Journal Article

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

Santiago, André da Silva, Couñago, Rafael Miguez, Silva, Suélen Fernandes, Klippel, Angélica Hollunder, Ramos, Priscila Zonzini, Valentini, Sandro Roberto, Bengtson, Mario Henrique, Zanelli, Cleslei Fernando, Bilsland, Elizabeth, Massirer, Katlin Brauer

2020

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Amino Acid Sequence / Amino acids / Animals / Anthelmintics - chemistry / Anthelmintics - pharmacology / Antiprotozoal Agents - chemistry / Antiprotozoal Agents - pharmacology / Assaying / Biology and Life Sciences / Brugia malayi / Brugia malayi - drug effects / Brugia malayi - enzymology / Brugia malayi - genetics / Brugia malayi - growth & development / Cell viability / Cells / Cloning / Consortia / Crystal structure / Cutaneous leishmaniasis / Drug Evaluation, Preclinical / Ectopic expression / Enzyme Inhibitors - chemistry / Enzyme Inhibitors - pharmacology / Enzymes / Filariasis / Genetic aspects / Genetic engineering / Genomes / Genomics / Health aspects / Helminth Proteins - antagonists & inhibitors / Helminth Proteins - chemistry / Helminth Proteins - genetics / Helminth Proteins - metabolism / High-throughput screening / High-Throughput Screening Assays / Homology / Inhibitors / Leishmania / Leishmania major / Leishmania major - drug effects / Leishmania major - enzymology / Leishmania major - genetics / Leishmania major - growth & development / Molecular biology / NAD / Nematoda / Oxidoreductases Acting on CH-NH Group Donors - antagonists & inhibitors / Oxidoreductases Acting on CH-NH Group Donors - chemistry / Oxidoreductases Acting on CH-NH Group Donors - genetics / Oxidoreductases Acting on CH-NH Group Donors - metabolism / Parasites / Parasitic diseases / Pathogens / Pharmaceutical sciences / Physical Sciences / Plasmids / Post-translation / Proteins / Protozoan Proteins - antagonists & inhibitors / Protozoan Proteins - chemistry / Protozoan Proteins - genetics / Protozoan Proteins - metabolism / Regulatory mechanisms (biology) / Research and Analysis Methods / Sequence Alignment / Structure / Therapeutic applications / Transcription factors / Tropical diseases / Vector-borne diseases / Vulnerability / Yeast / Yeasts