A Cysteine Protease Inhibitor Rescues Mice from a Lethal Cryptosporidium parvum Infection

Abstract

h Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-␥R-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

Recent discovery and development of inhibitors targeting coronaviruses

“…Cathepsins are cysteine proteases that are involved in the endosomal pathway and can be inhibited by cathepsin inhibitors such as K11777 (95) and its related vinyl sulfone analogs [149]. These compounds seem to be safe and effective against various parasitic infections in animal models and have broad-spectrum activities against enveloped RNA viruses such as CoVs (SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63), filoviruses (Ebola and Marburg viruses) and paramyxoviruses [149][150][151][152].…”

Treatment of Cryptosporidium: What We Know, Gaps, and the Way Forward

“…Nevertheless, research focused on anti-cryptosporidials continues to make substantial advancements. Several enzymes have been identified as potential drug targets, including calcium-dependent protein kinases [53], Clan CA cysteine proteases [55], IMPDH [57] and the folate biosynthesis pathway [56]. Other studies are testing compounds repurposed, which were developed for other indications.…”

“…Clan CA cysteine proteases are thought to be vital for host cell invasion and has been found to be structurally different than analogous enzymes in humans [54]. This protease can be inhibited utilizing N-methyl-piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits growth of Cryptospordium in vitro and has been shown to rescue immunocompromised mice from lethal infection [55]. …”

Cryptosporidium in humans and animals—a one health approach to prophylaxis

“…Other drug targets include long‐chain fatty acyl‐coenzyme A synthetases (LC‐ACS), which are essential in fatty acid metabolism, and a recent study reported good efficacy of the ACS inhibitor triacsin C against cryptosporidial infection in mice . A parasite cysteine protease inhibitor was also effective in vitro and in an animal model . Other studies have focused on repurposing existing drugs to overcome the prohibitive costs of de novo drug development (estimated to be between $500 million and $2 billion per compound successfully brought to market) .…”