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

Author(s)

Silva, Suelen Fernandes ^[1] ; Klippel, Angelica Hollunder ^[2] ; Ramos, Priscila Zonzini ^{[3, 4]} ; Santiago, Andree da Silva ^{[3, 4]} ; Valentini, Sandro Roberto ^[2] ; Bengtson, Mario Henrique ^{[3, 4]} ; Massirer, Katlin Brauer ^{[3, 4]} ; Bilsland, Elizabeth ; Counago, Rafael Miguez ^[4] ; Zanelli, Cleslei Fernando ^[2]

Affiliation

^[1] Sao Paulo State Univ UNESP, Chem Inst, Araraquara, SP - Brazil

^[2] Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Araraquara, SP - Brazil

^[3] Univ Campinas UNICAMP, Inst Biol, Dept Struct & Funct Biol, Campinas, SP - Brazil

^[4] Univ Campinas UNICAMP, Struct Genom Consortium SGC UNICAMP, Med Chem Ctr CQMED, Mol Biol & Genet Engn Ctr CBMEG, Campinas, SP - Brazil

• Document type: Journal article
• Source: PLoS Neglected Tropical Diseases; v. 14, n. 10 OCT 2020.
• Web of Science Citations: 1

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 fromBrugia malayiandLeishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enableB.malayi(Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD(+). We also reported anin vitrobiochemical assay for this enzyme that is amenable to a high-throughput screening format. TheL.majorgenome 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 complementeddys1 Delta yeast mutants can be used to screen for new inhibitors of theL.majorenzyme. We used the known human DHS inhibitor GC7 to validate bothin vitroand 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 againstB.malayiandL.majorinfections. Author summary Target-based drug discovery strategies hold the promise to discover safer and more effective treatments for Neglected Tropical Diseases (NTDs). Genetic manipulation techniques have been used to successfully identify essential genes in eukaryotic parasites. Unfortunately, the fact that a gene is essential under controlled laboratory conditions does not automatically make the corresponding gene-product vulnerable to pharmacological intervention in a clinical setting within the human host. To allow the discovery and development of small molecule tool compounds that can be used to validate pharmacologically vulnerable targets, one must first establish compound screening assays and obtain structural information for the candidate target. Eukaryotic cells lacking deoxyhypusine synthase (DHS) function are not viable. DHS catalyzes the first step in a post-translational modification that is critical for the function of eIF5A. Presence of mature eIF5A is also essential for eukaryotic cell viability. Here we reported compound screening assays (yeast-based forBrugia malayiandLeishmania major;in vitroforB.malayionly) and provided further regulatory and structural insights we hope will aid in the identification and development of inhibitors for the DHS enzymes from two NTD-causing organisms-B.malayi, the causative agent of lymphatic filariasis andL.major, the causative agent of cutaneous leishmaniasis. (AU)

• FAPESP's process: 13/50724-5 - Protein Kinase Chemical Biology Center: supporting drug development through open-access research
• Grantee: Paulo Arruda
• Support Opportunities: Research Grants - Research Partnership for Technological Innovation - PITE
• FAPESP's process: 14/50897-0 - INCT 2014: Open-acess Medicinal Chemistry Centre (OpenMedChem)
• Grantee: Katlin Brauer Massirer
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 16/16970-7 - Study of the impact of the role of eIF5A in the cell proteomic profile using Saccharomyces cerevisiae as a model
• Grantee: Cleslei Fernando Zanelli
• Support Opportunities: Regular Research Grants
• FAPESP's process: 15/03553-6 - Engineering yeast cells for drug discovery
• Grantee: Elizabeth Bilsland
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 18/16672-1 - Structural determination and search for inhibitors of the enzyme deoxy-hypusine synthase of eukaryotic organisms that cause neglected tropical diseases
• Grantee: Angélica Hollunder Klippel
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 19/14275-8 - Structural analysis of the kinase proteins PRPF4 and DYRK1B, from the CMGC family, and identification of small compound inhibitors
• Grantee: André da Silva Santiago
• Support Opportunities: Scholarships in Brazil - Post-Doctoral