Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives. Author summary: Nearly one-third of the global population is chronically infected with the apicomplexan parasite Toxoplasma gondii. It does not particularly have any drastic impacts on a healthy individual with a robust immune response. But in immune-compromised patients, the parasite has been found to wreak havoc. The necessity of developing novel antibiotic therapeutics against this ailment is reprised by the limited efficacy of the SP regimen generally prescribed to patients with toxoplasmosis–including suppression of active proliferation–but not the latent stage. This regime has its limitations in the clearance of chronic infection and in the fact that crossing over the blood-brain barrier is troublesome for small molecules, which might not be able to prevent ocular toxoplasmosis in severe cases. Targeting of molecular motors within the protein translation machinery such as prolyl tRNA synthetases with structure-based designed small molecules mimicking the natural substrate, ATP, forms the basis of the work published here as being an avenue for targeting the parasite selectively. [ABSTRACT FROM AUTHOR]