Abstract
Bacillus anthracis, which causes anthrax, has attracted attention because of its potential use as a biological weapon. The risk of multidrug resistance against B. anthracis increases the need for antibiotics with new molecular targets. Nucleoside analogs are well-known antiviral and anticancer prodrugs, and thymidine kinase catalyzes the rate-limiting step in the activation of pyrimidine nucleoside analogs used in chemotherapy. The thymidine kinase gene from B. anthracis Sterne strain (34F2) (Ba-TK) was cloned and expressed in E. coli, and the product was purified and characterized regarding its substrate specificity. Ba-TK phosphorylated pyrimidine nucleosides and all natural nucleoside triphosphates served as phosphate donors. Size exclusion chromatography indicated a dimeric form of Ba-TK, regardless of the presence of ATP. Thymidine was the most efficient substrate with a low Km value (0.6 μM) and a Vmax of 3.3 μmol dTMP mg-1 min-1, but deoxyuridine (Km=4.2 μM, Vmax=4.1 μmol dUMP mg-1 min-1) was also a good substrate. Several pyrimidine analogs were also tested and analogs with 5-position modifications showed higher activities compared to analogs with 3′- and N3-position modifications. Deoxyuridine analogs were the most potent inhibitors of B. anthracis growth in vitro. These results may be used to guide future development of nucleoside analogs against B. anthracis.
References
Al-Madhoun, A., Tjarks, W., and Eriksson, S. (2004). The role of thymidine kinase in the activation of pyrimidine nucleoside analogues. Mini Rev. Med. Chem.4, 341–350.10.2174/1389557043403963Search in Google Scholar
Auerbach, S. and Wright, G. (1955). Studies on immunity in anthrax. VI. Immunizing activity of protective antigen against various strains of Bacillus anthracis. J. Immunol.75, 129–133.10.4049/jimmunol.75.2.129Search in Google Scholar
Balzarini, J., Herdewijn, P., and De Clercq, E. (1989). Differential patterns of intracellular metabolism of 2′,3′-didehydro2′,3′-dideoxythymidine and 3′-azido-2′,3′-dideoxythymidine, two potential anti-human immunodeficiency virus compounds. J. Biol. Chem.264, 6127–6133.10.1016/S0021-9258(18)83322-1Search in Google Scholar
Bandyopadhyaya, A., Johnsamuel, J., Al-Madhoun, A., Eriksson, S., and Tjarks, W. (2005). Comparative molecular field analysis and comparative molecular similarity indices analysis of human thymidine kinase 1 substrates. Bioorg. Med. Chem.13, 1681–1689.10.1016/j.bmc.2004.12.008Search in Google Scholar
Black, M. and Hruby, D. (1990). Identification of the ATP-binding domain of vaccinia virus thymidine kinase. J. Biol. Chem.265, 17584–17592.10.1016/S0021-9258(18)38204-8Search in Google Scholar
Blondin, C., Serina, L., Wiesmuller, L., Gilles, A., and Barzu, O. (1994). Improved spectrophotometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase/lactate dehydrogenase coupling system. Anal. Biochem.220, 219–221.10.1006/abio.1994.1326Search in Google Scholar
Brook, I., Elliott, T., Pryor, H.N., Sautter, T., Gnade, B., Thakar, J., and Knudson, G. (2001). In vitro resistance of Bacillus anthracis Sterne to doxycycline, macrolides and quinolones. Int. J. Antimicrob. Agents18, 559–562.10.1016/S0924-8579(01)00464-2Search in Google Scholar
Carnrot, C., Wehelie, R., Eriksson, S., Bölske, G., and Wang, L. (2003). Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth. Mol. Microbiol.50, 771–780.10.1046/j.1365-2958.2003.03717.xSearch in Google Scholar PubMed
Cole, L. (1996). The specter of biological weapons. Sci. Am.275, 60–65.10.1038/scientificamerican1296-60Search in Google Scholar PubMed
Dawson, L. and Lawrence, T. (2004). The role of radiotherapy in the treatment of liver metastases. Cancer J.10, 139–144.10.1097/00130404-200403000-00009Search in Google Scholar PubMed
Eriksson, S., Munch-Petersen, B., Johansson, K., and Eklund, H. (2002). Structure and function of cellular deoxyribonucleoside kinases. Cell. Mol. Life Sci.59, 1327–1346.10.1007/s00018-002-8511-xSearch in Google Scholar PubMed
Friedlander, A. (2000). Anthrax: clinical features, pathogenesis, and potential biological warfare threat. Curr. Clin. Top. Infect. Dis.20, 335–349.Search in Google Scholar
Friedlander, A., Welkos, S., Pitt, M., Ezzell, J., Worsham, P., Rose, K., Ivins, B., Lowe, J., Howe, G., Mikesell, P., et al. (1993). Postexposure prophylaxis against experimental inhalation anthrax. J. Infect. Dis.167, 1239–1243.10.1093/infdis/167.5.1239Search in Google Scholar
Furman, P., Fyfe, J., St Clair, M., Weinhold, K., Rideout, J., Freeman, G., Nusinoff Lehrmann, S., Bolognesi, D., Broder, S., et al. (1986). Phosphorylation of 3′-azido-3′-deoxythymidine and selective interaction of the 5′-triphosphate with human immunodeficiency virus reverse transcriptase. Proc. Natl. Acad. Sci. USA83, 8333–8337.10.1073/pnas.83.21.8333Search in Google Scholar
Henderson, D., Peacock, S., and Belton, F. (1956). Observations on the prophylaxis of experimental pulmonary anthrax in the monkey. J. Hyg.54, 28–36.10.1017/S0022172400044272Search in Google Scholar
Inglesby, T., Henderson, D., Bartlett, J., Ascher, M., Eitzen, E., Friedlander, A., Hauer, J., McDade, J., Osterholm, M., O'Toole, T., et al. (1999). Anthrax as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. J. Am. Med. Assoc.281, 1735–1745.10.1001/jama.281.18.1735Search in Google Scholar
Jernigan, J. (2001). Clinical trials report. Curr. Infect. Dis. Rep.3, 505–511.Search in Google Scholar
Johansson, N. and Eriksson, S. (1996). Structure-activity relationships for phosphorylation of nucleoside analogs to monophosphates by nucleoside kinases. Acta Biochim. Pol.43, 143–160.10.18388/abp.1996_4573Search in Google Scholar
Kosinska, U., Carnrot, C., Eriksson, S., Wang, L., and Eklund, H. (2005). Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme. FEBS J.272, 6365–6372.10.1111/j.1742-4658.2005.05030.xSearch in Google Scholar
Lunato, A., Wang, J., Woollard, J., Anisuzzaman, A., Ji, W., Rong, F., Ikeda, S., Soloway, A., Eriksson, S., Ives, D., et al. (1999). Synthesis of 5-(carboranylalkylmercapto)-2′-deoxyuridines and 3-(carboranylalkyl)thymidines and their evaluation as substrates for human thymidine kinases 1 and 2. J. Med. Chem.42, 3378–3389.10.1021/jm990125iSearch in Google Scholar
Machover, D. (1991). Developments of the fluoropyrimidines as inhibitors of thymidylate synthase: pharmacologic and clinical aspects. J. Surg. Oncol. Suppl.2, 42–50.10.1002/jso.2930480511Search in Google Scholar
Marsh, S. and McLeod, H. (2001). Thymidylate synthase pharmacogenetics in colorectal cancer. Clin. Colorectal Cancer1, 175–178.10.3816/CCC.2001.n.018Search in Google Scholar
Munch-Petersen, B., Tyrsted, G., and Cloos, L. (1993). Reversible ATP-dependent transition between two forms of human cytosolic thymidine kinase with different enzymatic properties. J. Biol. Chem.268, 15621–15625.10.1016/S0021-9258(18)82301-8Search in Google Scholar
Okazaki, R. and Kornberg, A. (1964). Deoxythymidine kinase of Escherichia coli. II. Kinetics and feedback control. J. Biol. Chem.239, 269–274.Search in Google Scholar
Sambrook, J. and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press).Search in Google Scholar
Sandrini, M. and Piskur, J. (2005). Deoxyribonucleoside kinases: two enzyme families catalyze the same reaction. Trends Biochem. Sci.30, 225–228.10.1016/j.tibs.2005.03.003Search in Google Scholar PubMed
Welin, M., Kosinska, U., Mikkelsen, N.-E., Carnrot, C., Zhu, C., Wang, L., Eriksson, S., Munch-Petersen, B., and Eklund, H. (2004). Structures of thymidine kinase 1 of human and mycoplasmic origin. Proc. Natl. Acad. Sci. USA101, 17970–17975.10.1073/pnas.0406332102Search in Google Scholar PubMed PubMed Central
©2006 by Walter de Gruyter Berlin New York
