TY - JOUR
T1 - Prodrug activation by Cryptosporidium thymidine kinase
AU - Sun, Xin E.
AU - Sharling, Lisa
AU - Muthalagi, Mani
AU - Mudeppa, Devaraja G.
AU - Pankiewicz, Krzysztof W.
AU - Felczak, Krzysztof
AU - Rathod, Pradipsinh K.
AU - Mead, Jan
AU - Striepen, Boris
AU - Hedstrom, Lizbeth
PY - 2010/5/21
Y1 - 2010/5/21
N2 - Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of kcat= 0.28 s-1 and Km,ATP = 140 μM are similar to those of human TK1, the value of Km(thymidine) = 48 μM is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.
AB - Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of kcat= 0.28 s-1 and Km,ATP = 140 μM are similar to those of human TK1, the value of Km(thymidine) = 48 μM is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.
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U2 - 10.1074/jbc.M110.101543
DO - 10.1074/jbc.M110.101543
M3 - Article
C2 - 20231284
AN - SCOPUS:77952364996
SN - 0021-9258
VL - 285
SP - 15916
EP - 15922
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 21
ER -