1. An experimental target-based platform in yeast for screening Plasmodium vivax deoxyhypusine synthase inhibitors.
- Author
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Fernandes Silva S, Hollunder Klippel A, Sigurdardóttir S, Mahdizadeh SJ, Tiukova I, Bourgard C, Salazar-Alvarez LC, do Amaral Prado HM, de Araujo RV, Costa FTM, Bilsland E, King RD, Brauer Massirer K, Eriksson LA, Bengtson MH, Zanelli CF, and Sunnerhagen P
- Subjects
- Humans, Oxidoreductases Acting on CH-NH Group Donors genetics, Oxidoreductases Acting on CH-NH Group Donors antagonists & inhibitors, Oxidoreductases Acting on CH-NH Group Donors metabolism, Enzyme Inhibitors pharmacology, Eukaryotic Translation Initiation Factor 5A, Protozoan Proteins genetics, Protozoan Proteins metabolism, Protozoan Proteins antagonists & inhibitors, Drug Evaluation, Preclinical methods, Peptide Initiation Factors genetics, Peptide Initiation Factors metabolism, Plasmodium vivax drug effects, Plasmodium vivax genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae drug effects, Antimalarials pharmacology
- Abstract
The enzyme deoxyhypusine synthase (DHS) catalyzes the first step in the post-translational modification of the eukaryotic translation factor 5A (eIF5A). This is the only protein known to contain the amino acid hypusine, which results from this modification. Both eIF5A and DHS are essential for cell viability in eukaryotes, and inhibiting DHS is a promising strategy to develop new therapeutic alternatives. DHS proteins from many are sufficiently different from their human orthologs for selective targeting against infectious diseases; however, no DHS inhibitor selective for parasite orthologs has previously been reported. Here, we established a yeast surrogate genetics platform to identify inhibitors of DHS from Plasmodium vivax, one of the major causative agents of malaria. We constructed genetically modified Saccharomyces cerevisiae strains expressing DHS genes from Homo sapiens (HsDHS) or P. vivax (PvDHS) in place of the endogenous DHS gene from S. cerevisiae. Compared with a HsDHS complemented strain with a different genetic background that we previously generated, this new strain background was ~60-fold more sensitive to an inhibitor of human DHS. Initially, a virtual screen using the ChEMBL-NTD database was performed. Candidate ligands were tested in growth assays using the newly generated yeast strains expressing heterologous DHS genes. Among these, two showed promise by preferentially reducing the growth of the PvDHS-expressing strain. Further, in a robotized assay, we screened 400 compounds from the Pathogen Box library using the same S. cerevisiae strains, and one compound preferentially reduced the growth of the PvDHS-expressing yeast strain. Western blot revealed that these compounds significantly reduced eIF5A hypusination in yeast. The compounds showed antiplasmodial activity in the asexual erythrocyte stage; EC50 in high nM to low μM range, and low cytotoxicity. Our study demonstrates that this yeast-based platform is suitable for identifying and verifying candidate small molecule DHS inhibitors, selective for the parasite over the human ortholog., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fernandes Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2024
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