An experimental target-based platform in yeast for screening Plasmodium vivax deoxyhypusine synthase inhibitors

Journal article, 2024

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 similar to 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 mu M 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.