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

DOI: 10.1371/journal.pntd.0008762

Authors: Suélen Fernandes Silva (São Paulo State University—UNESP) , Angélica Hollunder Klippel (São Paulo State University—UNESP) , Priscila Zonzini Ramos (University of Campinas-UNICAMP,) , André Da Silva Santiago (University of Campinas-UNICAMP) , Sandro Roberto Valentini (São Paulo State University—UNESP) , Mario Henrique Bengtson (University of Campinas-UNICAMP) , Katlin Brauer Massirer (University of Campinas-UNICAMP) , Elizabeth Bilsland (University of Campinas—UNICAMP) , Rafael Miguez Counago (University of Campinas-UNICAMP) , Cleslei Fernando Zanelli (São Paulo State University—UNESP)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Plos Neglected Tropical Diseases , VOL 14

State: Published (Approved)
Published: October 2020
Diamond Proposal Number(s): 16171

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 DHS-encoding 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.

Journal Keywords: Saccharomyces cerevisiae; Yeast; Crystal structure; Brugia malayi; Enzymes; Biochemical cofactors; Trypanosoma brucei gambiense; Enzyme assays

Diamond Keywords: Leishmaniasis;Enzymes

Subject Areas: Biology and Bio-materials, Chemistry, Medicine


Instruments: I24-Microfocus Macromolecular Crystallography

Added On: 21/10/2020 11:52

Discipline Tags:

Pathogens Infectious Diseases Disease in the Developing World Health & Wellbeing Biochemistry Catalysis Chemistry Structural biology Drug Discovery Life Sciences & Biotech Parasitology

Technical Tags:

Diffraction Macromolecular Crystallography (MX)