The protein translation initiation machinery is targetable for developing fast-killing therapeutics against the zoonotic Cryptosporidium parvum

Abstract

Cryptosporidium parvum is a zoonotic apicomplexan parasite that causes moderate to severe watery diarrhea in humans and animals. It is one of the two major cryptosporidial species that infect humans, particularly those with weak or compromised immunity (e.g., children and AIDS patients). C. parvum is also one of the major pathogens causing diarrhea in neonatal farm ruminants (e.g., calves). However, fully effective drugs are still unavailable for medical or veterinary use to treat cryptosporidiosis. In the past decade or two, a number of key enzymes in the core metabolic pathways of Cryptosporidium have been explored as drug targets (e.g., energy metabolism, fatty acid synthesis, nucleotide synthesis, protein synthesis and modification), while the protein translation initiation machinery has been unexplored. Here, we report that C. parvum eukaryotic initiation factor 4A (CpeIF4A), a DEAD-box RNA helicase and part of the eIF4F complex (composed of eIF4A, eIF4E, and eIF4G), can be targeted for developing anti-cryptosporidial therapeutics. CpeIF4A can be inhibited by the selective inhibitor rocaglamide-A (Roc-A; a natural product from Aglaia plants). The inhibition of CpeIF4A activity by Roc-A impedes protein synthesis in the sporozoites of C. parvum (IC50 = 3.65-3.80 M) via tight binding to the CpeIF4A-RNA complex (Kd = 33.7 nM). This efficaciously lowers parasite growth in vitro (EC50 = 1.77 nM; selectivity index >1000) and oocyst production in vivo in IFN-{gamma} knockout mice (with a rapid drop on the second day post-administration at 0.5 mg/kg/day). These findings indicate that CpeIF4A can serve as a novel drug target, and that Roc-A is a promising anti-cryptosporidial lead compound for therapeutic development and structural optimization. Author summaryCryptosporidium parvum is a globally distributed protozoan parasite that infect both humans and animals. Cryptosporidial infection in hosts with weak or compromised immunity (e.g., AIDS patients, children or calves) can be severe or deadly, but fully effective treatments are yet unavailable. In the past decade or two, a number of potential drug targets in the core metabolic pathways in Cryptosporidium have been explored, but the protein translation machinery has been unexplored. Here, we report that the C. parvum eukaryotic initiation factor 4A (CpeIF4A), one of the key components in the protein translational machinery, can be targeted for developing fast-killing anti-cryptosporidial therapeutics. We show that CpeIF4A can be inhibited by the selective inhibitor rocaglamide-A (Roc-A), consequently suppressing the protein synthesis in the parasite via tight binding to the CpeIF4A-RNA complex. This efficaciously inhibits the parasite growth both in vitro and in vivo. The killing of C. parvum by Roc-A is fast and irreversible (parasiticidal). Our data indicate that the protein translational machinery is a novel drug target for developing fast-killing anti-cryptosporidial therapeutics, and that Roc-A is a promising lead compound for therapeutic development and structural optimization.