Evaluation of Bacillus anthracis thymidine kinase as a potential target for the development of antibacterial nucleoside analogs.
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 K(m) value (0.6 microM) and a V(max) of 3.3 micromol dTMP mg(-1) min(-1), but deoxyuridine (K(m)=4.2 microM, V(max)=4.1 micromol 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.
Carnrot
Cecilia
C
Department of Molecular Biosciences, The Swedish University of Agricultural Biosciences, Biomedical Center, P.O. Box 575, S-751 23 Uppsala, Sweden.
Vogel
Susan R
SR
Byun
Youngjoo
Y
Wang
Liya
L
Tjarks
Werner
W
Eriksson
Staffan
S
Phipps
Andrew J
AJ
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D015195:Drug Design; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009705:Nucleosides / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D009705:Nucleosides; D011994:Recombinant Proteins; D013937:Thymidine Kinase