Nucleotide metabolism has been highlighted as a source of enzymes for target‐based drug development in
Plasmodium [
25].
Plasmodium falciparum dUTPase had been extensively studied in the search of potent inhibitors showing anti-malarial properties [
7,
8,
10] yet, while presumed to be indispensable for growth considering its central role in providing dUMP for thymidylate biosynthesis, its essential character has not been demonstrated. Trimeric dUTPases have been shown to be essential for viability in several organisms, such as
Saccharomyces cerevisiae [
5],
Escherichia coli [
6], or
Mycobacterium smegmatis [
26]. Likewise, knockout mutants for the dimeric enzymes present a growth defective phenotype [
27]. The loss of viability has been associated with an imbalance in the dUTP/dTTP ratio. Thus in the absence of dUTPase an increase in this ratio results in massive incorporation of uracil during replication due to an expansion of the dUTP pool. Indeed
Saccharomyces cerevisiae [
5] and
Trypanosoma brucei [
28] knockout mutants are thymidine auxotrophs. Human and
Plasmodium dUTPases share a similar overall fold yet selective inhibition has been shown to be feasible. Thus, a series of trityl and deoxyuridine derivatives and their acyclic analogues can inhibit
P. falciparum dUTPase and show anti-malarial activity [
8]. Structural data obtained for enzyme–inhibitor complexes evidenced that the triphenylmethane group of these compounds interacts with the side chains of residues Phe46 and Ile117 that are part of a hydrophobic pocket present in
Plasmodium dUTPase different from the phosphate binding site [
13]. These residues are replaced by Val42 and Gly87 in the human enzyme [
13]. The present study provides genetic evidence suggesting that PfdUTPase is indeed indispensable for erythrocytic stages of
P. falciparum. The
dut locus could not be disrupted, yet could be correctly targeted. The inability to knockout the gene using a simple crossover strategy has been understood as evidence that supports the essentiality of the gene [
29]. Alternative strategies such as the complementation by HsdUTPase in a KO background were not feasible. Several reasons may explain this observation. Thus, it is possible that levels of human enzyme are inadequate to sustain dTMP biosynthesis. In addition, the existence of protein–protein interactions specific to PfdUTPase and that are essential for parasite viability cannot be discarded. In support of the essential character of
Pfdut, a recent study using transposon mutagenesis has defined the mutability and fitness costs for over 87% of
P. falciparum genes and established 2680 genes as essential for optimal growth of asexual blood stages in vitro [
30]. The coding sequence for PfdUTPase appeared in this study as non-mutable. Since the absence of insertions in the CDS was considered as an indicator that disruptions are lethal, the data is also indicative of
Pfdut being essential [
30]. In addition, the enzyme appears to be essential in
P. berghei since deletion of dUTPase failed after several attempts suggesting a crucial role during intraerythrocytic development [
15].
While multiple studies have shown that PfdUTPase can be efficiently inhibited in vitro and that enzyme inhibitors also exhibit antiplasmodial activity, no study has been performed in order to validate that indeed the intracellular target of these compounds is dUTPase. Most inhibitors discovered to date are uracil-based compounds that interact with the substrate binding site. Specifically, 5′-tritylated nucleosides are selective inhibitors of the
P. falciparum enzyme versus the HsdUTPase [
31]. Further modifications of 5′-tritylated deoxyuridine derivatives gave rise to a generation of acyclic analogues that showed a good correlation between enzyme inhibition and antiparasitic activity [
8,
10].
For chemical validation, different compounds that exhibit inhibition of both PfdUTPase and antiplasmodial activity were chosen. Mutants overexpressing PfdUTPase or HsdUTPase are expected to confer resistance if the enzyme is the primary target. When comparing the fold change in IC50 of the different compounds tested, the action of compounds 3 and 4 was clearly dependent on enzyme levels. Compound 3 is a 3′carbamate and a potent inhibitor of Plasmodium dUTPase while compound 4 is an acyclic 3′ urea that appears to be eightfold less active against the enzyme than compound 3 although both share the characteristic of exhibiting a bulky carboxybenzene substituent in the 3′ position. Both exhibit significant antiplasmodial activity in vitro and are selective versus the human enzyme. The lower ability of HsdUTPase to counteract the effect of the inhibitor, although still doubling the original IC50, can be due to low protein levels or a reduced ability of HsdUTPase to substitute the Plasmodium enzyme. While not performed in the present study, overexpression of a catalytic mutant would not confer resistance to the inhibitors thus reinforcing the concept that dUTPase is the target of compounds 3 and 4.
In the case of compounds 1 and 2 (3′ urea derivatives) dUTPase inhibition does not appear to relate to the antiplasmodial activity. Indeed, the Ki values for PfdUTPase for compounds 1 and 2 are respectively nearly two and one orders of magnitude higher than their anti-malarial activity in vitro pointing towards the existence of other intracellular targets. Hence, while certain compounds clearly involve inhibition of dUTPase as their main target within the cells, for others additional modes of action should be invoked, although these remain to be established. The pronounced decrease in dTTP and increase in dUTP in treated
Plasmodium cultures further reinforces the idea that compounds 3 and 4 are acting through inhibition of dUTPase. Depletion of nucleotide pools upon incubation with specific inhibitors also underscores not only the importance of dUTPase in keeping low levels of dUTP, but also its key role in providing dUMP for dTTP biosynthesis (Additional file
3).