Equilibrative transporters are potential drug targets, however most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64,560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2–2 µM). These nine compounds completely blocked [3H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5–50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5–50 µM). Wild-type (WT) parasite IC50 values were up to four-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.
BACKGROUND For transgender individuals taking hormone therapy (HT), data on laboratory values are limited, and the effects on laboratory values cannot be easily predicted. We evaluated the impact on common laboratory analytes in transgender individuals before and after initiation of HT. METHODS We conducted a retrospective chart review of transgender patients identified at transgender-specific clinics at an urban county hospital and community clinic. Laboratory data were collected on hormone concentrations, hematologic parameters, electrolytes, lipids, and liver and renal markers before and after initiation of HT. RESULTS We identified 183 transgender women (TW) and 119 transgender men (TM) for whom laboratory data were available. In all, 87 TW and 62 TM had baseline laboratory data, and data were also available for 133 TW and 89 TM on HT for >6 months. The most significant changes were seen in red blood cell count, hemoglobin concentration, hematocrit, and creatinine levels after >6 months of HT, which increased in TM and decreased in TW after HT (P < 0.005; d index > 0.6). Alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase levels increased in TM; however, the effect size was small (d index < 0.5). Calcium, albumin, and alkaline phosphatase levels significantly decreased in TW (P < 0.001; d > 0.6). Additionally, TM were found to have increased triglycerides and decreased HDL levels (P < 0.005; d > 0.6). CONCLUSIONS Changes occur in several common laboratory parameters for patients on HT. Some laboratory values changed to match the gender identity, whereas others remained unchanged or were intermediate from the baseline values. These findings will help guide interpretation of laboratory test results in transgender patients taking HT.
Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage–pathway enzymes to form the required purine nucleotides. The P. falciparum genome encodes four putative ENTs (PfENT1–4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine-salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (< 10 µM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target.
Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate K m . We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an ␣-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60 o arc suggesting that TM11 is largely ␣ helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure.
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