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Mutational, inhibitory and microcalorimetric analyses of Plasmodium falciparum TMP kinase. Implications for drug discovery

Published online by Cambridge University Press:  07 January 2009

M. KANDEEL ,
T. ANDO ,
Y. KITAMURA ,
M. ABDEL-AZIZ  and
Y. KITADE
M. KANDEEL
Affiliation:
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan Department of Pharmacology, Faculty of Veterinary Medicine, Kafr El-Shikh University, Kafr El-Shikh, Egypt
T. ANDO
Affiliation:
Center for Emerging Infectious Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
Y. KITAMURA
Affiliation:
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
M. ABDEL-AZIZ
Affiliation:
Department of Pharmacology, Faculty of Veterinary Medicine, Kafr El-Shikh University, Kafr El-Shikh, Egypt
Y. KITADE*
Affiliation:
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan Center for Emerging Infectious Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan Center for Advanced Drug Research, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
*
*Corresponding author: Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan. Tel/Fax: +81 58 293 2640. E-mail: [email protected]
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Summary

Plasmodium falciparum thymidylate kinase (PfTMK) can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. The enzyme not only phosphorylates TMP and dUMP but can also tolerate bulkier purines, namely, dGMP, GMP, and dIMP. In order to probe the flexibility of PfTMK in accommodating ligands of various sizes, we developed 6 mutant enzymes and subjected these to thermodynamic, inhibitory and catalytic evaluation. Kinase activity was markedly affected by introducing a larger lysine residue instead of A111. The lack of the hydroxyl group after inducing mutation of Y107F affected enzyme activity, and had a more severe impact on dGMP kinase activity. PfTMK can be inhibited by both purine and pyrimidine nucleosides, raising the possibility of developing highly selective drugs. Thermodynamic analysis revealed that enthalpic forces govern both purine and pyrimidine nucleoside monophosphate binding, and the binding affinity of both substrates was highly comparable. The heat produced due to dGMP binding is lower than that attributable to TMP. This indicates that additional interactions occur with TMP, which may be lost with larger dGMP. Targeting PfTMK not only affects thymidine nucleotide synthesis but may also affect purine nucleotides, and thus the enzyme represents an attractive antimicrobial target.

Keywords

Plasmodium falciparumthymidylate kinaseTMP kinasesite-directed mutagenesisdrug targets
Type
Research Article
Information
Parasitology , Volume 136 , Issue 1 , January 2009 , pp. 11 - 25
Copyright
Copyright © 2009 Cambridge University Press

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References

REFERENCES

Andrade, M. A., Chacón, P., Merelo, J. J. and Morán, F. (1993). Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Engineering 6, 383–390.CrossRefGoogle ScholarPubMed
Blondin, C., Serina, L., Wiesmüller, L., Gilles, A. M. and Bârzu, O. (1994). Improved spectrophotometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase/lactate dehydrogenase coupling system. Analytical Biochemistry 220, 219–221.CrossRefGoogle ScholarPubMed
Breman, J. G., Egan, A. and Keusch, G. T. (2001). The intolerable burden of malaria: a new look at the numbers. American Journal of Tropical Medicine and Hygiene 64, 4–7.Google Scholar
Brown, D. G., Visse, R., Sandhu, G., Davies, A., Rizkallah, P. J., Melitz, C., Summers, W. C. and Sanderson, M. R. (1995). Crystal structures of the thymidine kinase from herpes simplex virus type-1 in complex with deoxythymidine and ganciclovir. Nature Structural Biology 2, 876–881.Google Scholar
Brundiers, R., Lavie, A., Veit, T., Reinstein, J., Schlichting, I., Ostermann, N., Goody, R. S. and Konrad, M. (1999). Modifying human thymidylate kinase to potentiate azidothymidine activation. Journal of Biological Chemistry 274, 35289–35292.CrossRefGoogle ScholarPubMed
Champness, J. N., Bennett, M. S., Wien, F., Visse, R., Summers, W. C., Herdewijn, P., de Clerq, E., Ostrowski, T., Jarvest, R. L. and Sanderson, M. R. (1998). Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands. Proteins 32, 350–361.Google Scholar
De Winter, H. and Herdewijn, P. (1996). Understanding the binding of 5-substituted 2′-deoxyuridine substrates to thymidine kinase of Herpes simplex virus type-1. Journal of Medicinal Chemistry 39, 4727–4737.Google Scholar
El Omari, K., Liekens, S., Bird, L. E., Balzarini, J. and Stammers, D. K. (2006). Mutations distal to the substrate site can affect varicella zoster virus thymidine kinase activity: implications for drug design. Molecular Pharmacology 69, 1891–1896.Google Scholar
Guerin, P. J., Olliaro, P., Nosten, F., Druilhe, P., Laxminarayan, R., Binka, F., Kilama, W. L., Ford, F. and White, N. J. (2002). Malaria: current status of control, diagnosis, treatment, and a proposed agenda for research and development. The Lancet Infectious Diseases 2, 564–573.Google Scholar
Haouz, A., Vanheusden, V., Munier-Lehmann, H., Froeyen, M., Herdewijn, P., Van Calenbergh, S. and Delarue, M. (2003). Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism. Journal of Biological Chemistry 14, 4963–4971.Google Scholar
Kandeel, M. and Kitade, Y. (2008). Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase. Journal of Biochemistry 144, 245–250.Google Scholar
Kandeel, M., Nakanishi, M., Ando, T., El-Shazly, K., Yosef, T., Ueno, Y. and Kitade, Y. (2008) Molecular cloning, expression, characterization and mutation of Plasmodium falciparum guanylate kinase. Molecular and Biochemical Parasitology 159, 130–133.CrossRefGoogle ScholarPubMed
Ke, S. H. and Madison, E. L. (1997) Rapid and efficient site-directed mutagenesis by single-tube ‘megaprimer’ PCR method. Nucleic Acids Research 25, 3371–3372.Google Scholar
Kotaka, M., Dhaliwal, B., Ren, J., Nichols, C. E., Angell, R., Lockyer, M., Hawkins, A. R. and Stammers, D. K. (2006). Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding. Protein Science 15, 774–784.Google Scholar
Ladbury, J. E. (2001). Isothermal titration calorimetry: application to structure-based drug design. Thermochimica Acta 380, 209–215.Google Scholar
Lavie, A., Ostermann, N., Brundiers, R., Goody, R. S., Reinstein, J., Konrad, M. and Schlichting, I. (1998). Structural basis for efficient phosphorylation of 3′-azidothymidine monophosphate by Escherichia coli thymidylate kinase. Proceedings of the National Academy of Sciences, USA 95, 14045–14050.Google Scholar
Liang, Y. (2008). Applications of isothermal titration calorimetry in protein science. Acta Biochimica et Biophysica Sinica (Shanghai) 40, 565–576.Google Scholar
Li de la Sierra, I., Munier-Lehmann, H., Gilles, A. M., Bârzu, O. and Delarue, M. (2001). X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1·95 A resolution. Journal of Molecular Biology 311, 87–100.CrossRefGoogle ScholarPubMed
Manallack, D. T., Pitt, W. R., Herdewijn, P., Balzarini, J., De Clercq, E., Sanderson, M. R., Sohi, M., Wien, F., Munier-Lehmann, H., Haouz, A. and Delarue, M. (2002). Database searching for thymidine and thymidylate kinase inhibitors using three-dimensional structure-based methods. Journal of Enzyme Inhibition and Medicinal Chemistry 17, 167–174.CrossRefGoogle ScholarPubMed
Ostermann, N., Segura-Peña, D., Meier, C., Veit, T., Monnerjahn, C., Konrad, M. and Lavie, A. (2003). Structures of human thymidylate kinase in complex with prodrugs: implications for the structure-based design of novel compounds. Biochemistry 42, 2568–2577.Google Scholar
Pierce, M. M., Raman, C. S. and Nall, B. T. (1999). Isothermal titration calorimetry of protein-protein interactions. Methods 19, 213–221.Google Scholar
Pochet, S., Dugué, L., Labesse, G., Delepierre, M. and Munier-Lehmann, H. (2003). Comparative study of purine and pyrimidine nucleoside analogues acting on the thymidylate kinases of Mycobacterium tuberculosis and of humans. Chembiochem 4, 742–747.CrossRefGoogle ScholarPubMed
Reyes, P., Rathod, P. K., Sanchez, D. J., Mrema, J. E., Rieckmann, K. H. and Heidrich, H. G. (1982). Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Molecular and Biochemical Parasitology 5, 275–290.Google Scholar
Velázquez Campoy, A. and Freire, E. (2005). ITC in the post-genomic era…? Priceless. Biophysical Chemistry 115, 115–124.Google Scholar
Vogt, J., Perozzo, R., Pautsch, A., Prota, A., Schelling, P., Pilger, B., Folkers, G., Scapozza, L. and Schulz, G. E. (2000). Nucleoside binding site of herpes simplex type 1 thymidine kinase analyzed by X-ray crystallography. Proteins 41, 545–553.3.0.CO;2-8>CrossRefGoogle ScholarPubMed
Weber, P. C. and Salemme, F. R. (2003). Applications of calorimetric methods to drug discovery and the study of protein interactions. Current Opinion in Structural Biology 13, 115–121.CrossRefGoogle Scholar
Zhang, Y. (2005). The magic bullets and tuberculosis drug targets. Annual Review of Pharmacology and Toxicology 45, 529–564.Google Scholar