Computer modeling studies of the structural role of NADPH binding to active site mutants of human dihydrofolate reductase in complex with piritrexim.

• Authors: Wiesław Nowak , Vivian Cody , Andrzej Wojtczak
• Languages of publication: EN

Abstracts

EN

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays an important role in DNA synthesis. Inhibition of DHFR is a key element in the treatment of many diseases, including cancer and AIDS related infections. A search for new selective inhibitors is motivated by the resistance to common drugs observed in the course of treatment. In this paper, results of a detailed computer analysis of human DHFR interactions with the lipophilic inhibitor piritrexim (PTX) are presented. It was found that the NADPH cofactor contributes 30% of the total PTX-enzyme interaction energy. Substitution of the highly conserved Glu30 with alanine does not lead to the release of the inhibitor from the hDHFR pocket. The important L22F point mutation does affect PTX orientation but does not change the binding energy. Simulations of the dynamics of binary hDHFR-TX complexes were performed with the use of Extensible Systematic Force Field (ESFF) and the results indicate structural changes in the enzyme induced by NADPH binding.

Keywords

EN

ESFF forcefield; NADPH; dihydrofolate reductase; enzyme structure; chemotherapy; piritrexim; computer modeling

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2001
• Volume: 48
• Issue: 4
• Pages: 903-916

Dates

published

2001

received

2001-11-19

accepted

2001-12-03

Contributors

author

Wiesław Nowak

• Institute of Physics, Nicholas Copernicus University, Toruń, Poland

author

Vivian Cody

• Hauptman-Woodward Medical Research Institute, Buffalo, U.S.A.

author

Andrzej Wojtczak

• Institute of Chemistry, Nicholas Copernicus University, Toruń, Poland

References

• 1. Blakley, R.L. (1995) Advances in Enzymology and Related Areas of Molecular Biology; vol. 70, pp. 23-102, John Wiley & Sons, Inc.
• 2. Takimoto, C.H. (1996) New antifolates: Pharmacology and clinical applications. Oncologist 1, 68-81.
• 3. Patel, M., Sleep, S.E., Lewis, W.S., Spencer, H.T., Mareya, S.M., Sorrentino, B.P. & Blakley, R.L. (1997) Comparison of the protection of cells from antifolates by transduced human dihydrofolate reductase mutants. Hum. Gene Ther. 8, 2069-2077.
• 4. Chunduru, S. K., Cody, V., Luft, J.R., Pangborn, W., Appleman, J.R. & Blakley, R.L. (1994) Methotrexate-resistant variants of human dihydrofolate reductase. Effects of Phe31 substitutions. J. Biol. Chem. 269, 9547-9555.
• 5. Lewis, W.S., Cody, V., Galitsky, N., Luft, J.R., Pangborn, W., Chunduru, S.K., Spencer, H.T., Appleman, J.R. & Blakley, R.L. (1995) Methotrexate-resistant variants of human dihydrofolate reductase with substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers. J. Biol. Chem. 270, 5057-5064.
• 6. Grivsky, E.M., Lee, S., Sigel, C.W. & Nichol, C.A. (1980) Synthesis and antitumor activity of 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidine. J. Med. Chem. 23, 327-329.
• 7. Khorsand, M., Lange, J., Feun, L., Clendeninn, N.J., Collier, M. & Wilding, G. (1997) Phase II trial of oral piritrexim in advanced, previously treated transitional cell cancer of bladder. Invest. New Drugs 15, 157-163.
• 8. Graffner-Nordberg, M., Kolmodin, K., Aqvist, J., Queener, S.F. & Hallberg, A. (2001) Design, synthesis, computational prediction, and biological evaluation of ester soft drugs as inhibitors of dihydrofolate reductase from Pneumocystis carinii. J. Med. Chem. 44, 2391-2402.
• 9. Cinquina, C.C., Grogan, E., Sun, R., Lin, S.F., Beardsley, G.P. & Miller, G. (2000) Dihydrofolate reductase from Kaposi's sarcoma-associated herpesvirus. Virology 268, 201-217.
• 10. Cody, V., Wojtczak, A., Kalman, T.I., Freisheim, J.H. & Blakley, R.L. (1993) in: Chemistry and Biology of Pteridines and Folates (Ayling, J.E., Nair, M.G. & Baugh, C.M., eds.) pp. 481-486, Plenum Press.
• 11. Nowak, W., Wojtczak, A. & Cody, V. (1999) Computer modeling of human dihydrofolate reductase ternary complex with NADPH and piritreim (PTX) inhibitor. Internet J. Chem. 12, www.ijc.com/articles/1999v2/50/.
• 12. Sawaya, M.R. & Kraut, J. (1997) Loop and subdomain movements in mechanism of Escherichia coli dihydrofolate reductase: Crystallographic evidence. Biochemistry 36, 586-603.
• 13. Miller, P.G. & Benkovic, S.J. (1998) Stretching exercises - flexibility in dihydrofolate reductase catalysis. Chem. Biol. 5, R105-R113.
• 14. Osborne, M.J., Schnell, J., Benkovic, S.J., Dyson, H.J. & Wright, P.E. (2001) Backbone dynamics in dihydrofolate reductase complexes: Role of flexibility in the catalytic mechanism. Biochemistry 40, 9846-9859.
• 15. Johnson, J.M., Meiering, E.M., Wright, J.E., Pardo, J., Rosowsky, A. & Wagner, G. (1997) NMR solution structure of the antitumor compound PT523 and NADPH in the ternary complex with human dihydrofolate reductase. Biochemistry 36, 4399-4411.
• 16. Cody, V., Galitsky, N., Luft, J.R., Pangborn, W., Rosowsky, A. & Blakley, R.L. (1997) Comparison of two independent crystal structures of human dihydrofolate reductase ternary complexes reduced with nicotinamide adenine dinucleotide phosphate and the very tight-binding inhibitor PT523. Biochemistry 36, 13897-13903.
• 17. Pan, H., Lee, J.C. & Hilser, V.J. (2000) Binding sites in Escherichia coli dihydrofolate reductase communicate by modulating the conformational ensemble. Proc. Natl. Acad. Sci. U.S.A. 97, 12020-12025.
• 18. Bystroff, C. & Kraut, J. (1991) Crystal structure of unliganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding. Biochemistry 30, 2227-2239.
• 19. Carugo, O. & Argos, P. (1997) NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding. Proteins 28,10-28.
• 20. Whitlow, M., Howard, A.J., Stewart, D., Hardman, K.D, Chan, J.H., Baccanari, D.P., Tansik, R.L., Hong, J.S. & Kuyper, L.F. (2001) X-Ray crystal structures of Candida albicans dihydrofolate reductase: High resolution ternary complexes in which the dihydronicotinamide moiety of NADPH is displaced by an inhibitor. J. Med. Chem. 44, 2928-2932.
• 21. Verma, C.S., Caves, L.D.S., Hubbard, R.E. & Roberts, G.C.K. (1997). Domain motions in dihydrofolate reductase: A molecular dynamics study. J. Mol. Biol. 266, 776-796.
• 22. Graffner-Nordberg, M., Marelius, J., Ohlsson, S., Persson, A., Swedberg, G., Andersson, P., Andersson, S.E., Aqvist, J. & Hallberg, A. (2000) Computational predictions of binding affinities to dihydrofolate reductase: Synthesis and biological evaluation of methotrexate analogues. J. Med. Chem. 43, 3852-3861.
• 23. Pitts, C., Bowen, D. & Southerland, W.M. (2000) Interaction energy analyses of folate analog binding to human dihydrofolate reductase: Contribution of the antifolate substructural regions to complex stability. Drug Metabol. Drug Interact. 16, 99-121.
• 24. Cummins, P.L. & Gready, J.E. (2001) Energetically most likely substrate and active-site protonation sites and pathways in the catalytic mechanism of dihydrofolate reductase. J. Am. Chem. Soc. 123, 3418-3428.
• 25. Verkhivker, G.M., Rejto, P.A., Bouzida, D., Arthurs, S., Colson, A.B., Freer, S.T., Gehlhaar, D.K., Larson, V., Luty, B.A., Marrone, T. & Rose, P.W. (2001) Navigating ligand-protein binding free energy landscapes: Universality and diversity of protein folding and molecular recognition mechanisms. Chem. Phys. Lett. 336, 495-503.
• 26. Sham, Y.Y., Ma, B., Tsai, C.J. & Nussinov, R. (2001) Molecular dynamics simulation of Escherichia coli dihydrofolate reductase and its protein fragments: Relative stabilities in experiment and simulations. Protein Sci. 10, 135-348.
• 27. Barlow, S., Rohl, A.L., Shi, S.G., Freeman, C.M. & O'Hare, D. (1996) Molecular mechanics study of oligomeric models for poly(ferrocenylsilanes) using the Extensible Systematic Forcefield (ESFF). J. Am. Chem. Soc. 118, 7578-7592.
• 28. Forcefield-Based Simulations (1997) Molecular Simulations Inc., San Diego.
• 29. Andersen, H.C. (1983) RATTLE: A velocity version of the SHAKE algorithm for molecular dynamics. J. Comp. Physics 52, 24-34.
• 30. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. (1993) PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283-291.
• 31. Brunger, A.T. (1992) X-PLOR Version 3.1: A System for X-Ray Crystallography and NMR, Yale University Press, New Haven.
• 32. Cody, V., Galitsky, N., Rak, D., Luft, J.R., Pangborn, W. & Queener, S.F. (1999) Ligand- induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+. Biochemistry 38, 4303-4312.
• 33. Howell, E.E., Villafranca, J.E., Warren, M.S., Oatley, S.J. & Kraut, J. (1986) Functional role of aspartic acid-27 in dihydrofolate aeductase revealed by mutagenesis. Science 231, 1123-1128.
• 34. Li, R., Sirawaraporn, R., Chitnumsub, P., Sirawaraporn, W., Wooden, J., Athappilly, F., Turley, S. & Hol, W.G. (2000) Three-dimensional structure of M.tuberculosis dihydrofolate reductase reveals opportunities for the design of novel tuberculosis drugs. J. Mol. Biol. 295, 307-323.
• 35. Gargaro, A.R., Soteriou, A., Frenkiel, T.A., Bauer, C.J., Birdsall, B., Polshakov, V.I., Barsukov, I.L., Roberts, G.C.K. & Feeney, J. (1998) The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate. J. Mol. Biol. 277, 119-134.
• 36. Evans, S.V. (1993) SETOR: Hardware lighted three-dimensional solid model representations of macromolecules. J. Mol. Graph 11, 134-138.