Validation of deoxyhypusine synthase as an antimalarial target.

Background: Hypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs.

Methods: Transgenic parasites with modification of the PF3D7_1412600 gene encoding DHS enzyme were created by insertion of the riboswitch or the M9 inactive variant. The DHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of DHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling.

Results: Clonal transgenic parasites with integration of riboswitch downstream of the DHS gene were established. DHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The DHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas DHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of DHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated DHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (DHFR-TS) expression. DHS-attenuated parasites showed increased sensitivity to -guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes.

Discussion: Loss of DHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated DHS expression suggests that this gene is essential. However, the slower decline of DHS mutants compared with DHFR-TS mutants in competitive growth assays suggests that DHS is less vulnerable as an antimalarial target. Nevertheless, the data validate DHS as an antimalarial target which can be inhibited by spermidine-like compounds.