Medicinal Chemistry Research

, Volume 23, Issue 10, pp 4464–4481 | Cite as

Screening of potent antibacterial agents targeting Clostridium difficile virulence factor toxin B: an in silico approach

  • Vijayalakshmi Ezhilarasan
  • Ankush Jadhav
  • Archana Pan
Original Research

Abstract

Toxin B is the key determinant of virulence in Clostridium difficile. Cysteine protease domain (CPD) of toxin B plays a crucial role in host cell intoxication thereby making it a potential target for drug discovery. The present study is aimed at identifying the promising lead compounds targeting C. difficile toxin B CPD. Initial screening of the compounds was done using topomer search and drug-likeness properties. The subsequent molecular docking study yielded a set of lead compounds having better docking score and binding mode of interactions compared to the known inhibitor. Molecular dynamics simulations were performed to explore the stability of protein–ligand complexes. The identified promising lead molecules can be used for the development of therapeutics targeting C. difficile toxin B.

Graphical Abstract

Large scale screening protocol for identification of novel promising lead candidates against virulence factor (toxin B) of Clostridium difficile.

Keywords

Human pathogen Therapeutic target Topomer search Drug-likeness Docking MD simulation 

Notes

Acknowledgments

The author V.E. is grateful to Pondicherry University, India, for the pre-doctoral fellowship and A.J. is thankful to the University Grant Commission (UGC), New Delhi, Govt. of India, for providing research fellowship. Authors are grateful to A. Murali, Pondicherry University, Pondicherry, India and Aditya Sharma, Department of Genetics, University of Delhi, South Campus, New Delhi, India for critical reading of the manuscript and providing valuable suggestions. Authors are also thankful to Kannan M., Pondicherry University, Pondicherry, for his valuable suggestions to analysis ‘conformational changes in protein during MD simulation’.

Supplementary material

44_2014_1017_MOESM1_ESM.pdf (1.7 mb)
Fig. S1Chemical structural information of all 475 screened potential lead compounds. (PDF 1700 kb)
44_2014_1017_MOESM2_ESM.pdf (1.2 mb)
Fig. S2Chemical structure, 3D and 2D docking interactions of all 31 identified lead compounds in the active site of toxin B CPD. (PDF 1217 kb)
44_2014_1017_MOESM3_ESM.tif (9.9 mb)
Fig. S3Superimposition of all 31 identified promising lead compounds and reference inhibitor into the active site of toxin B CPD. Protein is shown in surface and ligand in stick representation with pink and element colour code, respectively. (TIFF 10088 kb)
44_2014_1017_MOESM4_ESM.tif (3.7 mb)
Fig. S4Final conformation of the binding site of four lead compounds, namely Akos_505 (1), Ambinter_1332 (2), Biocyc_473 (3), DrugBank_89 (4) and reference inhibitor (5) after 25 ns of molecular dynamics simulation. Respective 2D interactions are shown in the right panel. (TIFF 3789 kb)
44_2014_1017_MOESM5_ESM.tif (140 kb)
Fig. S5The three dimensional structure of toxin B CPD with the structural components of active site (α2-Helix, β10-strand and loops) (TIFF 141 kb)
44_2014_1017_MOESM6_ESM.jpg (194 kb)
Fig. S6Conformational changes of β10-strand at each 1 ns interval up to 25 ns during MD simulation for Akos-505 (a), Ambinter-1332 (JPEG 195 kb)
44_2014_1017_MOESM7_ESM.jpg (189 kb)
Fig. S6Conformational changes of β10-strand at each 1 ns interval up to 25 ns during MD simulation for Akos-505 (b), Biocyc-473 (JPEG 189 kb)
44_2014_1017_MOESM8_ESM.jpg (93 kb)
Fig. S6Conformational changes of β10-strand at each 1 ns interval up to 25 ns during MD simulation for Akos-505 (c), DrugBank-89 (JPEG 94 kb)
44_2014_1017_MOESM9_ESM.jpg (199 kb)
Fig. S6Conformational changes of β10-strand at each 1 ns interval up to 25 ns during MD simulation for Akos-505 (d) and reference inhibitor (e). (JPEG 199 kb)
44_2014_1017_MOESM10_ESM.tif (2.2 mb)
Fig. S7Backbone Hydrogen bonds between β10 and β11-strand. Both strands and ligand are shown in stick representation with brown and cyan colour, respectively, whereas hydrogen bond is shown in red dotted lines. (TIFF 2270 kb)
44_2014_1017_MOESM11_ESM.tif (354 kb)
Fig. S8Area and volume of the toxin B CPD active site are shown in the column chart. Blue and red colour represents before and after simulation, respectively (TIFF 354 kb)
44_2014_1017_MOESM12_ESM.tif (2.3 mb)
Fig. S9U shaped ligand orientation of Akos-505 (a), Ambinter-1332 (b), Biocyc-473 (c), DrugBank-89 (d) and reference inhibitor (e) formed during the period of simulation (TIFF 2306 kb)
44_2014_1017_MOESM13_ESM.pdf (840 kb)
Table S1Screened 475 potential lead compounds and reference inhibitor with their compound ID, G_score and G_energy and different QikProp descriptors (PDF 841 kb)

References

  1. Aktories K, Barbieri JT (2005) Bacterial cytotoxins: targeting eukaryotic switches. Nat Rev Microbiol 3:397–410CrossRefPubMedGoogle Scholar
  2. Aktories K, Just I (2005) Clostridial Rho-inhibiting protein toxins. Curr Top Microbiol Immunol 291:113–145PubMedGoogle Scholar
  3. Andrews KM, Cramer RD (2000) Toward general methods of targeted library design: topomer shape similarity searching with diverse structures as queries. J Med Chem 43:1723–1740CrossRefPubMedGoogle Scholar
  4. Arfken G (1985) The method of steepest descents, n§7.4. In: Mathematical methods for physicists. FL Academic Press, OrlandoGoogle Scholar
  5. Banks JL, Beard HS, Cao Y, Cho AE, Damm W, Farid R, Felts AK, Halgren TA, Mainz DT, Maple JR, Murphy R, Philipp DM, Repasky MP, Zhang LY, Berne BJ, Friesner RA, Gallicchio E, Levy RM (2005) Integrated modeling program, applied chemical theory (IMPACT). J Comput Chem 26:1752–1780PubMedCentralCrossRefPubMedGoogle Scholar
  6. Berendsen HJCGJ, Traatsma TP (1987) The missing term in effective pair potentials. J Phys Chem 91:6269–6271CrossRefGoogle Scholar
  7. Berendsen HJC, Postma JPM, Gunsteren WF, DiNola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684–3690CrossRefGoogle Scholar
  8. Berk H, Henk B, Herman JCB, Johannes GEMF (1997) LINCS: a linear constraint solver for molecular simulations. J Comput Chem 18:1463–1472CrossRefGoogle Scholar
  9. Chowdhuri S, Tan ML, Ichiye TJ (2006) Dynamical properties of the soft sticky dipole–quadrupole–octupole water model: a molecular dynamics study. J Chem Phys 125:14451–14453CrossRefGoogle Scholar
  10. Cramer RD (2006) Leadhopping—and beyond. Expert Opin Drug Discov 1:311–321CrossRefPubMedGoogle Scholar
  11. Cramer RD, Poss MA, Hermsmeier MA, Caulfield TJ, Kowala MC, Valentine MT (1999) Prospective identification of biologically active structures by topomer shape similarity searching. J Med Chem 42:3919–3933CrossRefPubMedGoogle Scholar
  12. Cramer RD, Jilek RJ, Andrews KM (2002) Dbtop: topomer similarity searching of conventional structure databases. J Mol Graph Model 20:447–462CrossRefPubMedGoogle Scholar
  13. Cramer RD, Jilek RJ, Guessregen S, Clark SJ, Wendt B, Clark RD (2004) “Lead hopping”. Validation of topomer similarity as a superior predictor of similar biological activities. J Med Chem 47:6777–6791CrossRefPubMedGoogle Scholar
  14. Cramer RD, Soltanshahi F, Jilek R, Campbell B (2007) AllChem: generating and searching 10(20) synthetically accessible structures. J Comput Aided Mol Des 21:341–350CrossRefPubMedGoogle Scholar
  15. Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an N-log(N) method for Ewald sums in large systems. J Chem Phys 98:10089–10092CrossRefGoogle Scholar
  16. Dove CH, Wang SZ, Price SB, Phelps CJ, Lyerly DM, Wilkins TD, Johnson JL (1990) Molecular characterization of the Clostridium difficile toxin A gene. Infect Immun 58:480–488PubMedCentralPubMedGoogle Scholar
  17. Duffy EM, Jorgensen WL (2000) Prediction of properties from simulations: free energies of solvation in hexadecane, octanol, and water. J Am Chem Soc 122:2878–2888CrossRefGoogle Scholar
  18. Egerer M, Giesemann T, Jank T, Satchell KJ, Aktories K (2007) Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity. J Biol Chem 282:25314–25321CrossRefPubMedGoogle Scholar
  19. 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 47:1739–1749CrossRefPubMedGoogle Scholar
  20. 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 49:6177–6196CrossRefPubMedGoogle Scholar
  21. Frisch C, Gerhard R, Aktories K, Hofmann F, Just I (2003) The complete receptor-binding domain of Clostridium difficile toxin A is required for endocytosis. Biochem Biophys Res Commun 300:706–711CrossRefPubMedGoogle Scholar
  22. Fuhrmans MSB, Marrink SJ, de Vries AH (2010) Effects of bundling on the properties of the SPC water mode. Theor Chem Acc 125:335–344CrossRefGoogle Scholar
  23. Genth H, Huelsenbeck J, Hartmann B, Hofmann F, Just I, Gerhard R (2006) Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B. FEBS Lett 580:3565–3569CrossRefPubMedGoogle Scholar
  24. Genth H, Dreger SC, Huelsenbeck J, Just I (2008) Clostridium difficile toxins: more than mere inhibitors of Rho proteins. Int J Biochem Cell Biol 40:592–597CrossRefPubMedGoogle Scholar
  25. Gerding DN (2005) Metronidazole for Clostridium difficile-associated disease: is it okay for Mom? Clin Infect Dis 40:1598–1600CrossRefPubMedGoogle Scholar
  26. Gerhard R, Nottrott S, Schoentaube J, Tatge H, Olling A, Just I (2008) Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells. J Med Microbiol 57:765–770CrossRefPubMedGoogle Scholar
  27. Goorhuis A, Van der Kooi T, Vaessen N, Dekker FW, Van den Berg R, Harmanus C, van den Hof S, Notermans DW, Kuijper EJ (2007) Spread and epidemiology of Clostridium difficile polymerase chain reaction ribotype 027/toxinotype III in The Netherlands. Clin Infect Dis 45:695–703CrossRefPubMedGoogle Scholar
  28. 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 47:1750–1759CrossRefPubMedGoogle Scholar
  29. Halsey J (2008) Current and future treatment modalities for Clostridium difficile-associated disease. Am J Health Syst Pharm 65:705–715CrossRefPubMedGoogle Scholar
  30. Hammond GA, Johnson JL (1995) The toxigenic element of Clostridium difficile strain VPI 10463. Microb Pathog 19:203–213CrossRefPubMedGoogle Scholar
  31. Hayes MJ, Stein M, Weiser J (2004) Accurate calculations of ligand binding free energies. J Phys Chem 108:3572–3580CrossRefGoogle Scholar
  32. Hermans J, Berendsen HJC, Van Gunsteren WF, Postma JPM (1984) A consistent empirical potential for water–protein interactions. Biopolymers 23:1513–1518CrossRefGoogle Scholar
  33. Hess B, Kutzner C, Van der Spoel D, Lindahl E (2008) GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4:435–447CrossRefGoogle Scholar
  34. Hubert B, Loo VG, Bourgault AM, Poirier L, Dascal A, Fortin E, Dionne M, Lorange M (2007) A portrait of the geographic dissemination of the Clostridium difficile North American pulsed-field type 1 strain and the epidemiology of C. difficile-associated disease in Quebec. Clin Infect Dis 44:238–244CrossRefPubMedGoogle Scholar
  35. Hundsberger T, Braun V, Weidmann M, Leukel P, Sauerborn M, von Eichel-Streiber C (1997) Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile. Eur J Biochem 244:735–742CrossRefPubMedGoogle Scholar
  36. Jilek RJ, Cramer RD (2004) Topomers: a validated protocol for their self-consistent generation. J Chem Inf Comput Sci 44:1221–1227CrossRefPubMedGoogle Scholar
  37. Just I, Gerhard R (2004) Large clostridial cytotoxins. Rev Physiol Biochem Pharmacol 152:23–47PubMedGoogle Scholar
  38. Just I, Selzer J, Wilm M, Von Eichel-Streiber C, Mann M, Aktories K (1995) Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375:500–503CrossRefPubMedGoogle Scholar
  39. Kato D, Boatright KM, Berger AB, Nazif T, Blum G, Ryan C, Chehade KA, Salvesen GS, Bogyo M (2005) Activity-based probes that target diverse cysteine protease families. Nat Chem Biol 1:33–38CrossRefPubMedGoogle Scholar
  40. Kelly CP, LaMont JT (2008) Clostridium difficile—more difficult than ever. N Engl J Med 359:1932–1940CrossRefPubMedGoogle Scholar
  41. Krovat EM, Steindl T, Langer T (2005) Recent advances in docking and scoring. Curr Comput Aided Drug Des 1:93–102CrossRefGoogle Scholar
  42. Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP (2010) The role of toxin A and toxin B in Clostridium difficile infection. Nature 467:711–713CrossRefPubMedGoogle Scholar
  43. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 46:3–26CrossRefPubMedGoogle Scholar
  44. Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S, Bourgault AM, Nguyen T, Frenette C, Kelly M, Vibien A, Brassard P, Fenn S, Dewar K, Hudson TJ, Horn R, Rene P, Monczak Y, Dascal A (2005) A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med 353:2442–2449CrossRefPubMedGoogle Scholar
  45. Lupardus PJ, Shen A, Bogyo M, Garcia KC (2008) Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain. Science 322:265–268PubMedCentralCrossRefPubMedGoogle Scholar
  46. Lyras D, O’Connor JR, Howarth PM, Sambol SP, Carter GP, Phumoonna T, Poon R, Adams V, Vedantam G, Johnson S, Gerding DN, Rood JI (2009) Toxin B is essential for virulence of Clostridium difficile. Nature 458:1176–1179PubMedCentralCrossRefPubMedGoogle Scholar
  47. Mani N, Dupuy B (2001) Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci USA 98:5844–5849PubMedCentralCrossRefPubMedGoogle Scholar
  48. McDonald LC, Killgore GE, Thompson A, Owens RC Jr, Kazakova SV, Sambol SP, Johnson S, Gerding DN (2005) An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 353:2433–2441CrossRefPubMedGoogle Scholar
  49. Mooney H (2007) Annual incidence of MRSA falls in England, but C difficile continues to rise. BMJ 335:958PubMedCentralCrossRefPubMedGoogle Scholar
  50. Pelaez T, Alcala L, Alonso R, Rodriguez-Creixems M, Garcia-Lechuz JM, Bouza E (2002) Reassessment of Clostridium difficile susceptibility to metronidazole and vancomycin. Antimicrob Agents Chemother 46:1647–1650PubMedCentralCrossRefPubMedGoogle Scholar
  51. Pfeifer G, Schirmer J, Leemhuis J, Busch C, Meyer DK, Aktories K, Barth H (2003) Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells. J Biol Chem 278:44535–44541CrossRefPubMedGoogle Scholar
  52. Prochazkova K, Shuvalova LA, Minasov G, Voburka Z, Anderson WF, Satchell KJ (2009) Structural and molecular mechanism for autoprocessing of MARTX toxin of Vibrio cholerae at multiple sites. J Biol Chem 284:26557–26568PubMedCentralCrossRefPubMedGoogle Scholar
  53. Pruitt RN, Chagot B, Cover M, Chazin WJ, Spiller B, Lacy DB (2009) Structure–function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A. J Biol Chem 284:21934–21940PubMedCentralCrossRefPubMedGoogle Scholar
  54. Puri AW, Lupardus PJ, Deu E, Albrow VE, Garcia KC, Bogyo M, Shen A (2010) Rational design of inhibitors and activity-based probes targeting Clostridium difficile virulence factor TcdB. Chem Biol 17:1201–1211PubMedCentralCrossRefPubMedGoogle Scholar
  55. Qa’Dan M, Spyres LM, Ballard JD (2000) pH-induced conformational changes in Clostridium difficile toxin B. Infect Immun 68:2470–2474PubMedCentralCrossRefPubMedGoogle Scholar
  56. Qa’Dan M, Ramsey M, Daniel J, Spyres LM, Safiejko-Mroczka B, Ortiz-Leduc W, Ballard JD (2002) Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells. Cell Microbiol 4:425–434CrossRefPubMedGoogle Scholar
  57. Redelings MD, Sorvillo F, Mascola L (2007) Increase in Clostridium difficile-related mortality rates, United States, 1999–2004. Emerg Infect Dis 13:1417–1419PubMedCentralCrossRefPubMedGoogle Scholar
  58. Reinert DJ, Jank T, Aktories K, Schulz GE (2005) Structural basis for the function of Clostridium difficile toxin B. J Mol Biol 351:973–981CrossRefPubMedGoogle Scholar
  59. Rolfe RD, Song W (1993) Purification of a functional receptor for Clostridium difficile toxin A from intestinal brush border membranes of infant hamsters. Clin Infect Dis 16(Suppl 4):S219–S227CrossRefPubMedGoogle Scholar
  60. Rupnik M, Wilcox MH, Gerding DN (2009) Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7:526–536CrossRefPubMedGoogle Scholar
  61. Schirmer J, Aktories K (2004) Large clostridial cytotoxins: cellular biology of Rho/Ras-glucosylating toxins. Biochim Biophys Acta 1673:66–74CrossRefPubMedGoogle Scholar
  62. Schuttelkopf AW, Van Aalten DM (2004) PRODRG: a tool for high-throughput crystallography of protein–ligand complexes. Acta Crystallogr D Biol Crystallogr 60:1355–1363CrossRefPubMedGoogle Scholar
  63. Shelley JC, Cholleti A, Frye LL, Greenwood JR, Timlin MR, Uchimaya M (2007) Epik: a software program for pK(a) prediction and protonation state generation for drug-like molecules. J Comput Aided Mol Des 21:681–691CrossRefPubMedGoogle Scholar
  64. Shen A, Lupardus PJ, Albrow VE, Guzzetta A, Powers JC, Garcia KC, Bogyo M (2009) Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin. Nat Chem Biol 5:469–478PubMedCentralCrossRefPubMedGoogle Scholar
  65. Shen A, Lupardus PJ, Gersch MM, Puri AW, Albrow VE, Garcia KC, Bogyo M (2011) Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins. Nat Struct Mol Biol 18:364–371PubMedCentralCrossRefPubMedGoogle Scholar
  66. Stabler RA, He M, Dawson L, Martin M, Valiente E, Corton C, Lawley TD, Sebaihia M, Quail MA, Rose G, Gerding DN, Gibert M, Popoff MR, Parkhill J, Dougan G, Wren BW (2009) Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol 10:R102PubMedCentralCrossRefPubMedGoogle Scholar
  67. Thornberry NA, Peterson EP, Zhao JJ, Howard AD, Griffin PR, Chapman KT (1994) Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry 33:3934–3940CrossRefPubMedGoogle Scholar
  68. TRIPOS (2010) SYBYL user’s manual. Tripos International, St. LouisGoogle Scholar
  69. Van Aalten DMF, Bywater R, Findlay JBC, Hendlich M, Hooft RWW, Vriend G (1996) PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates of small molecules. J Comput Aided Mol Des 10:255–262CrossRefPubMedGoogle Scholar
  70. Van der Spoel D, Van Buuren AR, Peter Tieleman D, Berendsen HJC, Grigera JR, Traatsma TP (1996) Molecular dynamics simulations of peptides from BPTI: a closer look at amide–aromatic interactions. J Biomol NMR 8:229–238CrossRefPubMedGoogle Scholar
  71. Von Eichel-Streiber C, Laufenberg-Feldmann R, Sartingen S, Schulze J, Sauerborn M (1992a) Comparative sequence analysis of the Clostridium difficile toxins A and B. Mol Gen Genet 233:260–268CrossRefGoogle Scholar
  72. Von Eichel-Streiber C, Sauerborn M, Kuramitsu HK (1992b) Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases. J Bacteriol 174:6707–6710Google Scholar
  73. Von Eichel-Streiber C, Boquet P, Sauerborn M, Thelestam M (1996) Large clostridial cytotoxins—a family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol 4:375–382CrossRefGoogle Scholar
  74. Voth DE, Ballard JD (2005) Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 18:247–263PubMedCentralCrossRefPubMedGoogle Scholar
  75. Warny M, Pepin J, Fang A, Killgore G, Thompson A, Brazier J, Frost E, McDonald LC (2005) Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 366:1079–1084CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Vijayalakshmi Ezhilarasan
    • 1
  • Ankush Jadhav
    • 1
  • Archana Pan
    • 1
  1. 1.Centre for Bioinformatics, School of Life SciencesPondicherry UniversityPondicherryIndia

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