS  Article  Google Scholar 

  14. Lomenick B, Olsen RW, Huang J. Identification of direct protein targets of small molecules. ACS Chem Biol. 2011;6:34–46.

    CAS  Article  Google Scholar 

  15. Sato S, Murata A, Shirakawa T, Uesugi M. Biochemical target isolation for novices: affinity-based strategies. Chem Biol. 2010;17:616–23.

    CAS  Article  Google Scholar 

  16. Lomenick B, Hao R, Jonai N, Chin RM, Aghajan M, Warburton S, et al. Target identification using drug affinity responsive target stability (DARTS). Proc Natl Acad Sci USA 2009;106:21984–9.

    CAS  Article  Google Scholar 

  17. Eichler J, Rinard K, Wickner W. Endogenous SecA catalyzes preprotein translocation at SecYEG. J Biol Chem. 1998;273:21675–81.

    CAS  Article  Google Scholar 

  18. van der Wolk JP, de Wit JG, Driessen AJ. The catalytic cycle of the escherichia coli SecA ATPase comprises two distinct preprotein translocation events. EMBO J 1997;16:7297–304.

    Article  Google Scholar 

  19. Segers K, Anne J. Traffic jam at the bacterial sec translocase: targeting the SecA nanomotor by small-molecule inhibitors. Chem Biol. 2011;18:685–98.

    CAS  Article  Google Scholar 

  20. Lamut A, Peterlin Mašič L, Kikelj D, Tomašič T. Efflux pump inhibitors of clinically relevant multidrug resistant bacteria. Med Res Rev. 2019;39:2460–504.

    CAS  Article  Google Scholar 

  21. Schindler BD, Kaatz GW. Multidrug efflux pumps of Gram-positive bacteria. Drug Resist Updat. 2016;27:1–13.

    CAS  Article  Google Scholar 

  22. Chen X, Xu H, Tai PC. A significant fraction of functional SecA is permanently embedded in the membrane. SecA cycling on and off the membrane is not essential during protein translocation. J Biol Chem. 1996;271:29698–706.

    CAS  Article  Google Scholar 

  23. Chen X, Brown T, Tai PC. Identification and characterization of protease-resistant SecA fragments: secA has two membrane-integral forms. J Bacteriol. 1998;180:527–37.

    CAS  Article  Google Scholar 

  24. Zhang Y, Eric Ballard C, Zheng SL, Gao X, Ko KC, Yang H, et al. Design, synthesis, and evaluation of efflux substrate-metal chelator conjugates as potential antimicrobial agents. Bioorg Med Chem Lett. 2007;17:707–11.

    CAS  Article  Google Scholar 

  25. Nikaido H, Zgurskaya HI. Antibiotic efflux mechanisms. Curr Opin Infect Dis. 1999;12:529–36.

    CAS  Article  Google Scholar 

  26. Van Bambeke F, Balzi E, Tulkens PM. Antibiotic efflux pumps. Biochem Pharm. 2000;60:457–70.

    Article  Google Scholar 

  27. Markham PN, Neyfakh AA. Efflux-mediated drug resistance in Gram-positive bacteria. Curr Opin Microbiol. 2001;4:509–14.

    CAS  Article  Google Scholar 

  28. Levy SB. Active efflux, a common mechanism for biocide and antibiotic resistance. J Appl Microbiol. 2002;92:65S–71S.

    Article  Google Scholar 

  29. Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H, Kobayashi I, et al. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 2001;357:1225–40.

    CAS  Article  Google Scholar 

  30. Kaatz GW, McAleese F, Seo SM. Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein. Antimicrob Agents Chemother. 2005;49:1857–64.

    CAS  Article  Google Scholar 

  31. Chitlaru T, Gat O, Gozlan Y, Ariel N, Shafferman A. Differential proteomic analysis of the Bacillus anthracis secretome: distinct plasmid and chromosome CO2-dependent cross talk mechanisms modulate extracellular proteolytic activities. J Bacteriol. 2006;188:3551–71.

    CAS  Article  Google Scholar 

  32. Sibbald MJ, Ziebandt AK, Engelmann S, Hecker M, de Jong A, Harmsen HJ, et al. Mapping the pathways to staphylococcal pathogenesis by comparative secretomics. Microbiol Mol Biol Rev. 2006;70:755–88.

    CAS  Article  Google Scholar 

  33. Walz A, Mujer CV, Connolly JP, Alefantis T, Chafin R, Dake C, et al. Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins. Proteome Sci. 2007;5:11.

    Article  Google Scholar 

  34. Price CT, Kaatz GW, Gustafson JE. The multidrug efflux pump NorA is not required for salicylate-induced reduction in drug accumulation by Staphylococcus aureus. Int J Antimicrob Agents. 2002;20:206–13.

    CAS  Article  Google Scholar 

  35. Kaatz GW, Moudgal VV, Seo SM. Identification and characterization of a novel efflux-related multidrug resistance phenotype in Staphylococcus aureus. J Antimicrob Chemother. 2002;50:833–8.

    CAS  Article  Google Scholar 

  36. Cui J, Jin J, Hsieh YH, Yang H, Ke B, Damera K, et al. Design, synthesis and biological evaluation of rose bengal analogues as SecA inhibitors. ChemMedChem. 2013;8:1384–93.

    CAS  Article  Google Scholar 

  37. Jin J, Cui J, Chaudhary AS, Hsieh YH, Damera K, Zhang H, et al. Evaluation of small molecule SecA inhibitors against methicillin-resistant Staphylococcus aureus. Bioorg Med Chem. 2015;23:7061–8.

    CAS  Article  Google Scholar 

  38. Cui J, Jin J, Hsieh Y-hH, Yang H, Ke B, Tai PC et al. Design, synthesis and biological evaluation of rose bengal analogues as SecA inhibitors. ChemMedChem. 2013;8:1384–93.

  39. Cui J, Jin J, Chaudhary AS, Hsieh YH, Zhang H, Dai C, et al. Design, synthesis and evaluation of triazole-pyrimidine analogues as SecA inhibitors. ChemMedChem. 2016;11:43–56.

    CAS  Article  Google Scholar 

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Acknowledgements

J.J., A.C., and H.Y. were recipients of fellowships of Molecular Basis of Diseases Program at Georgia State University. We acknowledge the financial support to P.C.T. and B.W. (AI104168) by the National Institutes of Health. We thank Drs. C.D. Lu and George Pierce of Georgia State University and Dr. G.W. Kaatz from Wayne State University School of Medicine for providing strains. F.B. was a visiting scholar at GSU when conducting the lab research with the partial financial support from the Islamic Development Bank (IDB) under a merit scholarship program.

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Correspondence to Binghe Wang or Phang C. Tai.

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Jin, J., Chaudhary, A., Hsieh, YH. et al. Thiouracil SecA inhibitors: bypassing the effects of efflux pumps and attenuating virulence factor secretion in MRSA and Bacillus anthracis. Med Chem Res 30, 1341–1347 (2021). https://doi.org/10.1007/s00044-021-02750-5

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Keywords

  • SecA inhibitor
  • Antimicrobial
  • MRSA
  • B. anthracis
  • Toxin secretion
  • Efflux pump