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Thymidine kinase and uridine-cytidine kinase from Entamoeba histolytica: cloning, characterization, and search for specific inhibitors

Published online by Cambridge University Press:  16 April 2009

A. LOSSANI ,
A. TORTI ,
S. GATTI ,
A. BRUNO ,
R. MASERATI ,
G. E. WRIGHT  and
F. FOCHER
A. LOSSANI
Affiliation:
Istituto di Genetica Molecolare, CNR, Pavia, Italy
A. TORTI
Affiliation:
Istituto di Genetica Molecolare, CNR, Pavia, Italy
S. GATTI
Affiliation:
Laboratorio di Parassitologia, Servizio Virologia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
A. BRUNO
Affiliation:
Dipartimento di Malattie Infettive, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
R. MASERATI
Affiliation:
Dipartimento di Malattie Infettive, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
G. E. WRIGHT*
Affiliation:
GLSynthesis Inc., Worcester, MA, USA
F. FOCHER
Affiliation:
Istituto di Genetica Molecolare, CNR, Pavia, Italy
*
*Corresponding author: GLSynthesis Inc., One Innovation Drive, Worcester, MA 01605USA. E-mail: [email protected]
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Summary

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0·27 μm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0·74 and 0·22 mm, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.

Keywords

Entamoebaamoebiasiskinasesinhibitorsdrugs
Type
Research Article
Information
Parasitology , Volume 136 , Issue 6 , May 2009 , pp. 595 - 602
Copyright
Copyright © Cambridge University Press 2009

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References

REFERENCES

Bansal, D., Sehgal, R., Chawla, Y., Mahajan, R. C. and Malla, N. (2004). In vitro activity of antiamoebic drugs against clinical isolates of Entamoeba histolytica and Entamoeba dispar. Annals of Clinical Microbiology and Antimicrobials 21, 3–27. doi: 10.1186/1476-0711-3-27.Google Scholar
Bansal, D., Malla, N. and Mahajan, R. C. (2006). Drug resistance in amoebiasis. Indian Journal of Medical Research 123, 115–118.Google ScholarPubMed
Gatti, S., Swierczynski, G., Robinson, F., Anselmi, M., Corrales, J., Moreira, J., Montalvo, G., Bruno, A., Maserati, R., Bisoffi, Z. and Scaglia, M. (2002). Amebic infections due to the Entamoeba histolytica-Entamoeba dispar complex: a study of the incidence in a remote rural area of Ecuador. American Journal of Tropical Medicine and Hygiene 67, 123–127.CrossRefGoogle Scholar
Leitsch, D., Kolarich, D., Wilson, I. B. H., Altmann, F. and Duchene, M. (2007). Metronidazole action in Entamoeba histolytica: a central role for thioredoxin reductase. PLoS Biology 5, 1820–1834.Google Scholar
Loftus, B., Anderson, I., Davies, R., Alsmark, U. C., Samuelson, J., Amedeo, P., Roncaglia, P., Berriman, M., Hirt, R. P., Mann, B. J., Nozaki, T., Suh, B., Pop, M., Duchene, M., Ackers, J., Tannich, E., Leippe, M., Hofer, M., Bruchhaus, I., Willhoeft, U., Bhattacharya, A., Chillingworth, T., Churcher, C., Hance, Z., Harris, B., Harris, D., Jagels, K., Moule, S., Mungall, K., Ormond, D., Squares, R., Whitehead, S., Quail, M. A., Rabbinowitsch, E., Norbertczak, H., Price, C., Wang, Z., Guillén, N., Gilchrist, C., Stroup, S. E., Bhattacharya, S., Lohia, A., Foster, P. G., Sicheritz-Ponten, T., Weber, C., Singh, U., Mukherjee, C., El-Sayed, N. M., Petri, W. A. Jr, Clark, C. G., Embley, T. M., Barrell, B., Fraser, C. M. and Hall, N. (2005). The genome of the protist parasite Entamoeba histolytica. Nature, London 433, 865–868. doi: 10.1038/nature03291.Google Scholar
Makioka, A., Kumagai, M., Kobayashi, S. and Takeuchi, T. (2002). Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin. Journal of Parasitology 88, 994–999.CrossRefGoogle ScholarPubMed
Maga, G., Spadari, S., Wright, G. E. and Focher, F. (1994). Identification, partial purification and inhibition by guanine analogues of a novel enzymic activity which phosphorylates guanosine to GMP in the protozoan parasite Eimeria tenella. The Biochemical Journal 298, 289–294.Google Scholar
Ranque, S., Molet, B., Christmann, D. and Candolfi, E. (2004). In vitro activity of azithromycin and dirithromycin against axenic Entamoeba histolytica. European Journal of Clinical Microbiology & Infectious Diseases 23, 932–933. doi: 10.1007/s10096-004-1250-1.Google ScholarPubMed
Rossignol, J. F., Kabil, S. M., El-Gohary, Y. and Younis, A. M. (2007). Nitazoxanide in the treatment of amoebiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 101, 1025–1031. doi: 10.1016/j.trstmh.2007.04.001.Google Scholar
Roy, B., Verri, A., Lossani, A., Spadari, S., Focher, F., Aubertin, A. M., Gosselin, G., Mathé, C. and Périgaud, C. (2004). Enantioselectivity of ribonucleotide reductase: a first study using stereoisomers of pyrimidine 2′-azido-2′-deoxynucleosides. Biochemical Pharmacology 68, 711–718. doi: 10.1016/j.bcp.2004.05.002.CrossRefGoogle Scholar
Samarawickrema, N. A., Brown, D. M., Upcroft, J. A., Thammapalerd, N. and Upcroft, P. (1997). Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole resistance in Entamoeba histolytica. Journal of Antimicrobial Chemotherapy 40, 833–840.Google Scholar
Sargeaunt, P. G., Williams, J. E. and Grene, J. D. (1978). The differentiation of invasive and non-invasive Entamoeba histolytica by isoenzyme electrophoresis. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 519–520.Google Scholar
Seifert, K., Duchene, M., Wernsdorfer, W. H., Kollaritsch, H., Scheiner, O., Wiedermann, G., Hottkowitz, T. and Eibl, H. (2001). Effects of miltefosine and other alkylphosphocholines on human intestinal parasite Entamoeba histolytica. Antimicrobial Agents and Chemotherapy 45, 1505–1510. doi: 10.1128/AAC.45.5.1505-1510.2001.Google Scholar
Spadari, S., Maga, G., Focher, F., Ciarrocchi, G., Manservigi, R., Arcamone, F., Capobianco, M., Carcuro, A., Colonna, F., Iotti, S. and Garbesi, A. (1992). L-thymidine is phosphorylated by Herpes simplex virus type 1 thymidine kinase and inhibits viral growth. Journal of Medicinal Chemistry 35, 4214–4220.CrossRefGoogle ScholarPubMed
Spadari, S., Maga, G., Verri, A. and Focher, F. (1998). Molecular basis for the antiviral and anticancer activities of unnatural L-β-nucleosides. Expert Opinion on Investigational Drugs 7, 1285–1300.Google Scholar
Stanley, S. L. Jr. (2003). Amoebiasis. Lancet 361, 1025–1034.Google Scholar
Striepen, B., Pruijssers, A. J. P., Huang, J., Li, C., Gubbels, M.-J., Umejiego, N. N., Hedstrom, L. and Kissinger, J. C. (2004). Gene transfer in the evolution of parasite nucleoside biosynthesis. Proceedings of the National Academy of Sciences, USA 101, 3154–3159. doi: 10.1073/pnas.0304686101.Google Scholar
Strosselli, S., Spadari, S., Walker, R. T., Basnak, I. and Focher, F. (1998). Trichomonas vaginalis thymidine kinase: purification, characterization and search for inhibitors. The Biochemical Journal 334, 15–22.Google Scholar
Suzuki, N. N., Koizumi, K., Fukushima, M., Matsuda, A. and Inagaki, F. (2004). Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase. Structure 12, 751–764. doi: 10.1016/j.cell.2006.11.023.CrossRefGoogle ScholarPubMed
Wassmann, C., Hellberg, A., Tannich, E. and Bruchhaus, I. (1999). Metronidazole resistance in the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase. Journal of Biological Chemistry 274, 26051–26056.Google Scholar
World Health Organization (1997). A consultation with experts on amoebiasis. Epidemiological Bulletin 18, 13–14.Google Scholar