Skip to main content
Log in

Design of Ligand Binding to an Engineered Protein Cavity Using Virtual Screening and Thermal Up-shift Evaluation

  • Published:
Journal of Computer-Aided Molecular Design Aims and scope Submit manuscript

Summary

Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores, a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds. In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes. To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable protein cavities for the hosting of ligands of interest.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. R. Abagyan M. Totrov (2001) Curr. Opin. Chem. Biol. 5 375 Occurrence Handle10.1016/S1367-5931(00)00217-9 Occurrence Handle11470599

    Article  PubMed  Google Scholar 

  2. P. Bamborough F.E. Cohen (1996) Curr. Opin. Struct. Biol. 6 236 Occurrence Handle10.1016/S0959-440X(96)80081-9 Occurrence Handle8728658

    Article  PubMed  Google Scholar 

  3. G.M. Morris D.S. Goodsel R.S. Halliday R. Huey W.E. Hart R.K Belew A.J. Olson (1998) J. Comput. Chem. 19 1639 Occurrence Handle10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-B

    Article  Google Scholar 

  4. M. Totrov R. Abagyan (1997) Proteins (Suppl.) 1 215 Occurrence Handle10.1002/(SICI)1097-0134(1997)1+<215::AID-PROT29>3.0.CO;2-Q

    Article  Google Scholar 

  5. J.H. McKie K.T. Douglas C. Chan S.A. Roser R. Yates M. Read J.E. Hyde M.J. Dascombe Y. Yuthavong W. Sirawaraporn (1998) J. Med. Chem. 41 1367 Occurrence Handle10.1021/jm970845u Occurrence Handle9554869

    Article  PubMed  Google Scholar 

  6. S. Iyer D. Kontoyiannis D. Chevrier J. Woo N. Mori M. Cornejo G. Kollias R. Buelow (2000) J. Biol. Chem. 275 17051 Occurrence Handle10.1074/jbc.M909219199 Occurrence Handle10748117

    Article  PubMed  Google Scholar 

  7. S. Gruneberg M.T. Stubbs G. Klebe (2002) J. Med. Chem. 45 3588 Occurrence Handle10.1021/jm011112j Occurrence Handle12166932

    Article  PubMed  Google Scholar 

  8. M. Schapira R. Abagyan M. Totrov (2003) J. Med. Chem. 46 3045 Occurrence Handle10.1021/jm0300173 Occurrence Handle12825943

    Article  PubMed  Google Scholar 

  9. M. Itzstein Particlevon J.C. Dyason S.W. Oliver H.F. White W.Y. Wu G.B. Kok M.S. Pegg (1996) J. Med. Chem. 39 388 Occurrence Handle10.1021/jm950294c Occurrence Handle8558506

    Article  PubMed  Google Scholar 

  10. PerrierV. A.C. Wallace K. Kaneko J. Safar S.B. Prusiner F.E. Cohen (2000) Proc. Natl. Acad. Sci. U.S.A. 97 6073 Occurrence Handle10.1073/pnas.97.11.6073 Occurrence Handle10823951

    Article  PubMed  Google Scholar 

  11. M.O.F. Khan S.E. Austin C. Chan H. Yin D. Marks S.N. Vaghjiani H. Kendrick V. Yardley S.L. Croft K.T. Douglas (2001) J. Med. Chem. 43 3148 Occurrence Handle10.1021/jm000156+

    Article  Google Scholar 

  12. F. Pavao M.S. Castilho M.T. Pupo R.L. Dias A.G. Correa J.B. Fernandes M.F. da Silva J. Mafezoli P.C. Vieira G. Oliva (2002) FEBS Lett. 520 13 Occurrence Handle10.1016/S0014-5793(02)02700-X Occurrence Handle12044862

    Article  PubMed  Google Scholar 

  13. G.W.A. Milne M.C. Nicklaus J.S. Driscoll S. Wang D.J. Zaharevitz (1994) Chem. Inf. Comput. Sci. 34 1219 Occurrence Handle10.1021/ci00021a032

    Article  Google Scholar 

  14. M. Schapira B.M. Raaka H.H. Samuels R. Abagyan (2000) Proc. Natl. Acad. Sci. U.S.A. 97 1008 Occurrence Handle10.1073/pnas.97.3.1008 Occurrence Handle10655475

    Article  PubMed  Google Scholar 

  15. E.B. Gottlin R.E. Benson S. Conary B. Antonio K. Duke E.S. Payne S.S. Ashraf D.J. Christensen (2003) J. Biomol. Screen. 8 332 Occurrence Handle10.1177/1087057103008003011 Occurrence Handle12857387

    Article  PubMed  Google Scholar 

  16. B. Cox J.C. Denyer A. Binnie M.C. Donnelly B. Evans D.V. Green J.A. Lewis T.H. Mander A.T. Merritt M.J. Valler S.P. Watson (2000) Prog. Med. Chem. 37 83 Occurrence Handle10845248

    PubMed  Google Scholar 

  17. B. Waszkowycz T.D.J. Perkins R.A. Sykes J. Li (2001) IBM Syst. J. 40 360

    Google Scholar 

  18. S.D. Pickett B.S. Sherborne T. Wilkinson J. Bennett N. Borkakoti M. Broadhurst D. Hurst I. Kilford M. McKinnell P.S. Jones (2003) Bioorg. Med. Chem. Lett. 13 1691 Occurrence Handle10.1016/S0960-894X(03)00237-3 Occurrence Handle12729643

    Article  PubMed  Google Scholar 

  19. J.L. Jenkins R.Y. Kao R. Shapiro (2003) Proteins 50 81 Occurrence Handle10.1002/prot.10270 Occurrence Handle12471601

    Article  PubMed  Google Scholar 

  20. A.E. Eriksson W.A. Baase X.J. Zhang D.W. Heinz M. Blaber E.P. Baldwin B.W Matthews (1992) Science 255 178 Occurrence Handle1553543

    PubMed  Google Scholar 

  21. J. Xu W.A. Baase E. Baldwin B.W. Matthews (1998) Protein Sci 7 158 Occurrence Handle9514271

    PubMed  Google Scholar 

  22. E. Baldwin W.A. Baase X.J. Zhang V. Feher B.W. Matthews (1998) J. Mol. Biol. 277 468 Occurrence Handle10.1006/jmbi.1997.1606

    Article  Google Scholar 

  23. A. Morton B.W. Matthews (1995) Biochemistry 34 8576 Occurrence Handle10.1021/bi00027a007 Occurrence Handle7612599

    Article  PubMed  Google Scholar 

  24. C. Machicado M. Bueno J. Sancho (2002) Prot. Eng. 15 669 Occurrence Handle10.1093/protein/15.8.669

    Article  Google Scholar 

  25. A. Morton W.A. Baase B.W. Matthews (1995) Biochemistry 34 8564 Occurrence Handle10.1021/bi00027a006 Occurrence Handle7612598

    Article  PubMed  Google Scholar 

  26. P. Dauber-Ogusthorpe V.A. Roberts D.J. Ogusthorpe J. Wolff M. Genest A.T. Hagler (1998) ArticleTitleProteins Struct Funct. Genet 4 31

    Google Scholar 

  27. A. Pedretti L. Villa G. Vistoli (2002) J. Mol. Graph. 21 47 Occurrence Handle10.1016/S1093-3263(02)00123-7

    Article  Google Scholar 

  28. A.K. Ghose G.M. Crippen (1987) J. Chem. Inf. Comput. Sci. 27 21 Occurrence Handle10.1021/ci00053a005 Occurrence Handle3558506

    Article  PubMed  Google Scholar 

  29. T.L. Hill (Eds) (1960) Introduction to Statistical Thermodynamics Addison-Wesley New-York

    Google Scholar 

  30. J. Gasteiger M. Marsali (1980) Tetrahedron 36 3219 Occurrence Handle10.1016/0040-4020(80)80168-2

    Article  Google Scholar 

  31. S.L. Mayo B.D. Olafson W.A. Goddard SuffixIII (1990) J. Phys. Chem. 94 8897 Occurrence Handle10.1021/j100389a010

    Article  Google Scholar 

  32. J.R. Maple M.J. Hwang K.J. Jalkanen T.P. Stockæsch A.T. Hagler (1998) J. Comput. Chem. 19 430 Occurrence Handle10.1002/(SICI)1096-987X(199803)19:4<430::AID-JCC5>3.0.CO;2-T

    Article  Google Scholar 

  33. A.T. Hagler E. Huler S. Lifson (1974) J. Am. Chem. Soc. 96 5319 Occurrence Handle10.1021/ja00824a004 Occurrence Handle4851860

    Article  PubMed  Google Scholar 

  34. C.M. Venkatachalam X. Jiang T. Oldfield M.J. Waldman (2003) Mol. Graphics Modell. 21 289 Occurrence Handle10.1016/S1093-3263(02)00164-X

    Article  Google Scholar 

  35. D.K. Gehlhaar D. Bouzida P.A. Rejto (Eds) (1999) Rational Drug Design: Novel Methodology and Practical Applications American Chemical Society Washington, D.C 292–311

    Google Scholar 

  36. A.M. Jain (1996) J. Comput. Aided Mol. Design 10 427 Occurrence Handle10.1007/BF00124474

    Article  Google Scholar 

  37. I. Muegge Y.C. Martin (1999) J. Med. Chem. 42 791 Occurrence Handle10.1021/jm980536j Occurrence Handle10072678

    Article  PubMed  Google Scholar 

  38. P.S. Charifson J.J. Corkery M.A. Murcko W.P. Walters (1999) J. Med. Chem. 42 5100 Occurrence Handle10.1021/jm990352k Occurrence Handle10602695

    Article  PubMed  Google Scholar 

  39. B.A. Luty Z.R. Wasserman P.F.W. Stouten C.N. Hodge M. Zacharias J.A. McCammon (1995) J. Comput. Chem. 16 454 Occurrence Handle10.1002/jcc.540160409

    Article  Google Scholar 

  40. P.F.W. Stouten C. Frommel H. Nakamura C. Sander (1993) Mol. Simul. 10 97

    Google Scholar 

  41. Z. Li H.A. Scheraga (1987) Proc. Natl. Acad. Sci. U.S.A. 84 6611 Occurrence Handle3477791

    PubMed  Google Scholar 

  42. N. Guex M.C. Peitsch (1996) Protein Data Bank Quat. Newslett. 77 77

    Google Scholar 

  43. W.D. Cornell P. Cieplak C.I. Bayly I.R. Gould K.M. Merz D.M. Ferguson SuffixJr. D.C. Spellmeyer T. Fo J.W. Caldwell P.A. Kollman (1995) J. Am. Chem. Soc. 117 5179 Occurrence Handle10.1021/ja00124a002

    Article  Google Scholar 

  44. J. Florián A. Warshel (1997) J. Phys. Chem. B 101 5583

    Google Scholar 

  45. A.A. Rashin K. Namboodiri (1987) J. Phys. Chem. 91 6003 Occurrence Handle10.1021/j100307a038

    Article  Google Scholar 

  46. M.L. Connolly (1983) J. Appl. Cryst. 16 548 Occurrence Handle10.1107/S0021889883010985

    Article  Google Scholar 

  47. A. Finkelstein J. Janin (1989) Protein Eng. 3 1 Occurrence Handle2813338

    PubMed  Google Scholar 

  48. A.V. Jain (2003) J. Med. Chem. 46 499 Occurrence Handle10.1021/jm020406h Occurrence Handle12570372

    Article  PubMed  Google Scholar 

  49. J. Novotny R.E. Bruccoleri F.A. Saul (1989) Biochemistry 28 4735 Occurrence Handle10.1021/bi00437a034 Occurrence Handle2475171

    Article  PubMed  Google Scholar 

  50. H.P. Erickson (1989) J. Mol. Biol. 206 465 Occurrence Handle10.1016/0022-2836(89)90494-4 Occurrence Handle2716058

    Article  PubMed  Google Scholar 

  51. N. Horton M. Lewis (1992) Protein Sci. 1 169 Occurrence Handle1339024

    PubMed  Google Scholar 

  52. J.A. D’Aquino J. Gomez V.J. Hilser K.H. Lee L.M. Amzel E. Freire (1996) Proteins Struct. Funct. Genet. 25 143 Occurrence Handle10.1002/(SICI)1097-0134(199606)25:2<143::AID-PROT1>3.0.CO;2-J Occurrence Handle8811731

    Article  PubMed  Google Scholar 

  53. B. Lee F.M. Richards (1971) J. Mol. Biol. 55 379 Occurrence Handle10.1016/0022-2836(71)90324-X Occurrence Handle5551392

    Article  PubMed  Google Scholar 

  54. D. Eisenberg A.D. McLachlan (1989) Nature 319 199 Occurrence Handle10.1038/319199a0

    Article  Google Scholar 

  55. L. Wesson D. Eisenberg (1992) Protein Sci. 2 227

    Google Scholar 

  56. R. Fraczkiewicz W. Braun (1998) J. Comput. Chem. 19 319 Occurrence Handle10.1002/(SICI)1096-987X(199802)19:3<319::AID-JCC6>3.0.CO;2-W

    Article  Google Scholar 

  57. F. Sanger S. Nicklen A.R. Coulson (1977) Proc. Natl. Acad. Sci. U.S.A. 74 5463 Occurrence Handle271968

    PubMed  Google Scholar 

  58. C.G. Genzor A. Beldarrain C. Gomez-Moreno J.L. Lopez-Lacomba M. Cortijo J. Sancho (1996) Protein Sci. 5 1376 Occurrence Handle8819170

    PubMed  Google Scholar 

  59. D.E. Edmonson G. Tollin (1971) Biochemistry 10 124 Occurrence Handle10.1021/bi00777a019 Occurrence Handle5538602

    Article  PubMed  Google Scholar 

  60. M.P. Irun S. Maldonado J. Sancho (2001) Protein. Eng. 14 173 Occurrence Handle10.1093/protein/14.3.173 Occurrence Handle11342714

    Article  PubMed  Google Scholar 

  61. Pace , C.N., Shirley, B.A., and Thompson J.A. (Eds), Protein Structure, a Practical Approach, IRL Press, Oxford, 1989

  62. M.P. Irun M.M. Garcia-Mira J.M. Sanchez-Ruiz J. Sancho (2001) J. Mol. Biol. 306 877 Occurrence Handle10.1006/jmbi.2001.4436 Occurrence Handle11243795

    Article  PubMed  Google Scholar 

  63. L.A. Campos M. Bueno J. Lopez-Llano M.A. Jimenez J. Sancho (2004) J. Mol. Biol. 344 239 Occurrence Handle10.1016/j.jmb.2004.08.081 Occurrence Handle15504414

    Article  PubMed  Google Scholar 

  64. J.A. Schellman (1976) Biopolymers 15 999 Occurrence Handle10.1002/bip.1976.360150513

    Article  Google Scholar 

  65. M. Pantoliano E. Petrella J. Kwasnoski V. Lobanov J. Myslik E. Graf T. Carver E. Asel B. Springer P. Lane F. Salemme (2001) J. Biomol. Screen. 6 429 Occurrence Handle10.1177/108705710100600609 Occurrence Handle11788061

    Article  PubMed  Google Scholar 

  66. A. Lostao C. Gomez-Moreno S.G. Mayhew J. Sancho (1997) Biochemistry 36 14334 Occurrence Handle10.1021/bi971384h Occurrence Handle9398151

    Article  PubMed  Google Scholar 

  67. A.I. Su D.M. Lorber G.S. Weston W.A. Baase B.W. Matthews B.K. Shoichet (2001) Proteins Struct. Funct. Genet. 42 279 Occurrence Handle10.1002/1097-0134(20010201)42:2<279::AID-PROT150>3.0.CO;2-U Occurrence Handle11119652

    Article  PubMed  Google Scholar 

  68. B.Q. Wei W.A. Baase L.H. Weaver B.W. Matthews B.K. Shoichet (2002) J. Mol. Biol. 322 339 Occurrence Handle10.1016/S0022-2836(02)00777-5 Occurrence Handle12217695

    Article  PubMed  Google Scholar 

  69. B.Q. Wei L.H. Weaver A.M. Ferrari B.W. Matthews B.K. Shoichet (2004) J. Mol. Biol. 337 1161 Occurrence Handle10.1016/j.jmb.2004.02.015 Occurrence Handle15046985

    Article  PubMed  Google Scholar 

  70. E. Akaho G.M. Morris D.S. Goodsell D. Wong A.J. Olson (2001) J. Chem. Sofware 7 103

    Google Scholar 

  71. J. Apostolakis A. Pluckthun A. Caflisch (1998) J. Comput. Chem. 19 21 Occurrence Handle10.1002/(SICI)1096-987X(19980115)19:1<21::AID-JCC2>3.0.CO;2-0

    Article  Google Scholar 

  72. K. Holloway J. Wai T. Halgren P. Fitzgerald J. Vacca B. Dorsey R. Levin W. Thompson L. Chen S. deSolms N. Gaffin A. Ghosh E. Giuliani S. Graham J. Guare R. Hungate T. Lyle W. Sanders T. Tucker M. Wiggins C. Wiscount O. Woltersdorf S. Young P. Darke J. Zugay (1995) J. Med. Chem. 38 305 Occurrence Handle10.1021/jm00002a012 Occurrence Handle7830273

    Article  PubMed  Google Scholar 

  73. M. Rarey B. Kramer T. Lengauer (1997) Proteins (Suppl) 1 221

    Google Scholar 

  74. E.A. Sudbeck C. Mao R. Vig T.K. Venkatachalam L. Tuel-Ahlgren F.M. Uckun (1998) Antimicrob. Agents Chemother. 42 3225 Occurrence Handle9835518

    PubMed  Google Scholar 

  75. S.T. Rao F. Shaffie C. Yu K.A. Satyshur B.J. Stockman J.L. Markley M. Sundarlingam (1992) Protein Sci. 11 1413

    Google Scholar 

  76. A.M. Buckle P. Cramer A.R. Fersht (1996) Biochemistry 35 4298 Occurrence Handle10.1021/bi9524676 Occurrence Handle8605178

    Article  PubMed  Google Scholar 

  77. MC Lo A Aulabaugh G Jin R Cowling J Bard M Malamas G. Ellestad (2004) Anal. Biochem. 332 153 Occurrence Handle10.1016/j.ab.2004.04.031 Occurrence Handle15301960

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Javier Sancho.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Machicado, C., López-Llano, J., Cuesta-López, S. et al. Design of Ligand Binding to an Engineered Protein Cavity Using Virtual Screening and Thermal Up-shift Evaluation. J Comput Aided Mol Des 19, 421–443 (2005). https://doi.org/10.1007/s10822-005-7969-7

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10822-005-7969-7

Key words

Navigation