Structural features and development of an assay platform of the parasite target deoxyhypusine synthase of Brugia malayi and Leishmania major

Abstract

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD +. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHSencoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.

Development of an activity assay for characterizing deoxyhypusine synthase and its diverse reaction products

“…Indeed, it has been shown that in Trypanosoma brucei DHS and its paralog can form a tetramer with a highly increased enzyme activity [30,31]. A similar heterotetrameric architecture is suggested for paralogous DHS proteins from Leishmania major [11].…”

“…Deoxyhypusine synthase with its central function in eukaryotic organisms has long been a topic of biochemical research and has also come into prominence for drug development in the treatment of cancer and other diseases [7–11]. The biologically active unit of DHS is a homotetramer (Figs.…”

The eukaryotic initiation factor 5A (eIF5A1), the molecule, mechanisms and recent insights into the pathophysiological roles

“…Despite conservation of some of the active site amino acid residues between the human and leishmanial DHPS, GC7 inhibited only slightly L. donovani proliferation. This suggests a topological difference in the spermidine binding sites between the human and the leishmanial enzymes and opens up the possibility that the differences between the two enzymes could be exploited for drug development for visceral leishmaniasis [ 67 , 112 ]. The importance of eIF5A in the fight against Leishmania has also been reported by Duarte et al [ 113 ] who engineered a polyproteins vaccine raised against eIF5A and another hypothetical protein named LbHyp cloned from Leishmania braziliensis .…”

An experimental target-based platform in yeast for screeningPlasmodium vivaxdeoxyhypusine synthase inhibitors

“…While continuous culture of P. falciparum in its intraerythrocytic stage is possible, there is currently no established continuous in vitro culturing system for P. vivax. This prompts the exploration of alternative approaches for assaying potential P. vivax drug target proteins, surrogate genetics systems in the yeast Saccharomyces cerevisiae emerging as an attractive option due to the facile genetic engineering in this organism [5][6][7].…”

“…Target-based drug discovery campaigns typically rely on in vitro biochemical assays, which require protein purification and lack a cellular context [8,31,32]. Alternatively, designing systems with genetically modified S. cerevisiae strains can provide efficient platforms for target-based drug discovery within a eukaryotic cell [5][6][7][33][34][35].…”