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

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.

Fig 1. Structure-based sequence alignment of H. sapiens DHS (HsDHS), P.vivax DHS (PvDHS) and S. cerevisiae DHS (ScDHS).

Residues indicated by a light-colored background participate in the orthosteric binding site (blue), allosteric binding site (yellow), or both (green). Background coloring for PvDHS and ScDHS follows that of the HsDHS proteins. Residues indicated by a black background or framed in a box are conserved in all DHSs analyzed here. The secondary structure (α-helices and β sheets) shown in the top line are for HsDHS. Protein sequences and structures used in the alignment were: HsDHS (UniProt ID P49366, PDB ID 6P4V), PvDHS (UniProt ID Q0KHM1) and ScDHS (UniProt ID P38791).

Fig 2. GC7 inhibits growth of yeast strains, wt or dys1Δ complemented by HsDHS or PvDHS.

(A) Comparison of inhibition caused by different concentrations of GC7 (as indicated) in dys1Δ yeast complemented by HsDHS (SFS04, S2 Table). The concentration of methionine used to regulate HsDHS expression is indicated in the figure. (B) Western blot detection of hypusination levels after 12 h of treatment with 50 μM GC7 in the wild-type (wt) and PvDHS and HsDHS-complemented strains. The methionine concentrations used in precultures (as mentioned in Material and Methods) are indicated in the figure. (C) Graph reporting the ratio between hypusinated eIF5A and total eIF5A under the conditions indicated in the figure. The bars represent mean of relative hypusination values ((hypusinated eIF5A/ eEF2) / total eIF5A/ eEF2)) ± standard deviation (SD; n = 3). A Student’s t-test was performed comparing the compound condition and the solvent control condition where an asterisk (*) indicates that the differences are statistically significant with 95% confidence (p < 0.05); two asterisks (**), 99% confidence (p < 0.01); three asterisks (***), 99.9% confidence (p < 0.001); ns, not statistically significant (p > 0.05).

Fig 3. N2 and N7 detected as potential PvDHS selective inhibitors in the yeast-based assay.

Relative area under the curve (AUC) of S. cerevisiae dys1Δ strains complemented by the indicated DHS enzymes, or of isogenic wt (ScDHS). The concentrations of the compounds range from 25 to 200 μM. Statistical significance levels indicated as in Fig 2. Each point represents the mean ± SD of experimental quadruplicates.

Fig 4. Relative growth of PvDHS and HsDHS strains in the presence of compound PB1 Growth curves were generated using different concentrations of PB1 (indicated in the figure) in the PvDHS (A) and HsDHS (B) strains.

Each point represents the mean ± SD of four experimental replicates. (C) Relative growth score, calculated by the AUC with the compound in relation to the DMSO curve. Statistical significance levels indicated as in Fig 2.

Fig 5. Hypusination of eIF5A in the presence of PvDHS inhibitor hits.

(A) Detection by western blot of hypusination levels after 12 h of treatment with compounds indicated in the figure, at the following concentrations: GC7 (50 μM), N7 (50 μM) N2 (100 μM) and PB1 (100 μM). The methionine concentrations used in pre-cultures were 130 μM for HsDHS and 0 μM for PvDHS and WT. (B) Graph reporting the ratio between eIF5A hypusinated and total eIF5A under the conditions indicated in the figure. The bars represent the mean relative hypusination values ((hypusinated eIF5A / eEF2) / total eIF5A / eEF2)) ± SD (n = 3); except for GC7 where the bar represents the value for one experiment (n = 1). NT, not treated. A Student’s t-test was performed comparing the compound treated condition and the solvent control condition. Statistical significance levels indicated as in Fig 2.