On-target inhibition of Cryptosporidium parvum by nitazoxanide (NTZ) and paclitaxel (PTX) validated using a novel MDR1-transgenic host cell model and algorithms to quantify on/off-target rates

Abstract

Cryptosporidium parvum is a globally distributed zoonotic protozoan parasite that causes moderate to severe, sometime deadly, watery diarrhea in humans and animals, for which fully effective treatments are yet unavailable. In the study of mechanism of action of drugs against intracellular pathogens, it is important to validate whether the observed anti-infective activity is attributed to the drug action on the pathogen (on-target effect) or host cells (off-target effect). For the epicellular Cryptosporidium, we have previously developed a concept that the host cells with significantly increased drug tolerance by transient overexpression of the multidrug resistance protein-1 (MDR1) could be utilized to evaluate whether an observed anti-cryptosporidial activity of an inhibitor was attributed to the action of the inhibitor on the parasite or host cell targets. However, the transient transfection model was only applicable to evaluating inhibitors that were MDR1 substrates. Here we report an advanced model using stable MDR1-transgenic HCT-8 cells that allowed continuous application of drug pressure for rapid development of novel resistance to non-MDR1 substrates. Using the new model, we successfully validated that nitazoxanide, the only FDA-approved drug to treat human cryptosporidiosis and non-MDR1 substrate, killed C. parvum by fully acting on the parasite target (100% on-target). We also confirmed that paclitaxel acted fully on-target, while several other inhibitors including mitoxantrone, doxorubicin, vincristine and ivermectin acted partially on-target. Additionally, we developed mathematical models to quantify the proportional contributions of on-target and off-target effects to the observed anti-cryptosporidial activity and to evaluate the relationships between antiparasitic efficacy (ECi), cytotoxicity (TCi), safety interval (SI) and Hill slope (h) parameters. Owning to the promiscuity of the MDR1 efflux pump, the MDR1 -transgenic host cell model could be applied to assess the on/off-target effects of newly hits/leads, either substrates or non-substrates of MDR1, against Cryptosporidium or other epicellular pathogens.