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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: ykkitade@gifu-u.ac.jp
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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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