Bicyclic Pyrrolidine Inhibitors of Toxoplasma gondii Phenylalanine t-RNA Synthetase with Antiparasitic Potency In Vitro and Brain Exposure.

Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular, to achieve optimal exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency and ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analogue series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.

Effects of unstable ß-PheRS on food avoidance, growth, and development are suppressed by the appetite hormone CCHa2.

Amino acyl-tRNA synthetases perform diverse non-canonical functions aside from their essential role in charging tRNAs with their cognate amino acid. The phenylalanyl-tRNA synthetase (PheRS/FARS) is an α2ß2 tetramer that is needed for charging the tRNAPhe for its translation activity. Fragments of the α-subunit have been shown to display an additional, translation-independent, function that activates growth and proliferation and counteracts Notch signalling. Here we show in Drosophila that overexpressing the ß-subunit in the context of the complete PheRS leads to larval roaming, food avoidance, slow growth, and a developmental delay that can last several days and even prevents pupation. These behavioural and developmental phenotypes are induced by PheRS expression in CCHa2+ and Pros+ cells. Simultaneous expression of ß-PheRS, α-PheRS, and the appetite-inducing CCHa2 peptide rescued these phenotypes, linking this ß-PheRS activity to the appetite-controlling pathway. The fragmentation dynamic of the excessive ß-PheRS points to ß-PheRS fragments as possible candidate inducers of these phenotypes. Because fragmentation of human FARS has also been observed in human cells and mutations in human ß-PheRS (FARSB) can lead to problems in gaining weight, Drosophila ß-PheRS can also serve as a model for the human phenotype and possibly also for obesity.