Novel MscL agonists that allow multiple antibiotics cytoplasmic access activate the channel through a common binding site

Autoři: Robin Wray aff001;  Junmei Wang aff002;  Irene Iscla aff001;  Paul Blount aff001
Působiště autorů: Department of Physiology, UT Southwestern Medical Center, Dallas, Texas, United States of America aff001;  Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center, University of Pittsburgh School of Pharmacy, Pittsburg, Pennsylvania, United States of America aff002
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0228153


The antibiotic resistance crisis is becoming dire, yet in the past several years few potential antibiotics or adjuvants with novel modes of action have been identified. The bacterial mechanosensitive channel of large conductance, MscL, found in the majority of bacterial species, including pathogens, normally functions as an emergency release valve, sensing membrane tension upon low-osmotic stress and discharging cytoplasmic solutes before cell lysis. Opening the huge ~30Å diameter pore of MscL inappropriately is detrimental to the cell, allowing solutes from and even passage of drugs into to cytoplasm. Thus, MscL is a potential novel drug target. However, there are no known natural agonists, and small compounds that modulate MscL activity are just now being identified. Here we describe a small compound, K05, that specifically modulates MscL activity and we compare results with those obtained for the recently characterized MscL agonist 011A. While the structure of K05 only vaguely resembles 011A, many of the findings, including the binding pocket, are similar. On the other hand, both in vivo and molecular dynamic simulations indicate that the two compounds modulate MscL activity in significantly different ways.

Klíčová slova:

Antibiotic resistance – Antibiotics – Biochemical simulations – Cysteine – Free energy – Glutamate – Molecular dynamics – Staphylococcus aureus


1. Ventola CL. The antibiotic resistance crisis: part 2: management strategies and new agents. P T. 2015;40(5):344–52. 25987823.

2. Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. P T. 2015;40(4):277–83. 25859123.

3. Dukes MNG, editor. Side-Effects of Drugs—Annual 4—a Worldwide Yearly Survey of New Data and Trends 1980.

4. Butler MS, Cooper MA. Antibiotics in the clinical pipeline in 2011. The Journal of antibiotics. 2011;64(6):413–25. doi: 10.1038/ja.2011.44 21587262.

5. Piddock L. Antibiotic crisis. New Sci. 2013;217(2910):29-.

6. Booth IR, Blount P. Microbial Emergency Release Valves: the MscS and MscL Families of Mechanosensitive Channels. J Bacteriol. 2012;194(18):4802–9. Epub 2012/06/12.

7. Iscla I, Blount P. Sensing and Responding to Membrane Tension: The Bacterial MscL Channel as a Model System. Biophys J. 2012;103(2):169–74. Epub 2012/08/03. doi: 10.1016/j.bpj.2012.06.021 22853893.

8. Moe P, Blount P. Assessment of potential stimuli for mechano-dependent gating of MscL: effects of pressure, tension, and lipid headgroups. Biochemistry. 2005;44(36):12239–44. Epub 2005/09/07. doi: 10.1021/bi0509649 16142922.

9. Malcolm HR, Heo YY, Elmore DE, Maurer JA. Heteromultimerization of prokaryotic bacterial cyclic nucleotide-gated (bCNG) ion channels, members of the mechanosensitive channel of small conductance (MscS) superfamily. Biochemistry. 2014;53(51):8005–7. doi: 10.1021/bi501118c 25493556.

10. Li Y, Moe PC, Chandrasekaran S, Booth IR, Blount P. Ionic regulation of MscK, a mechanosensitive channel from Escherichia coli. EMBO J. 2002;21(20):5323–30. doi: 10.1093/emboj/cdf537 12374733.

11. Wray R, Herrera N, Iscla I, Wang J, Blount P. An agonist of the MscL channel affects multiple bacterial species and increases membrane permeability and potency of common antibiotics. Mol Microbiol. 2019. doi: 10.1111/mmi.14325 31177589.

12. Cox CD, Bavi N, Martinac B. Bacterial Mechanosensors. Annu Rev Physiol. 2018;80:71–93. doi: 10.1146/annurev-physiol-021317-121351 29195054.

13. Ajouz B, Berrier C, Garrigues A, Besnard M, Ghazi A. Release of thioredoxin via the mechanosensitive channel MscL during osmotic downshock of Escherichia coli cells. J Biol Chem. 1998;273(41):26670–4. doi: 10.1074/jbc.273.41.26670 9756908.

14. Berrier C, Garrigues A, Richarme G, Ghazi A. Elongation factor Tu and DnaK are transferred from the cytoplasm to the periplasm of Escherichia coli during osmotic downshock presumably via the mechanosensitive channel mscL. J Bact. 2000;182(1):248–51. doi: 10.1128/jb.182.1.248-251.2000 10613892

15. Ewis HE, Lu CD. Osmotic shock: a mechanosensitive channel blocker can prevent release of cytoplasmic but not periplasmic proteins. FEMS Microbiol Lett. 2005;253(2):295–301. doi: 10.1016/j.femsle.2005.09.046 16288836.

16. Cruickshank CC, Minchin RF, Le Dain AC, Martinac B. Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli. Biophys J. 1997;73(4):1925–31. doi: 10.1016/S0006-3495(97)78223-7 9336188.

17. Ou X, Blount P, Hoffman RJ, Kung C. One face of a transmembrane helix is crucial in mechanosensitive channel gating. Proc Nat Acad Sci USA. 1998;95(19):11471–5. doi: 10.1073/pnas.95.19.11471 9736761.

18. Maurer JA, Dougherty DA. A high-throughput screen for MscL channel activity and mutational phenotyping. Biochim Biophys Acta. 2001;1514(2):165–9. doi: 10.1016/s0005-2736(01)00390-x 11557017

19. Iscla I, Wray R, Wei S, Posner B, Blount P. Streptomycin potency is dependent on MscL channel expression. Nature communications. 2014;5:4891. doi: 10.1038/ncomms5891 25205267.

20. Wray R, Iscla I, Gao Y, Li H, Wang J, Blount P. Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore. PLoS Biol. 2016;14(6):e1002473. doi: 10.1371/journal.pbio.1002473 27280286.

21. Dubin DT, Hancock R, Davis BD. The Sequence of Some Effects of Streptomycin in Escherichia Coli. Biochim Biophys Acta. 1963;74:476–89. doi: 10.1016/0006-3002(63)91390-8 14071591.

22. Wray R, Iscla I, Kovacs Z, Wang J, Blount P. Novel compounds that specifically bind and modulate MscL: insights into channel gating mechanisms. FASEB J. 2019;33(3):3180–9. doi: 10.1096/fj.201801628R 30359098.

23. Stokes NR, Murray HD, Subramaniam C, Gourse RL, Louis P, Bartlett W, et al. A role for mechanosensitive channels in survival of stationary phase: Regulation of channel expression by RpoS. Proc Nat Acad Sci USA. 2003;100(26):15959–64. doi: 10.1073/pnas.2536607100 14671322

24. Blount P, Sukharev SI, Moe PC, Martinac B, Kung C. Mechanosensitive channels of bacteria. In: Conn PM, editor. Meth Enzymol. Ion Channels, Part C. 294. San Diego, CA: Academic Press; 1999. p. 458–82.

25. Kouwen TR, Trip EN, Denham EL, Sibbald MJ, Dubois JY, van Dijl JM. The large mechanosensitive channel MscL determines bacterial susceptibility to the bacteriocin sublancin 168. Antimicrob Agents Chemother. 2009;53(11):4702–11. Epub 2009/09/10. doi: 10.1128/AAC.00439-09 19738010.

26. Bartlett JL, Levin G, Blount P. An in vivo assay identifies changes in residue accessibility on mechanosensitive channel gating. Proc Natl Acad Sci U S A. 2004;101:10161–5. doi: 10.1073/pnas.0402040101 15226501.

27. Bartlett JL, Li Y, Blount P. Mechanosensitive channel gating transitions resolved by functional changes upon pore modification. Biophys J. 2006;91(10):3684–91. doi: 10.1529/biophysj.106.088062 16935962.

28. Iscla I, Levin G, Wray R, Blount P. Disulfide Trapping the Mechanosensitive Channel MscL into a Gating-Transition State. Biophys J. 2007;92(4):1224–32. doi: 10.1529/biophysj.106.090316 17114217.

29. Iscla I, Wray R, Blount P. The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface. Channels (Austin, Tex). 2012;6(4):255–61. doi: 10.4161/chan.20756 22874845.

30. Iscla I, Wray R, Eaton C, Blount P. Scanning MscL Channels with Targeted Post-Translational Modifications for Functional Alterations. PloS one. 2015;10(9):e0137994. Epub 2015/09/15. doi: 10.1371/journal.pone.0137994 26368283.

31. Levin G, Blount P. Cysteine scanning of MscL transmembrane domains reveals residues critical for mechanosensitive channel gating. Biophys J. 2004;86(5):2862–70. doi: 10.1016/S0006-3495(04)74338-6 15111403.

32. Wang E, Sun H, Wang J, Wang Z, Liu H, Zhang JZH, et al. End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design. Chem Rev. 2019;119(16):9478–508. Epub 2019/06/28. doi: 10.1021/acs.chemrev.9b00055 31244000.

33. Hou T, Wang J, Li Y, Wang W. Assessing the performance of the MM/PBSA and MM/GBSA methods. 1. The accuracy of binding free energy calculations based on molecular dynamics simulations. J Chem Inf Model. 2011;51(1):69–82. Epub 2010/12/02. doi: 10.1021/ci100275a 21117705.

34. Hou T, Wang J, Li Y, Wang W. Assessing the performance of the molecular mechanics/Poisson Boltzmann surface area and molecular mechanics/generalized Born surface area methods. II. The accuracy of ranking poses generated from docking. J Comput Chem. 2011;32(5):866–77. Epub 2010/10/16. doi: 10.1002/jcc.21666 20949517.

35. Wang J, Hou T, Xu X. Recent Advances in Free Energy Calculations with a Combination of Molecular Mechanics and Continuum Models. Current Computer Aided-Drug Design. 2006;2(3):287–306. doi: 10.2174/157340906778226454

36. Wang J, Morin P, Wang W, Kollman PA. Use of MM-PBSA in Reproducing the Binding Free Energies to HIV-1 RT of TIBO Derivatives and Predicting the Binding Mode to HIV-1 RT of Efavirenz by Docking and MM-PBSA. Journal of the American Chemical Society. 2001;123(22):5221–30. doi: 10.1021/ja003834q 11457384

37. Lee TS, Hu Y, Sherborne B, Guo Z, York DM. Toward Fast and Accurate Binding Affinity Prediction with pmemdGTI: An Efficient Implementation of GPU-Accelerated Thermodynamic Integration. J Chem Theory Comput. 2017;13(7):3077–84. Epub 2017/06/16. doi: 10.1021/acs.jctc.7b00102 28618232.

38. Wang L, Wu Y, Deng Y, Kim B, Pierce L, Krilov G, et al. Accurate and reliable prediction of relative ligand binding potency in prospective drug discovery by way of a modern free-energy calculation protocol and force field. J Am Chem Soc. 2015;137(7):2695–703. Epub 2015/01/28. doi: 10.1021/ja512751q 25625324.

39. Ge X, Roux B. Absolute binding free energy calculations of sparsomycin analogs to the bacterial ribosome. J Phys Chem B. 2010;114(29):9525–39. Epub 2010/07/09. doi: 10.1021/jp100579y 20608691.

40. Woo HJ, Roux B. Calculation of absolute protein-ligand binding free energy from computer simulations. Proc Natl Acad Sci U S A. 2005;102(19):6825–30. Epub 2005/05/04. doi: 10.1073/pnas.0409005102 15867154.

41. Singh N, Warshel A. Absolute binding free energy calculations: on the accuracy of computational scoring of protein-ligand interactions. Proteins. 2010;78(7):1705–23. Epub 2010/02/27. 20186976.

42. Wang J, Hou T, Xu X. Recent Advances in Free Energy Calculations with a Combination of Molecular Mechanics and Continuum Models. Current Computer-Aided Drug Design. 2006;2(3):287–306. doi: 10.2174/157340906778226454

43. Wang J, Morin P, Wang W, Kollman PA. Use of MM-PBSA in reproducing the binding free energies to HIV-1 RT of TIBO derivatives and predicting the binding mode to HIV-1 RT of efavirenz by docking and MM-PBSA. J Am Chem Soc. 2001;123(22):5221–30. Epub 2001/07/18. doi: 10.1021/ja003834q 11457384.

44. Taylor RK, Hall MN, Silhavy TJ. Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine. J Mol Biol. 1983;166(3):273–82. Epub 1983/05/25. doi: 10.1016/s0022-2836(83)80085-0 6304323.

45. Rampersaud A, Inouye M. Procaine, a local anesthetic, signals through the EnvZ receptor to change the DNA binding affinity of the transcriptional activator protein OmpR. J Bacteriol. 1991;173(21):6882–8. Epub 1991/11/01. doi: 10.1128/jb.173.21.6882-6888.1991 1718943.

46. Martinac B, Adler J, Kung C. Mechanosensitive ion channels of E. coli activated by amphipaths. Nature. 1990;348(6298):261–3. doi: 10.1038/348261a0 1700306.

47. Perozo E, Kloda A, Cortes DM, Martinac B. Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating. Nature Struct Biol. 2002;9(9):696–703. doi: 10.1038/nsb827 12172537.

48. Blount P, Li Y, Moe PC, Iscla I. Mechanosensitive channels gated by membrane tension: Bacteria and beyond. In: Kamkin A, Kiseleva I, editors. Mechanosensitive ion channels (a volume in the Mechanosensitivity in Cells and Tissues, Moscow Academia series). New York: Springer Press; 2008. p. 71–101.

49. Iscla I, Wray R, Blount P, Larkins-Ford J, Conery AL, Ausubel FM, et al. A new antibiotic with potent activity targets MscL. The Journal of antibiotics. 2015. doi: 10.1038/ja.2015.4 25649856.

50. Rao S, Prestidge CA, Miesel L, Sweeney D, Shinabarger DL, Boulos RA. Preclinical development of Ramizol, an antibiotic belonging to a new class, for the treatment of Clostridium difficile colitis. The Journal of antibiotics. 2016;69(12):879–84. Epub 2016/05/18. doi: 10.1038/ja.2016.45 27189122.

51. Iscla I, Wray R, Blount P. On the structure of the N-terminal domain of the MscL channel: helical bundle or membrane interface. Biophys J. 2008;95(5):2283–91. Epub 2008/06/03. doi: 10.1529/biophysj.107.127423 18515388.

52. Iscla I, Wray R, Blount P. An in vivo screen reveals protein-lipid interactions crucial for gating a mechanosensitive channel. FASEB J. 2011;25(2):694–702. Epub 2010/11/12. doi: 10.1096/fj.10-170878 21068398.

53. Diamond JM, Wright EM. Molecular forces governing non-electrolyte permeation through cell membranes. Proc R Soc Lond B Biol Sci. 1969;171(1028):273–316. Epub 1969/03/18. doi: 10.1098/rspb.1969.0022 4388931.

54. Baitinger WF, Schleyer PvR, Murty TSSR, Robinson L. Nitro groups as proton acceptors in hydrogen bonding. Tetrahedron. 1964;20(7):1635–47.

55. Nagakura S, Gouterman M. Effect of Hydrogen Bonding on the Near Ultraviolet Absorption of Naphthol. The Journal of Chemical Physics. 1957;26(4):881–6. doi: 10.1063/1.1743428

56. Nepali K, Lee HY, Liou JP. Nitro-Group-Containing Drugs. J Med Chem. 2019;62(6):2851–93. Epub 2018/10/09. doi: 10.1021/acs.jmedchem.8b00147 30295477.

57. Levina N, Totemeyer S, Stokes NR, Louis P, Jones MA, Booth IR. Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 1999;18(7):1730–7. doi: 10.1093/emboj/18.7.1730 10202137

58. Schumann U, Edwards MD, Rasmussen T, Bartlett W, van West P, Booth IR. YbdG in Escherichia coli is a threshold-setting mechanosensitive channel with MscM activity. Proc Natl Acad Sci U S A. 2010;107(28):12664–9. doi: 10.1073/pnas.1001405107 20616037.

59. Batiza AF, Kuo MM, Yoshimura K, Kung C. Gating the bacterial mechanosensitive channel MscL in vivo. Proc Nat Acad Sci USA. 2002;99(8):5643–8. doi: 10.1073/pnas.082092599 11960017

60. Sybyl-X. Molecular Modeling Software Package Version 2.1.1 Tripos International, 1699 South Hanley Rd., St. Louis, Missouri, 63144, USA2013.

61. Glide. version 6.8. Schrodinger, Inc New York. 2015.

62. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of Simple Potential Functions for Simulating Liquid Water. Journal of Chemical Physics. 1983;79(2):926–35. doi: 10.1063/1.445869

63. Bayly CI, Cieplak P, Cornell WD, Kollman PA. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges- the RESP model. Journal Of Physical Chemistry. 1993;97(40):10269–80.

64. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian 16 Rev. B.01. Wallingford, CT2016.

65. Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of a general amber force field. J Comput Chem. 2004;25(9):1157–74. Epub 2004/04/30. doi: 10.1002/jcc.20035 15116359.

66. Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J Chem Theory Comput. 2015;11(8):3696–713. Epub 2015/11/18. doi: 10.1021/acs.jctc.5b00255 26574453.

67. Dickson CJ, Madej BD, Skjevik AA, Betz RM, Teigen K, Gould IR, et al. Lipid14: The Amber Lipid Force Field. J Chem Theory Comput. 2014;10(2):865–79. doi: 10.1021/ct4010307 24803855

68. Darden T, Perera L, Li L, Pedersen L. New tricks for modelers from the crystallography toolkit: the particle mesh Ewald algorithm and its use in nucleic acid simulations. Structure. 1999;7(3):R55–60. Epub 1999/06/16. doi: 10.1016/s0969-2126(99)80033-1 10368306.

69. Wang J, Hou T. Develop and test a solvent accessible surface area-based model in conformational entropy calculations. J Chem Inf Model. 2012;52(5):1199–212. doi: 10.1021/ci300064d 22497310.

70. Hawkins GD, Cramer CJ, Truhlar DG. Parametrized Models of Aqueous Free Energies of Solvation Based on Pairwise Descreening of Solute Atomic Charges from a Dielectric Medium. The Journal of Physical Chemistry. 1996;100(51):19824–39. doi: 10.1021/jp961710n

71. Case DA, Berryman JT, Betz RM, Cerutti DS, Cheatham I, T.E., Darden TA, et al. AMBER 14. University of California, San Francisco. 2015.

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