Molecular Microbiology (2008) 69(2), 520–533 doi:10.1111/j.1365-2958.2008.06305.x First published online 16 June 2008 Chemical and genetic validation of dihydrofolate reductase–thymidylate synthase as a drug target in African trypanosomes OnlineOpen: This article is available free online at www.blackwell-synergy.com Natasha Sienkiewicz, Szymon Jarosławski,† Susan Wyllie and Alan H. Fairlamb* Division of Biological Chemistry & Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK. Summary The phenotypes of single- (SKO) and double-knockout (DKO) lines of dihydrofolate reductase–thymidylate synthase (DHFR–TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild-type (WT) cells, whereas DKO has an absolute requirement for thymi- dine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross mor- phological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR–TS, but not with Escherichia coli TS. As pteri- dine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotr- exate with structural similarity to folic acid are up to 300-fold more potent inhibitors of WT cultured in a novel low-folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypano- somes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000-fold for raltitrexed. These data demonstrate that DHFR–TS is essential for parasite survival and represents a promising target for drug discovery. Introduction The post-genomic era offers unparalleled opportunities Accepted 13 May, 2008. *For correspondence. E-mail a.h.fairlamb@ dundee.ac.uk; Tel. (+44) 1382 385155; Fax (+44) 1382 385542. †Present address: Institut Curie, UMR168-CNRS, 26 rue d’Ulm, 75248 Paris, France. Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd for the identiﬁcation, characterization and validation of novel molecular targets for drug discovery in order to replace the currently unsatisfactory therapies for human African trypanosomiasis. The initial selection of potential targets from the ‘druggable genome’ is of crucial impor- tance and known targets of current drugs in clinical use for other diseases are a useful starting point. Folic acid metabolism is one such area with clinical precedents in bacterial or protozoan infections and certain human malignancies (Blaney et al., 1984; McGuire, 2003; Gangjee et al., 2007). Folate and its derivatives are essential cofactors in one-carbon metabolism that are required for the biosyn- thesis of purines, thymidylate, serine and methionine in a wide variety of organisms. Most bacteria, some protozoa (e.g. malaria), fungi and plants synthesize folates de novo using a pterin (from GTP), p-aminobenzoate (via the chorismate pathway) and glutamate. In contrast, try- panosomatids and their mammalian hosts lack a de novo folate-synthesis pathway and thus require exogenous folate for these biosynthetic functions. Trypanosomatids have lost the ability to synthesize purines and therefore salvage them from their environ- ment, yet have retained the complete biosynthetic pathway to pyrimidines necessary for nucleic acid synthe- sis (Fig. 1). A key step in DNA synthesis is formation of thymidylate (dTMP) catalysed by thymidylate synthase (TS; EC 2.1.1.45) involving the reductive methylation of deoxyuridylate (dUMP) by 5, 10-methylene- tetrahydrofolate (CH2-H4F). The other product of this reac- tion, dihydrofolate (H2F), is converted into tetrahydrofolate (H4F) by dihydrofolate reductase (DHFR; EC 1.5.1.3). Finally CH2-H4F is regenerated from H4F via either serine hydroxymethyltransferase (EC 2.1.2.1) or the glycine cleavage system to complete the reaction cycle. In trypa- nosomatids and other parasites, DHFR and TS are fused to form a bifunctional protein, unlike their mammalian hosts. Selective inhibition of DHFR or TS in prokaryotic and eukaryotic cells results in ‘thymine-less death’ by necrosis or apoptosis as a consequence of thymidine starvation (Ahmad et al., 1998; Van Triest et al., 2000). However, DHFR–TS has not been successfully exploited so far for treatment of either trypanosomal or leishmanial infections