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.
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R. Abagyan M. Totrov (2001) Curr. Opin. Chem. Biol. 5 375 Occurrence Handle10.1016/S1367-5931(00)00217-9 Occurrence Handle11470599
P. Bamborough F.E. Cohen (1996) Curr. Opin. Struct. Biol. 6 236 Occurrence Handle10.1016/S0959-440X(96)80081-9 Occurrence Handle8728658
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
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
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
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
S. Gruneberg M.T. Stubbs G. Klebe (2002) J. Med. Chem. 45 3588 Occurrence Handle10.1021/jm011112j Occurrence Handle12166932
M. Schapira R. Abagyan M. Totrov (2003) J. Med. Chem. 46 3045 Occurrence Handle10.1021/jm0300173 Occurrence Handle12825943
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
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
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+
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
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
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
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
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
B. Waszkowycz T.D.J. Perkins R.A. Sykes J. Li (2001) IBM Syst. J. 40 360
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
J.L. Jenkins R.Y. Kao R. Shapiro (2003) Proteins 50 81 Occurrence Handle10.1002/prot.10270 Occurrence Handle12471601
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
J. Xu W.A. Baase E. Baldwin B.W. Matthews (1998) Protein Sci 7 158 Occurrence Handle9514271
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
A. Morton B.W. Matthews (1995) Biochemistry 34 8576 Occurrence Handle10.1021/bi00027a007 Occurrence Handle7612599
C. Machicado M. Bueno J. Sancho (2002) Prot. Eng. 15 669 Occurrence Handle10.1093/protein/15.8.669
A. Morton W.A. Baase B.W. Matthews (1995) Biochemistry 34 8564 Occurrence Handle10.1021/bi00027a006 Occurrence Handle7612598
P. Dauber-Ogusthorpe V.A. Roberts D.J. Ogusthorpe J. Wolff M. Genest A.T. Hagler (1998) ArticleTitleProteins Struct Funct. Genet 4 31
A. Pedretti L. Villa G. Vistoli (2002) J. Mol. Graph. 21 47 Occurrence Handle10.1016/S1093-3263(02)00123-7
A.K. Ghose G.M. Crippen (1987) J. Chem. Inf. Comput. Sci. 27 21 Occurrence Handle10.1021/ci00053a005 Occurrence Handle3558506
T.L. Hill (Eds) (1960) Introduction to Statistical Thermodynamics Addison-Wesley New-York
J. Gasteiger M. Marsali (1980) Tetrahedron 36 3219 Occurrence Handle10.1016/0040-4020(80)80168-2
S.L. Mayo B.D. Olafson W.A. Goddard SuffixIII (1990) J. Phys. Chem. 94 8897 Occurrence Handle10.1021/j100389a010
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
A.T. Hagler E. Huler S. Lifson (1974) J. Am. Chem. Soc. 96 5319 Occurrence Handle10.1021/ja00824a004 Occurrence Handle4851860
C.M. Venkatachalam X. Jiang T. Oldfield M.J. Waldman (2003) Mol. Graphics Modell. 21 289 Occurrence Handle10.1016/S1093-3263(02)00164-X
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
A.M. Jain (1996) J. Comput. Aided Mol. Design 10 427 Occurrence Handle10.1007/BF00124474
I. Muegge Y.C. Martin (1999) J. Med. Chem. 42 791 Occurrence Handle10.1021/jm980536j Occurrence Handle10072678
P.S. Charifson J.J. Corkery M.A. Murcko W.P. Walters (1999) J. Med. Chem. 42 5100 Occurrence Handle10.1021/jm990352k Occurrence Handle10602695
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
P.F.W. Stouten C. Frommel H. Nakamura C. Sander (1993) Mol. Simul. 10 97
Z. Li H.A. Scheraga (1987) Proc. Natl. Acad. Sci. U.S.A. 84 6611 Occurrence Handle3477791
N. Guex M.C. Peitsch (1996) Protein Data Bank Quat. Newslett. 77 77
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
J. Florián A. Warshel (1997) J. Phys. Chem. B 101 5583
A.A. Rashin K. Namboodiri (1987) J. Phys. Chem. 91 6003 Occurrence Handle10.1021/j100307a038
M.L. Connolly (1983) J. Appl. Cryst. 16 548 Occurrence Handle10.1107/S0021889883010985
A. Finkelstein J. Janin (1989) Protein Eng. 3 1 Occurrence Handle2813338
A.V. Jain (2003) J. Med. Chem. 46 499 Occurrence Handle10.1021/jm020406h Occurrence Handle12570372
J. Novotny R.E. Bruccoleri F.A. Saul (1989) Biochemistry 28 4735 Occurrence Handle10.1021/bi00437a034 Occurrence Handle2475171
H.P. Erickson (1989) J. Mol. Biol. 206 465 Occurrence Handle10.1016/0022-2836(89)90494-4 Occurrence Handle2716058
N. Horton M. Lewis (1992) Protein Sci. 1 169 Occurrence Handle1339024
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
B. Lee F.M. Richards (1971) J. Mol. Biol. 55 379 Occurrence Handle10.1016/0022-2836(71)90324-X Occurrence Handle5551392
D. Eisenberg A.D. McLachlan (1989) Nature 319 199 Occurrence Handle10.1038/319199a0
L. Wesson D. Eisenberg (1992) Protein Sci. 2 227
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
F. Sanger S. Nicklen A.R. Coulson (1977) Proc. Natl. Acad. Sci. U.S.A. 74 5463 Occurrence Handle271968
C.G. Genzor A. Beldarrain C. Gomez-Moreno J.L. Lopez-Lacomba M. Cortijo J. Sancho (1996) Protein Sci. 5 1376 Occurrence Handle8819170
D.E. Edmonson G. Tollin (1971) Biochemistry 10 124 Occurrence Handle10.1021/bi00777a019 Occurrence Handle5538602
M.P. Irun S. Maldonado J. Sancho (2001) Protein. Eng. 14 173 Occurrence Handle10.1093/protein/14.3.173 Occurrence Handle11342714
Pace , C.N., Shirley, B.A., and Thompson J.A. (Eds), Protein Structure, a Practical Approach, IRL Press, Oxford, 1989
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
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
J.A. Schellman (1976) Biopolymers 15 999 Occurrence Handle10.1002/bip.1976.360150513
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
A. Lostao C. Gomez-Moreno S.G. Mayhew J. Sancho (1997) Biochemistry 36 14334 Occurrence Handle10.1021/bi971384h Occurrence Handle9398151
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
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
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
E. Akaho G.M. Morris D.S. Goodsell D. Wong A.J. Olson (2001) J. Chem. Sofware 7 103
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
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
M. Rarey B. Kramer T. Lengauer (1997) Proteins (Suppl) 1 221
E.A. Sudbeck C. Mao R. Vig T.K. Venkatachalam L. Tuel-Ahlgren F.M. Uckun (1998) Antimicrob. Agents Chemother. 42 3225 Occurrence Handle9835518
S.T. Rao F. Shaffie C. Yu K.A. Satyshur B.J. Stockman J.L. Markley M. Sundarlingam (1992) Protein Sci. 11 1413
A.M. Buckle P. Cramer A.R. Fersht (1996) Biochemistry 35 4298 Occurrence Handle10.1021/bi9524676 Occurrence Handle8605178
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
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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
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DOI: https://doi.org/10.1007/s10822-005-7969-7