Structural determination and search for inhibitors of the enzyme deoxy-hypusine synthase of eukaryotic organisms that cause neglected tropical diseases

Author(s)

Angélica Hollunder Klippel

• Document type: Doctoral Thesis
• Press: Araraquara. 0000-00-00.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2023-02-27
• Advisor: Cleslei Fernando Zanelli

Abstract

The eukaryotic translation factor 5A (eIF5A) is highly conserved in eukaryotes and is essential for cell proliferation. eIF5A undergoes an extremely specific post-translational modification, called hypusination, required for eIF5A function. Hypusination occurs in two steps that involve the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOOH). Considering the essentiality of hypusination for eIF5A function, DHPS and DOHH are promising targets for developing new drugs to stop cell proliferation in various organisms. In this context, the aim of this work is to determine the three-dimensional structure and search for DHPS inhibitors of the following organisms that cause neglected tropical diseases: Paracoccidioides brasiliensis (Pb), Histoplasma capsulatum (Hc), Brugia malayi (Bm) and Leishmania sp. (two isoforms: A and B). For this purpose, the conditions for cloning, production in Escherichia coli and purification of these proteins were established. This enabled the adaptation of an easy-to-miniaturize biochemical assay for HcDHPSA, BmDHPSA and PbDHPSA, and the determination of the crystallographic structure of BmDHPSA and HcDHPSA. However, these structures present a very similar architecture to the human enzyme (HsDHPS), which hinders the development of selective inhibitors for the DHPSs from those pathogens. Therefore, all the efforts were directed towards obtaining Leishmania sp. DHPSs in a soluble form from E.coli, as these enzymes probably form a heterocomplex, unlike HsDHPS. After combining a series of strategies, it was possible to produce recombinant L. donovani (LdDHPSAB) and L. major (LmDHPSAB) DHPSs in a soluble and active form from E. coli. A high-throughput screening assay was established in vitro with the LdDHPSAB, in which the LdDHPSAB activity was quantified using NAD+/NADH-Glo™ assay coupled with the partial reaction of the DHPSs. Of the 26771 compounds initially screened at 10 μM, 44 compounds (hits) inhibited LdDHPSAB activity by over 60%. The secondary screening confirmed that 24 (57%) hits exhibited consistent inhibitory effects (inhibition above 60%) on LdDHPSAB activity. Then, 11 hit compounds that also inhibited the enzymes from the NAD+/NADH-Glo™ assay were excluded from further analysis. The remaining 13 hit compounds were analyzed for dose-response using the same coupled assay. Nine out of those 13 compounds presented IC50 against LdDHPSAB in the lower micromolar range. Among these, the compounds MMV1674024 and MMV904563 showed increased potential for future optimizations to improve the inhibition potency against Leishmania sp. DHPSs activity. Furthermore, the in vitro data suggested that inhibiting Leishmania sp. DHPS targets without inhibiting the human counterpart is a feasible task, confirming the hypothesis that DHPS is an interesting target to be explored in Leishmania sp. Also, using the assay based on the detection of NADH fluorescence, it was possible to determine that the compound MMV1674024 exerts a mixed-type inhibition on the activity of the LmDHPSAB in relation to NAD+ and spermidine. Finally, crystals were obtained for LmDHPSAB prepared in the presence of NAD+ and the compound MMV1674024. However, these crystals did not diffract during x-ray diffraction data collection, even after attempts to optimize the crystallization conditions. (AU)

• 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