Comparison of X-ray Crystallographic Structures and Docking Models of Dihydrofolate Reductase-Inhibitor Complexes

Dihydrofolate Reductase-저해제 복합체에 대한 X-선 결정체 구조와 docking model의 구조 비교

  • 안미현 (이화여자대학교 약학대학) ;
  • 최인희 (이화여자대학교 약학대학) ;
  • 김춘미 (이화여자대학교 약학대학)
  • Published : 2002.12.01


A comparative study to validate the reliability of a fully automated docking program, FlexiDock, was carried out to predict the binding modes of DHFR-inhibitor complex. The inhibitors were extracted from the crystallographically determined DHFR-NADP$^{+}$(H)-inhibitor ternary complexes of human, Escherichia coli and Candida albicans and then docked back into the remaining DHFR-NADP$^{+}$(H) binary complexes using FlexiDock. The resulting conformations and orientations were compared to the original crystal complex structures for reproducibility. Then, folate, the substrate, and known inhibitors such as methotrexate, piritrexim and trimethoprim were docked into the wild-type human DHFR and their binding modes were compared with X-ray crystallographic or other modeling data. The root mean square deviations (RMSDs) for ligands ranged from 1.14 to 1.57$\AA$, and the protein backbone RMSDs from 0.94 to 1.26$\AA$. FlexiDock reproduced the orientations and binding modes of all seven ligands in good agreement with the crystal structures. It proved to be a reliable and efficient program in studying binding modes of DHFR-inhibitor complexes of different species, and the information obtained from this work may provide additional insight into the design of new agents with improved activity.ity.


  1. Cody, V., Galitsky, N., Luft, J. R., Pangborn, W., Blakley, R. L. and Gangjee, A. : Comparison of ternary crystal complexes of F31 variants of human dihydrofolate reductase with NADPH and a classical antitumor furopyrimidine. Anti-Cancer Drug Design 13, 307 (1998)
  2. Meiering, E. M., Li, H., Delcamp, T. J, Freisheim, J. H. and Wagner, G. : Contributions of tryptophan 24 and glutamate 30 to binding long-lived water molecules in the ternary complex of human dihydrofolate reductase with methotrexate and NADPH studied by site-directed mutagenesis and nuclear magnetic resonance spectroscopy. J. Mol. Biol. 247, 309 (1995)
  3. Chowdhury, S. F., Villamor, V. B., Guerrero, R. H., Leal, I., Brun, R., Croft, S. L., Goodman, J. M., Maes, L., Ruiz-Perez, L. M., Pacanowska, D. G. and Gilbert, I. H. : Design, synthesis, and evaluation of inhibitors of trypanosomal and leishmanial dihydrofolate reductase. J. Med. Chem. 42, 4300 (1999)
  4. Lewis, W. S., Cody, V., Galitsky, N., Luft, J. R., Pangborn, W., Chundurn, S. K., Spencer, H. T., Appleman, J. R. and Blakley, R. L. : Methotrexate-resisteant variants of human dihydrofolate reductase with substitutions of leucine 22. J. Biol. Chem. 270, 5057 (1995)
  5. Walker, V. K., Tyshenko, M. G., Kuiper, M. J., Darger, R. V., Yuhas, D. A., Cruickshank, P. A. and Chaguturu, R. : Tobacco budworm dihydrofolate reductase is a promising target for insecticide discovery. Eur. J Biochem. 267, 394 (2000)
  6. Cody, V., Galitsky, N., Luft, J. R., Pangborn, W., Rosowsky, A. and Blakley, L. : Comparison of two independent crystal structures of human dinucleotide phosphate and the very tight-binding inhibitor PT523. Biochemistry 36, 13897 (1997)
  7. Graffner-Nordberg, M., Marelius. J., Ohlsson, S., Persson, A., Swedberg, G., Andersson, P., Andersson, S. E., Aqvist, J. and Hallberg, A. : Computational predictions of binding affinities to dihydrofolate reductase: synthesis and biological evaluation of methotrexate analogues. J. Med. Chem. 43, 3852 (2000)
  8. Gangjee, A., Vidwans, A. P., Vasudevan, A., Queener, S. F., Kisliuk, R. L., Cody, V., Li, R., Galitsky, N., Luft, J. R. and Pangborn, W. : Structure-based design and synthesis of lipophilic 2,4-diamino-6-substituted quinazolines and their evaluation as inhibitors of dihydrofolate reductases and potential antitumor agents. J. Med. Chem. 41, 3426 (1998)
  9. Barry, I. S., Adam, P. D. and Joseph, R. B. : Dihydrofolate reductase as a therapeutic target. Faseb 4, 2441 (1990)
  10. Oefner, C., D'Arcy, A. and Winkler, F. K. : Crystal structure of human dihydrofolate reductase complexed with folate. Eur. J. Biochem. 174, 377 (1988)
  11. Sybyl version 6.6. FlexiDock Manual, Tripos Co.: Receptor-Based Design Manual, Ligand-Based Design Manual, ForceField Manual, Getting started (1999)
  12. Kim, S. H. and Katzenellenbogen, J. A.: Triarylethylene bisphenols with a novel cycle are ligands for the estrogen receptor. Bioorganic & Med. Chem. 8, 785 (2000)
  13. Stauffer, S. R., Huang, Y., Coletta, C. J., Tedesco, R. and Katzenellenbogen, J. A. : Estrogen pyrazoles: Defining the pyrazole core structure and the orientation of substituents in the ligand binding pocket of the estrogen receptor. Bioorg. Med. Chem. 9, 141 (2001)
  14. Stauffer, S. R., Huang, Y. R., Aron, Z. D., Coletta, C. J., Sun, J., Katzenellenbogen, B. S. and Katzenellenbogen, J. A. : Triarylpyrazoles with basic side chains: development of pyrazole-based estrogen receptor antagonists. Bioorg. Med. Chem. 9, 151 (2001)
  15. Inoue, J., Cui, Y. S., Sakai, O., Nakamura, Y., Kogiso, H. and Kador, P. F. : Synthesis and aldose reductase inhibitory activities of novel N-nitromethylsulfonanilide derivatives. Bioorg. Med. Chem. 8, 2167 (2000)
  16. Chebib, M., McKeveney, D. and Quinn, R. J. : 1-Phenylpyrazolo[3,4-d]pyrimidines; structure-activity relationships for C6 substituents at A1 and A2A adenosine receptors. Bioorg. Med. Chem. 8, 2581 (2000).
  17. Dhar, A., Liu, S., Klucik, J., Berlin, K. D., Madler, M. M., Lu, S., lvey, R. T., Zacheis, D., Brown, C. W., Nelson, E. C., Birckbichler, P. J. and Benbrook, D. M. : Synthesis, structureactivity relationships, and RARgamma-ligand interactions of nitrogen heteroarotinoids. J. Med. Chem. 42, 3602 (1999)
  18. Volter, K. E., Embrey, K. J., Pierens, G. K. and Quinn, R. J. : A study of the binding requirements of calyculin A and dephosphonocalyculin A with PP1, development of a molecular recognition model for the binding interactions of the okadaic acid class of compounds with PP1. Eur. J. Pharm. Sci. 12, 181 (2001)
  19. Whitlow, M., Howard, A. J., Stewart, D., Hardman, K. D., Kuyper, L. F., Baccanari, D. P., Fling, M. E. and Tansik, R. L. : X-ray crystallographic studies of Candida albicans dihydrofolate reductase. High resolution structures of the holoenzyme and an inhibited ternary complex. J. Biol. Chem. 272, 30289 (1997)
  20. Sawaya, M. R. and Kraut, J.: Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crysrallographic evidence. Biochemistry 36, 586 (1997)
  21. Dauber-Osguthorpe, P., Roberts, V. A., Osguthorpe, D. J., Wolff, J., Genest, M. and Hagler, A. T. : Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system. Proteins. 4, 31 (1988)
  22. Klon, A. E., Heroux, A., Ross, L. J., Pathak, V., Johnson, C. A., Piper, J. R. and Borhani, D. W. : Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 $\AA$ and 1.05 $\AA$ resolution. J. Mol. Biol. 320, 677 (2002)
  23. Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J., Voltz, K. W. and Kraut, J. : Dihydrofolate reductase. The stereochemistry of inhibitor selectivity. J. Biol. Chem. 260, 392 (1985)
  24. Pan, R., Bowen, D. and Southerland, W. M. : Molecular modelling of trimethoprim complexes of human wild-type and mutant dihydrofolate reductases: identification of two subsets of binding residues in the antifolate binding site. Biopharm. Drug. Dispos. 20, 335 (1999)