Bicyclic azetidines kill the diarrheal pathogen <i>Cryptosporidium</i> in mice by inhibiting parasite phenylalanyl-tRNA synthetase.

Abstract

<i>Cryptosporidium</i> is a protozoan parasite and a leading cause of diarrheal disease and mortality in young children. Currently, there are no fully effective treatments available to cure infection with this diarrheal pathogen. In this study, we report a broad drug repositioning effort that led to the identification of bicyclic azetidines as a new anticryptosporidial series. Members of this series blocked growth in in vitro culture of three <i>Cryptosporidium parvum</i> isolates with EC₅₀ ^'s in 1% serum of <0.4 to 96 nM, had comparable potencies against <i>Cryptosporidium hominis</i> and <i>C. parvum</i>, and was effective in three of four highly susceptible immunosuppressed mice with once-daily dosing administered for 4 days beginning 2 weeks after infection. Comprehensive genetic, biochemical, and chemical studies demonstrated inhibition of <i>C. parvum</i> phenylalanyl-tRNA synthetase (<i>Cp</i>PheRS) as the mode of action of this new lead series. Introduction of mutations directly into the <i>C. parvum pheRS</i> gene by CRISPR-Cas9 genome editing resulted in parasites showing high degrees of compound resistance. In vitro, bicyclic azetidines potently inhibited the aminoacylation activity of recombinant <i>Ch</i>PheRS. Medicinal chemistry optimization led to the identification of an optimal pharmacokinetic/pharmacodynamic profile for this series. Collectively, these data demonstrate that bicyclic azetidines are a promising series for anticryptosporidial drug development and establish a broad framework to enable target-based drug discovery for this infectious disease.