Bicyclic azetidines kill the diarrheal pathogen Cryptosporidium in mice by inhibiting parasite phenylalanyl-tRNA synthetase

Abstract

Cryptosporidium 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 Cryptosporidium parvum isolates with EC50’s in 1% serum of <0.4 to 96 nM, had comparable potencies against Cryptosporidium hominis and C. parvum, 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 C. parvum phenylalanyl-tRNA synthetase (CpPheRS) as the mode of action of this new lead series. Introduction of mutations directly into the C. parvum pheRS 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 ChPheRS. 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.

Recent Development of Aminoacyl-tRNA Synthetase Inhibitors for Human Diseases: A Future Perspective

“…This study has served as an example of developing a cure for malaria as well as the potential to give prophylaxis and prevent disease transmission. Additionally, Vinayak et al reported that bicyclic azetidine 32 is a potent inhibitor of Cryptosporidium parvum PheRS (Cp PheRS) for diarrheal disease in young children [48]. The in vivo efficacy of 32 for cryptosporidiosis was shown in immunosuppressed mouse models.…”

“…Two major classes of compounds incorporating tetracyclic tetrahydro-beta-carboline and 4amino-2-piperidone were prepared to selectively inhibit the MetRS of T. brucei. The inhibitory activities of three tetracyclic carboline compounds(47)(48)(49) were tested for the T. brucei MetRS aminoacylation assay, showing submicromolar IC50 values as depicted inFigure 17. One of the tetracyclic compounds exhibited no off-target activity when screened against a panel of 45 CNS proteins.…”

“…One of the tetracyclic compounds exhibited no off-target activity when screened against a panel of 45 CNS proteins. Structures of three tetracyclic carbolines(47)(48)(49) and their inhibitory activities against the aminoacylation of Trypanosoma brucei MetRS.…”

“…Structures of three tetracyclic carbolines(47)(48)(49) and their inhibitory activities against the aminoacylation of Trypanosoma brucei MetRS.…”

Spontaneous selection ofCryptosporidiumdrug resistance in a calf model of infection

“…Phenylalanyl-tRNA synthetase (PheRS) and Lysyl-tRNA synthetase (LysRS) inhibitors with promising activity in several mouse models of infection are also under development (14,33).…”