Prodrug activation by Cryptosporidium thymidine kinase

Xin E. Sun, Lisa Sharling, Mani Muthalagi, Devaraja G. Mudeppa, Krzysztof W. Pankiewicz, Krzysztof Felczak, Pradipsinh K. Rathod, Jan Mead, Boris Striepen, Lizbeth Hedstrom

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)15916-15922
Number of pages7
JournalJournal of Biological Chemistry
Volume285
Issue number21
DOIs
StatePublished - May 21 2010

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