Preferences help
enabled [disable] Abstract
Number of results
2017 | 64 | 1 | 49-63
Article title

Design of small molecule inhibitors of type III secretion system ATPase EscN from enteropathogenic Escherichia coli

Title variants
Languages of publication
Enteropathogenic E. coli (EPEC) is a human pathogen using type III secretion system for delivery of proteins directly into the human host. The system contains a single ATPase, EscN, which is essential for uncoupling of proteins from their complexes with chaperones before the delivery. The structure of EscN ATPase (PDB code: 2obm) was used to screen computationally for small molecule inhibitors blocking its active site. Two lead candidates were examined but only one, Compound 54, was selected for further optimization. After extended QSAR optimization, two derivatives were found to be competitive inhibitors of EscN capable of blocking ATPase activity with a Ki below 50 µM. One candidate, WEN05-03, with a Ki=16±2 µM, was also minimally toxic to mammalian cells as determined by other assays. In the cell infection model of HeLa cells with EPEC, Compound WEN05-03 completely blocked actin cluster formation at 100 µM concentration, when analyzed by confocal microscopy. The second best inhibitor of EscN ATPase activity was WEN04-34 with a Ki=46±2 µM. However, the compound was highly toxic to the BALB/3T3 cell line. In summary, the work identifies a compound blocking bacterial ATPase in its active site without causing cellular toxicity to the host cells. It is the first report showing feasibility of using bacterial virulence system ATPase as a target for safe, non-toxic compounds and offering a proof-of-concept for non-antibiotic alternatives.
Physical description
  • Department of Biotechnology, Wroclaw Research Centre EIT+ Ltd., Wrocław, Poland
  • Department of Biotechnology, Wroclaw Research Centre EIT+ Ltd., Wrocław, Poland
  • Department of Biotechnology, Wroclaw Research Centre EIT+ Ltd., Wrocław, Poland
  • Department of Biotechnology, Wroclaw Research Centre EIT+ Ltd., Wrocław, Poland
  • Department of Biotechnology, Wroclaw Research Centre EIT+ Ltd., Wrocław, Poland
  • Department of Biotechnology, Wroclaw Research Centre EIT+ Ltd., Wrocław, Poland
  • Department of Biotechnology, Wroclaw Research Centre EIT+ Ltd., Wrocław, Poland
  • Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, Wrocław, Poland
  • Abdallah AM, Gey van Pittius NC, Champion PA, Cox J, Luirink J, Vandenbroucke-Grauls C M, Appelmelk BJ, Bitter W (2007) Type VII secretion - mycobacteria show the way. Nat Rev Microbiol 5: 883-891.
  • Andrade A, Pardo JP, Espinosa N, Perez-Hernandez G, Gonzalez-Pedrajo B (2007) Enzymatic characterization of the enteropathogenic Escherichia coli type III secretion ATPase EscN. Arch Biochem Biophys 468: 121-127.
  • Berger CN, Crepin VF, Jepson MA, Arbeloa A, Frankel G (2009) The mechanisms used by enteropathogenic Escherichia coli to control filopodia dynamics Cell Microbiol 11: 309-322. doi: 10.1111/j.1462-5822.2008.01254.x.
  • Blaylock B, Riordan KE, Missiakas DM, Schneewind O (2006) Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator, YscL. J Bacteriol 188: 3525-3534. doi: 10.1128/jb.188.10.3525-3534.2006.
  • Bozue J, Cote CK, Webster W, Bassett A, Tobery S, Little S, Swietnicki W (2012) A Yersinia pestis YscN ATPase mutant functions as a live attenuated vaccine against bubonic plague in mice. FEMS Microbiol Lett 332: 113-121. doi: 10.1111/j.1574-6968.2012.02583.x.
  • Chen L, Ai X, Portaliou AG, Minetti CA, Remeta DP, Economou A, Kalodimos CG (2013) Substrate-activated conformational switch on chaperones encodes a targeting signal in type III secretion. Cell Rep 3: 709-715. doi: 10.1016/j.celrep.2013.02.025.
  • Cornelis GR (2006) The type III secretion injectisome. Nat Rev Microbiol 4: 811-825. doi: 10.1038/nrmicro1526.
  • Cowan C, Philipovskiy AV, Wulff-Strobel CR, Ye Z, Straley SC (2005) Anti-LcrV antibody inhibits delivery of Yops by Yersinia pestis KIM5 by directly promoting phagocytosis. Infect Immun 73: 6127-6137. doi: 10.1128/iai.73.9.6127-6137.2005.
  • Davis AJ, Díaz DA, Mecsas J (2010) A dominant-negative needle mutant blocks type III secretion of early but not late substrates in Yersinia. Mol Microbiol 76: 236-259. doi: 10.1111/j.1365-2958.2010.07096.x.
  • Diepold A, Amstutz M, Abel S, Sorg I, Jenal U, Cornelis GR (2010) Deciphering the assembly of the Yersinia type III secretion injectisome. EMBO J 29: 1928-1940. doi: 10.1038/emboj.2010.84.
  • Diepold A, Wiesand U, Cornelis GR (2011) The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer. Mol Microbiol 82: 502-514. doi: 10.1111/j.1365-2958.2011.07830.x.
  • Dixon M (1953) The determination of enzyme inhibitor constants. Biochem J 55: 170-171. doi: 10.1042/bj0550170.
  • Duncan MC, Linington RG, Auerbuch V (2012) Chemical inhibitors of the type three secretion system: disarming bacterial pathogens. Antimicrob Agents Chemother 56: 5433-5441. doi: 10.1128/aac.00975-12.
  • Edwards PR, Ewing WH (1972) Identification of Enterobacteriaceae, 3rd edn, Burgess Publishing Co. MEDLINE
  • Eichelberg K, Ginocchio CC, Galán JE (1994) Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J Bacteriol 176: 4501-4510. doi: 10.1128/jb.176.15.4501-4510.1994.
  • Feltcher ME, Sullivan JT, Braunstein M (2010) Protein export systems of Mycobacterium tuberculosis: novel targets for drug development? Future Microbiol 5: 1581-1597. doi: 10.2217/fmb.10.112.
  • Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK, Shaw DE, Francis P, Shenkin PS (2004) Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem 47: 1739-1749. doi: 10.1021/jm0306430.
  • Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA, Sanschagrin PC, Mainz DT (2006) Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49: 6177-6196. doi: 10.1021/jm051256o.
  • Garrity-Ryan LK, Kim OK, Balada-Llasat JM, Bartlett VJ, Verma AK, Fisher ML, Castillo C, Songsungthong W, Tanaka SK, Levy SB, Mecsas J, Alekshun MN (2010) Small molecule inhibitors of LcrF, a Yersinia pseudotuberculosis transcription factor, attenuate virulence and limit infection in a murine pneumonia model. Infect Immun 78: 4683-4690. doi: 10.1128/iai.01305-09.
  • Gartner JF, Schmidt MA (2004) Comparative analysis of locus of enterocyte effacement pathogenicity islands of atypical enteropathogenic Escherichia coli. Infect Immun 72: 6722-6728. doi: 10.1128/iai.72.11.6722-6728.2004.
  • Gauthier A, Finlay BB (2003) Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli. J Bacteriol 185: 6747-6755. doi: 10.1128/jb.185.23.6747-6755.2003.
  • Germane KL, Spiller BW (2011) Structural and functional studies indicate that the EPEC effector, EspG, directly binds p21-activated kinase. Biochemistry 50: 917-919. doi: 10.1021/bi1020138.
  • Ghosh A, Hartung S, van der Does C, Tainer JA, Albers SV (2011) Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding. Biochem J 437: 43-52. doi: 10.1042/bj20110410.
  • Grier MC, Garrity-Ryan LK, Bartlett VJ, Klausner KA, Donovan PJ, Dudley C, Alekshun MN, Tanaka SK, Draper MP, Levy SB, Kim OK (2010) N-Hydroxybenzimidazole inhibitors of ExsA MAR transcription factor in Pseudomonas aeruginosa: In vitro anti-virulence activity and metabolic stability. Bioorg Med Chem Lett 20: 3380-3383. doi: 10.1016/j.bmcl.2010.04.014.
  • Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 100: 1541-1546. doi: 10.1073/pnas.0337542100.
  • Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, Banks JL (2004) Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem 47: 1750-1759. doi: 10.1021/jm030644s.
  • Hou T, Wang J, Li Y, Wang W (2011a) 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 51: 69-82. doi: 10.1021/ci100275a.
  • Hou T, Wang J, Li Y, Wang W (2011b) 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 32: 866-877. doi: 10.1002/jcc.21666.
  • Hu X, Vujanac M, Southall N, Stebbins CE (2013) Inhibitors of the Yersinia protein tyrosine phosphatase through high throughput and virtual screening approaches. Bioorg Med Chem Lett 23: 1056-1062. doi: 10.1016/j.bmcl.2012.12.018.
  • Iguchi A, Thomson NR, Ogura Y, Saunders D, Ooka T, Henderson IR, Harris D, Asadulghani M, Kurokawa K, Dean P, Kenny B, Quail MA, Thurston S, Dougan G, Hayashi T, Parkhill J, Frankel G (2009) Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69. J Bacteriol 191: 347-354. doi: 10.1128/jb.01238-08.
  • Izore T, Job V, Dessen A (2011) Biogenesis, regulation, and targeting of the type III secretion system. Structure 19: 603-612. doi: 10.1016/j.str.2011.03.015.
  • Jepson MA, Pellegrin S, Peto L, Banbury DN, Leard AD, Mellor H, Kenny B (2003) Synergistic roles for the Map and Tir effector molecules in mediating uptake of enteropathogenic Escherichia coli (EPEC) into non-phagocytic cells. Cell Microbiol 5: 773-783. doi: 10.1046/j.1462-5822.2003.00315.x.
  • Johnson JR, Tchesnokova V, Johnston B, Clabots C, Roberts PL, Billig M, Riddell K, Rogers P, Qin X, Butler-Wu S, Price LB, Aziz M, Nicolas-Chanoine MH, Debroy C, Robicsek A, Hansen G, Urban C, Platell J, Trott DJ, Zhanel G, Weissman SJ, Cookson BT, Fang FC, Limaye AP, Scholes D, Chattopadhyay S, Hooper DC, Sokurenko EV (2013) Abrupt emergence of a single dominant multidrug-resistant strain of Escherichia coli. J Infect Dis 207: 919-928. doi: 10.1093/infdis/jis933.
  • Kawamoto A, Morimoto YV, Miyata T, Minamino T, Hughes KT, Kato T, Namba K (2013) Common and distinct structural features of Salmonella injectisome and flagellar basal body. Sci Rep 3: 3369. doi: 10.1038/srep03369.
  • Keyser P, Elofsson M, Rosell S, Wolf-Watz H (2008) Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria. J Intern Med 264: 17-29. doi: 10.1111/j.1365-2796.2008.01941.x.
  • Li J, Abel R, Zhu K, Cao Y, Zhao S, Friesner RA (2011) The VSGB 2.0 model: a next generation energy model for high resolution protein structure modeling. Proteins 79: 2794-2812. doi: 10.1002/prot.23106.
  • Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56: 658-666. doi: 10.1021/ja01318a036
  • Marshall NC, Finlay BB (2014) Targeting the type III secretion system to treat bacterial infections. Expert Opin Ther Targets 18: 137-152. doi: 10.1517/14728222.2014.855199.
  • Martinez-Argudo I, Veenendaal AK, Liu X, Roehrich AD, Ronessen MC, Franzoni G, van Rietschoten KN, Morimoto YV, Saijo-Hamano Y, Avison MB, Studholme DJ, Namba K, Minamino T, Blocker AJ (2013) Isolation of Salmonella mutants resistant to the inhibitory effect of Salicylidene acylhydrazides on flagella-mediated motility. PloS One 8: e52179. doi: 10.1371/journal.pone.0052179.
  • Mills E, Baruch K, Charpentier X, Kobi S, Rosenshine I (2008) Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli. Cell Host Microbe 3: 104-113. doi: 10.1016/j.chom.2007.11.007.
  • Monjaras Feria J, Garcia-Gomez E, Espinosa N, Minamino T, Namba K, Gonzalez-Pedrajo B (2012) Role of EscP (Orf16) in injectisome biogenesis and regulation of type III protein secretion in enteropathogenic Escherichia coli. J Bacteriol 194: 6029-6045. doi: 10.1128/jb.01215-12.
  • Murphy TA, Catto LE, Halford SE, Hadfield AT, Minor W, Walsh TR, Spencer J (2006) Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-beta-lactamases. J Mol Biol 357: 890-903. doi: 10.1016/j.jmb.2006.01.003.
  • Nataro JP, Kaper JB (1998) Diarrheagenic Escherichia coli. Clin Microbiol Rev 11: 142-201.
  • Orskov F, Orskov I (1992) Escherichia coli serotyping and disease in man and animals. Can J Microbiol 38: 699-704. doi: 10.1139/m92-115.
  • Pozidis C, Chalkiadaki A, Gomez-Serrano A, Stahlberg H, Brown I, Tampakaki AP, Lustig A, Sianidis G, Politou AS, Engel A, Panopoulos NJ, Mansfield J, Pugsley AP, Karamanou S, Economou A (2003) Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization. J Biol Chem 278: 25816-25824. doi: 10.1074/jbc.m301903200.
  • Rastelli G, Del Rio A, Degliesposti G, Sgobba M (2010) Fast and accurate predictions of binding free energies using MM-PBSA and MM-GBSA. J Comput Chem 31: 797-810. doi: 10.1002/jcc.21372.
  • Robicsek A, Jacoby GA, Hooper DC (2006) The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis 6: 629-640. doi: 10.1016/s1473-3099(06)70599-0.
  • Rossier O, Van den Ackerveken G, Bonas U. (2000) HrpB2 and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant. Mol Microbiol 38: 828-838. doi: 10.1046/j.1365-2958.2000.02173.x.
  • Sastry GM, Adzhigirey M, Day T, Annabhimoju R, Sherman W (2013) Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des 27: 221-234. doi: 10.1007/s10822-013-9644-8.
  • Sorg JA, Blaylock B, Schneewind O (2006) Secretion signal recognition by YscN, the Yersinia type III secretion ATPase. Proc Natl Acad Sci U S A 103: 16490-16495. doi: 10.1073/pnas.0605974103.
  • Stone CB, Johnson DL, Bulir DC, Gilchrist JD, Mahony JB (2008) Characterization of the putative type III secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae. J Bacteriol 190: 6580-6588. doi: 10.1128/jb.00761-08.
  • Strahilevitz J, Jacoby GA, Hooper DC, Robicsek A (2009) Plasmid-mediated quinolone resistance: a multifaceted threat. Clin Microbiol Rev 22: 664-689. doi: 10.1128/cmr.00016-09.
  • Swietnicki W, Carmany D, Retford M, Guelta M, Dorsey R, Bozue J, Lee MS, Olson MA (2011) Identification of small-molecule inhibitors of Yersinia pestis Type III secretion system YscN ATPase. PloS One 6: e19716. doi: 10.1371/journal.pone.0019716.
  • Tautz L, Bruckner S, Sareth S, Alonso A, Bogetz J, Bottini N, Pellecchia M, Mustelin T (2005) Inhibition of Yersinia tyrosine phosphatase by furanyl salicylate compounds. J Biol Chem 280: 9400-9408. doi: 10.1074/jbc.m413122200.
  • Thomas NA, Deng W, Puente JL, Frey EA, Yip CK, Strynadka NC, Finlay BB (2005) CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli. Mol Microbiol 57: 1762-1779. doi: 10.1111/j.1365-2958.2005.04802.x.
  • Umanski T, Rosenshine I, Friedberg D (2002) Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli. Microbiology 148: 2735-2744. doi: 10.1099/00221287-148-9-2735.
  • Vaudaux P, Waldvogel FA (1979) Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes. Antimicrob Agents Chemotherapy 16: 743-749. doi: 10.1128/aac.16.6.743.
  • Welch RA, Burland V, Plunkett G 3rd, Redford P, Roesch P, Rasko D, Buckles EL, Liou SR, Boutin A, Hackett J, Stroud D, Mayhew GF, Rose DJ, Zhou S, Schwartz DC, Perna NT, Mobley HL, Donnenberg MS, Blattner FR (2002) Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc Natl Acad Sci U S A 99: 17020-17024. doi: 10.1073/pnas.252529799.
  • Wiles TJ, Kulesus RR, Mulvey MA (2008) Origins and virulence mechanisms of uropathogenic Escherichia coli. Exp Mol Pathol 85: 11-19. doi: 10.1016/j.yexmp.2008.03.007.
  • Wong AR, Pearson JS, Bright MD, Munera D, Robinson KS, Lee SF, Frankel G, Hartland EL (2011) Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol 80: 1420-1438. doi: 10.1111/j.1365-2958.2011.07661.x.
  • Yoshida Y, Miki T, Ono S, Haneda T, Ito M, Okada N. (2014) Functional characterization of the type III secretion ATPase SsaN encoded by Salmonella pathogenicity island 2. PLoS One 9: e94347. doi: 10.1371/journal.pone.0094347.
  • Zarivach R, Vuckovic M, Deng W, Finlay BB, Strynadka NC (2007) Structural analysis of a prototypical ATPase from the type III secretion system. Nat Struct Mol Biol 14: 131-137. doi: 10.1038/nsmb1196.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.