Molecular Biology

, Volume 53, Issue 4, pp 596–605 | Cite as

Reserpine Is the New Addition into the Repertoire of AcrB Efflux Pump Inhibitors

  • A. Shaheen
  • W. A. Afridi
  • S. Mahboob
  • M. Sana
  • N. Zeeshan
  • F. Ismat
  • O. Mirza
  • M. Iqbal
  • M. RahmanEmail author


Acriflavine resistance protein B (AcrB) serves as prototype for multidrug resistance (MDR) efflux transporters of resistance nodulation division (RND) superfamily. AcrB has been proven as potential drug target with many synthetic and natural inhibitors have been identified such as those belonging to pyranopyridine, naphthamide and pimozide classes. The plant derived alkaloid inhibitors represented by reserpine has been found to inhibit both ATP binding cassette and major facilitator efflux transporters. In this study we report the reserpine induced inhibition of RND transporter AcrB. The preliminary docking analysis hints that reserpine shares its binding site with ciprofloxacin, a known substrate of AcrB and could possibly act as competitive inhibitor. For in vitro validation, AcrB from Salmonellatyphi was cloned under the control of tac promoter and resulting vector was introduced into E. coli C41(DE3). Under autoinduced conditions, cells overexpressing AcrB transporter were subjected to combined dose of ciprofloxacin and reserpine. The combined exposure resulted in enhanced ciprofloxacin-induced growth inhibition of cells expressing AcrB transporter as compared to control cells transformed with vector of backbone sequence. Time kill analysis further confirmed these findings. To the best of our knowledge, this is first study to show that exposure to reserpine induces inhibition of AcrB. The assay developed in this study allows simple and reproducible detection of substrate/inhibitor effects upon AcrB and related efflux transporters.


acriflavine resistance protein resistance nodulation division transporter reserpine ciprofloxacin multidrug resistance Salmonellatyphi 



This research work was funded by the Higher Education Commission of Pakistan (grant no. 20-1504 to M. Rahman).


The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.


W.A. Afridia and S. Mahbooba are both equally contributed to this work.

Supplementary material

11008_2019_8097_MOESM1_ESM.pdf (804 kb)


  1. 1.
    Seeger M.A., Diederichs K., Eicher T., Brandstatter L., Schiefner A., Verrey F., Pos K.M. 2008. The AcrB efflux pump: Conformational cycling and peristalsis lead to multidrug resistance. Curr. Drug Targets. 9, 729–749.CrossRefGoogle Scholar
  2. 2.
    Seeger M.A., Schiefner A., Eicher T., Verrey F., Diederichs K., Pos K.M. 2006. Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313, 1295–1298.CrossRefGoogle Scholar
  3. 3.
    Shaheen A., Iqbal M., Mirza O., Rahman M. 2017. Structural biology meets drug resistance: An overview on multidrug resistance transporters. J. Indian Inst. Sci. 97, 165–175.CrossRefGoogle Scholar
  4. 4.
    Opperman T.J., Kwasny S.M., Kim H.S., Nguyen S.T., Houseweart C., D’Souza S., Walker G.C., Peet N.P., Nikaido H., Bowlin T.L. 2014. Characterization of a novel pyranopyridine inhibitor of the AcrAB efflux pump of Escherichia coli. Antimicrob. Agents Chemother. 58, 722–733.CrossRefGoogle Scholar
  5. 5.
    Wang Y., Mowla R., Ji S., Guo L., De Barros Lopes M.A., Jin C., Song D., Ma S., Venter H. 2018. Design, synthesis and biological activity evaluation of novel 4-subtituted 2-naphthamide derivatives as AcrB inhibitors. Eur. J. Med. Chem. 143, 699–709.CrossRefGoogle Scholar
  6. 6.
    Wang-Kan X., Blair J.M.A., Chirullo B., Betts J., La Ragione R.M., Ivens A., Ricci V., Opperman T.J., Piddock L.J.V. 2017. Lack of AcrB efflux function confers loss of virulence on Salmonella enterica serovar Typhimurium. MBio. 8 (4). pii: e00968-17. CrossRefGoogle Scholar
  7. 7.
    Vargiu A.V., Nikaido H. 2012. Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations. Proc. Natl. Acad. Sci. U. S. A. 109, 20637–20642.CrossRefGoogle Scholar
  8. 8.
    Takatsuka Y., Chen C., Nikaido H. 2010. Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 107, 6559–6565.CrossRefGoogle Scholar
  9. 9.
    Aparna V., Dineshkumar K., Mohanalakshmi N., Velmurugan D., Hopper W. 2014. Identification of natural compound inhibitors for multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation. PLoS One. 9, e101840.CrossRefGoogle Scholar
  10. 10.
    Pakzad I., Zayyen Karin M., Taherikalani M., Boustanshenas M., Lari A.R. 2013. Contribution of AcrAB efflux pump to ciprofloxacin resistance in Klebsiella pneumoniae isolated from burn patients. GMS Hyg. Infect. Control. 8 (2), Doc15. Google Scholar
  11. 11.
    Das D., Xu Q.S., Lee J.Y., Ankoudinova I., Huang C., Lou Y., DeGiovanni A., Kim R., Kim S.H. 2007. Crystal structure of the multidrug efflux transporter AcrB at 3.1A resolution reveals the N-terminal region with conserved amino acids. J. Struct. Biol. 158, 494–502.CrossRefGoogle Scholar
  12. 12.
    Berman H.M., Westbrook J., Feng Z., Gilliland G., Bhat T.N., Weissig H., Shindyalov I.N., Bourne P.E. 2000. The Protein Data Bank. Nucleic Acids Res. 28, 235–242.CrossRefGoogle Scholar
  13. 13.
    Murakami S., Nakashima R., Yamashita E., Matsumoto T., Yamaguchi A. 2006. Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443, 173–179.CrossRefGoogle Scholar
  14. 14.
    Soparkar K., Kinana A.D., Weeks J.W., Morrison K.D., Nikaido H., Misra R. 2015. Reversal of the drug binding pocket defects of the AcrB multidrug efflux pump protein of Escherichia coli. J. Bacteriol. 197, 3255–3264.CrossRefGoogle Scholar
  15. 15.
    Zeng B., Wang H., Zou L., Zhang A., Yang X., Guan Z. 2010. Evaluation and target validation of indole derivatives as inhibitors of the AcrAB-TolC efflux pump. Biosci. Biotechnol. Biochem. 74, 2237–2241.CrossRefGoogle Scholar
  16. 16.
    Li B., Yao Q., Pan X.C., Wang N., Zhang R., Li J., Ding G., Liu X., Wu C., Ran D., Zheng J., Zhou H. 2011. Artesunate enhances the antibacterial effect of beta-lactam antibiotics against Escherichia coli by increasing antibiotic accumulation via inhibition of the multidrug efflux pump system AcrAB-TolC. J. Antimicrob. Chemother. 66, 769–777.CrossRefGoogle Scholar
  17. 17.
    Vargiu A.V., Ruggerone P., Opperman T.J., Nguyen S.T., Nikaido H. 2014. Molecular mechanism of MBX2319 inhibition of Escherichia coli AcrB multidrug efflux pump and comparison with other inhibitors. Antimicrob. Agents Chemother. 58, 6224–6234.CrossRefGoogle Scholar
  18. 18.
    Bohnert J.A., Schuster S., Kern W.V. 2013. Pimozide inhibits the AcrAB-TolC efflux pump in Escherichia coli. Open Microbiol. J. 7, 83–86.CrossRefGoogle Scholar
  19. 19.
    Hemaiswarya S., Kruthiventi A.K., Doble M. 2008. Synergism between natural products and antibiotics against infectious diseases. Phytomedicine. 15, 639–652.CrossRefGoogle Scholar
  20. 20.
    Stermitz F.R., Tawara-Matsuda J., Lorenz P., Mueller P., Zenewicz L., Lewis K. 2000. 5'-Methoxyhydnocarpin-D and pheophorbide A: Berberis species components that potentiate berberine growth inhibition of resistant Staphylococcus aureus. J. Nat. Prod. 63, 1146–1149.CrossRefGoogle Scholar
  21. 21.
    Stermitz F.R., Beeson T.D., Mueller P.J., Hsiang J., Lewis K. 2001. Staphylococcus aureus MDR efflux pump inhibitors from a Berberis and a Mahonia (sensu strictu) species. Biochem. Syst. Ecol. 29, 793–798.CrossRefGoogle Scholar
  22. 22.
    Castaneda-Gomez J., Figueroa-Gonzalez G., Jacobo N., Pereda-Miranda R. 2013. Purgin II, a resin glycoside ester-type dimer and inhibitor of multidrug efflux pumps from Ipomoea purga. J. Nat. Prod. 76, 64–71.CrossRefGoogle Scholar
  23. 23.
    Shiu W.K., Malkinson J.P., Rahman M.M., Curry J., Stapleton P., Gunaratnam M., Neidle S., Mushtaq S., Warner M., Livermore D.M., Evangelopoulos D., Basavannacharya C., Bhakta S., Schindler B.D., Seo S.M., et al. 2013. A new plant-derived antibacterial is an inhibitor of efflux pumps in Staphylococcus aureus. Int. J. Antimicrob. Agents. 42, 513–518.CrossRefGoogle Scholar
  24. 24.
    Schmitz F.J., Fluit A.C., Luckefahr M., Engler B., Hofmann B., Verhoef J., Heinz H.P., Hadding U., Jones M.E. 1998. The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 42, 807–810.CrossRefGoogle Scholar
  25. 25.
    Holler J.G., Christensen S.B., Slotved H.C., Rasmussen H.B., Guzman A., Olsen C.E., Petersen B., Molgaard P. 2012. Novel inhibitory activity of the Staphylococcus aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees. J. Antimicrob. Chemother. 67, 1138–1144.CrossRefGoogle Scholar
  26. 26.
    Fiamegos Y.C., Kastritis P.L., Exarchou V., Han H., Bonvin A.M., Vervoort J., Lewis K., Hamblin M.R., Tegos G.P. 2011. Antimicrobial and efflux pump inhibitory activity of caffeoylquinic acids from Artemisia absinthium against Gram-positive pathogenic bacteria. PLoS One. 6, e18127.CrossRefGoogle Scholar
  27. 27.
    Neyfakh A.A., Bidnenko V.E., Chen L.B. 1991. Efflux-mediated multidrug resistance in Bacillus subtilis: Similarities and dissimilarities with the mammalian system. Proc. Natl. Acad. Sci. U. S. A. 88, 4781–4785.CrossRefGoogle Scholar
  28. 28.
    Gibbons S., Udo E.E. 2000. The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracycline against clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) possessing the tet(K) determinant. Phytother. Res. 14, 139–140.CrossRefGoogle Scholar
  29. 29.
    Neyfakh A.A., Borsch C.M., Kaatz G.W. 1993. Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter. Antimicrob. Agents Chemother. 37, 128–129.CrossRefGoogle Scholar
  30. 30.
    Klemm E.J., Shakoor S., Page A.J., Qamar F.N., Judge K., Saeed D.K., Wong V.K., Dallman T.J., Nair S., Baker S., Shaheen G., Qureshi S., Yousafzai M.T., Saleem M.K., Hasan Z., et al. 2018. Emergence of an extensively drug-resistant Salmonella enterica serovar Typhi clone harboring a promiscuous plasmid encoding resistance to fluoroquinolones and third-generation cephalosporins. MBio. 9(1), pii: e00105-18. CrossRefGoogle Scholar
  31. 31.
    Kumar S., Stecher G., Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874.CrossRefGoogle Scholar
  32. 32.
    Waterhouse A.M., Procter J.B., Martin D.M., Clamp M., Barton G.J. 2009. Jalview version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics. 25, 1189–1191.CrossRefGoogle Scholar
  33. 33.
    Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. 2004. UCSF chimera: A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612.CrossRefGoogle Scholar
  34. 34.
    Trott O., Olson A.J. 2010. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461.Google Scholar
  35. 35.
    Shaheen A., Ismat F., Iqbal M., Haque A., De Zorzi R., Mirza O., Walz T., Rahman M. 2015. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella typhi. J. Infect. Chemother. 21, 357–362.CrossRefGoogle Scholar
  36. 36.
    Miroux B., Walker J.E. 1996. Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.CrossRefGoogle Scholar
  37. 37.
    Studier F.W. 2005. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234.CrossRefGoogle Scholar
  38. 38.
    Hayashi K., Nakashima R., Sakurai K., Kitagawa K., Yamasaki S., Nishino K., Yamaguchi A. 2016. AcrB-AcrA fusion proteins that act as multidrug efflux transporters. J. Bacteriol. 198, 332–342.CrossRefGoogle Scholar
  39. 39.
    Ward A., Sanderson N.M., O’Reilly J., Rutherford N.G., Poolman B., Henderson P.J.F. 1999. The amplified expression, identification, purification, assay, and properties of hexahistidine-tagged bacterial membrane transport proteins. In: Membrane Transport–A Practical Approach. Oxford: Oxford Univ. Press.Google Scholar
  40. 40.
    Wink M. 2008. Ecological roles of alkaloids. In: Modern Alkaloids: Structure, Isolation, Synthesis, and Biology. Eds. Fattorusso E., Taglialatela-Scafati O. Wiley-VCH, vol. 1.Google Scholar
  41. 41.
    Roberts M.F. 2013. Enzymology of alkaloid biosynthesis. In: Alkaloids: Biochemistry, Ecology, and Medicinal Applications. Ed. Roberts M.F. Springer Science & Business Media.Google Scholar
  42. 42.
    Liu M., Heng J., Gao Y., Wang X. 2016. Crystal structures of MdfA complexed with acetylcholine and inhibitor reserpine. Biophys. Rep. 2, 78–85.CrossRefGoogle Scholar
  43. 43.
    Beyer R., Pestova E., Millichap J.J., Stosor V., Noskin G.A., Peterson L.R. 2000. A convenient assay for estimating the possible involvement of efflux of fluoroquinolones by Streptococcus pneumoniae and Staphylococcus aureus: Evidence for diminished moxifloxacin, sparfloxacin, and trovafloxacin efflux. Antimicrob. Agents Chemother. 44, 798–801.CrossRefGoogle Scholar
  44. 44.
    Abdelfatah S.A., Efferth T. 2015. Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells. Phytomedicine 22, 308–318.CrossRefGoogle Scholar
  45. 45.
    Ahmed M., Borsch C.M., Neyfakh A.A., Schuldiner S. 1993. Mutants of the Bacillus subtilis multidrug transporter Bmr with altered sensitivity to the antihypertensive alkaloid reserpine. J. Biol. Chem. 268, 11086–11089.Google Scholar
  46. 46.
    Lee E.W., Huda M.N., Kuroda T., Mizushima T., Tsuchiya T. 2003. EfrAB, an ABC multidrug efflux pump in Enterococcus faecalis. Antimicrob. Agents Chemother. 47, 3733–3738.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2019

Authors and Affiliations

  • A. Shaheen
    • 1
    • 2
  • W. A. Afridi
    • 1
  • S. Mahboob
    • 1
  • M. Sana
    • 1
  • N. Zeeshan
    • 2
  • F. Ismat
    • 1
  • O. Mirza
    • 3
  • M. Iqbal
    • 1
  • M. Rahman
    • 1
    Email author
  1. 1.Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE)FaisalabadPakistan
  2. 2.Department of Biochemistry and Biotechnology, University of Gujrat, Hafiz Hayat CampusGujratPakistan
  3. 3.Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of CopenhagenCopenhagenDenmark

Personalised recommendations