Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all ␣-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric ␤-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead antitrypanosomal compounds.Various members of the genus Leishmania cause leishmaniasis, which threatens ϳ350 million people worldwide and gives rise to about two million clinical cases each year, of which ϳ25% are of the fatal visceral form (1). The disease is largely endemic to developing countries, and current treatments are expensive and can result in undesirable side effects for the patient (1). This, combined with the increasing drug resistance that is developing in the Leishmania species, means that new and novel anti-parasitic drug targets are urgently required.Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) 2 represents such a target. dUTPases catalyze the hydrolysis of dUTP to dUMP and pyrophosphate (2). This provides the starting material for the synthesis of dTMP by thymidylate synthase and in addition maintains the ratio of dTTP:dUTP in the cell at a high enough level to prevent excessive misincorporation of dUMP into the genome during DNA replication (3). dUTPase activity is essential as was shown by gene knock-outs in Escherichia coli and Saccharomyces cerevisiae. Inhibition of dUTPase activity results in futile cycles of DNA repair that ultimately cause the fragmentation of DNA and cell death (4, 5). dUTPase activity also appears to be essential in L. major (6) and the related parasite Trypanosoma brucei, where an RNAi approach was used to knock down dUTPase expression, resulting in decreased cell proliferation and growth in both procyclic and bloodstream forms of the organism (7). dUTPases have been characterized extensively both biochemically and structurally in many species. Most organisms, including humans, have trimeric dUTPases with structures largely consisting of ␤-pleated sheet (8). Three active sites are formed at the trimer interfaces by...

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a potential drug target for malaria. We previously reported some 5'-tritylated deoxyuridine analogues (both cyclic and acyclic) as selective inhibitors of the Plasmodium falciparum dUTPase. Modelling studies indicated that it might be possible to replace the trityl group with a diphenyl moiety, as two of the phenyl groups are buried, whereas the third is exposed to solvent. Herein we report the synthesis and evaluation of some diphenyl analogues that have lower lipophilicity and molecular weight than the trityl lead compound. Co-crystal structures show that the diphenyl inhibitors bind in a similar manner to the corresponding trityl derivatives, with the two phenyl moieties occupying the predicted buried phenyl binding sites. The diphenyl compounds prepared show similar or slightly lower inhibition of PfdUTPase, and similar or weaker inhibition of parasite growth than the trityl compounds.

No abstract