Host Cells Overexpressing MDR1 As Novel In Vitro Models For Evaluating The On-target Effect Of Drugs Against The Epicellular Parasite Cryptosporidium Parvum

Cryptosporidium parvum is an intracellular,but extra-cytoplasmic protozoan parasite infecting humans and animals.In 1986,the World Health Organization designated human cryptosporidiosis as one of the suspected indicators of AIDS.Currently,only nitazoxanide has been approved by the U.S.Food and Drug Administration for use in immunocompetent people and halofuginone lactate approved by the European Union for use in animals.Therefore,drug development against Cryptosporidium is urgently needed.In drug discovery against an intracellular pathogen including Cryptosporidium parvum,there is a lack of an effective method to confirm the on-target effect of an identified hit or lead,as a regular in vitro assay is unable to differentiate the action of a compound on the parasite target or the host cell target,or both.One approach to solve this problem is to compare the antiparasitic efficacy of a compound against the parasite cultured with wild-type and derived cell lines with significantly increased drug tolerance,in which no or little change in antiparasitic efficacy is indicative of the action of the compound on parasite target(or on-target effect).However,it is usually a time-consuming process to generate drug-resistant cells.Here we report a novel approach for rapid generation of drug-tolerant host cells by the overexpression of a human multidrug resistance gene MDR1 in HCT-8 cells that could be applied to evaluating on-target effect of compounds against the epicellular C.parvum.We first used a transient transfection model,in which the overexpression of MDR1 in HCT-8 cells last for at least 72 h,thus giving a sufficient timeframe for evaluating drug efficacy on C.parvum.Among six tested compounds,transient overexpression of MDR1 significantly increased drug-tolerance of HCT-8 cells on paclitaxel,doxorubicin and vincristine(2.11-to 2.27-fold increase),but not on cyclosporin A,daunorubicin and nitazoxanide.Further drug efficacy assays showed that increased drug-tolerance in host cells had no effect on the anti-cryptosporidial efficacy of paclitaxel,but affected that of doxorubicin.These observations validated that,at efficacious concentrations,paclitaxel mainly acted on the parasite target,while doxorubicin might act on both parasite and host cell targets.However,the application of transicent infection system to the anti-cryptosporidial on-target effect is limited to selected compounds(i.e.,intrinsic substrates of MDR1).To overcome this limitation,we explored a stable transfection system(i.e.,MDR1-transgenic HCT-8 cells;or MDR1 cells for short)that allowed development of drug resistance to both intrinsic substrates and non-substrates of MDR1 by continuous application of drug pressure in a relatively short timeframe.Using the stable transgenic cells,drug resistance was successfully increased for paclitaxel(MDR1substrate;3.71-fold increase vs.blank vector negative control [NC])and developed for nitazoxanide(non-MDR1 substrate;2.02-fold increase)in three months.Using these two host cell lines,namely MDR1(PTX)and MDR1(NTZ)cells,we confirmed that the on-target effect of both paclitaxel and nitazoxanide for their anti-cryptosporidial activity in vitro.Additionally,MDR1(PTX)cells also showed increase of drug resistance to a number of other compounds,including mitoxantrone(2.73-fold increase vs.NC cells),doxorubicin(2.40-fold increase),vincristine(3.01-fold increase)and ivermectin(2.38-fold increase),allowing the evaluation of their on-target effects.Subsequent experiments confirmed that the anti-cryptosproidial efficacies of the four compounds were attributed partially to their on-target effects and the actions on the host cell targets(i.e.,off-target effects)also contributed to their anti-cryptosporidial efficacies.In conclusion,we have developed two in vitro models for the evaluation of on-target effect of anti-cryptosporidial drugs.The first model uses HCT-8 cells transciently transfected with MDR1 gene that is applicable to selected compounds(i.e.,MDR1 substrates).The second model uses a stable MDR1-transgenic HCT-8cells that is applicable to both substrates and non-substrates of MDR1.Using these models,we have confirmed that paclitaxel and nitazoxanide inhibit the growth of C.parvum in vitro by fully acting on the parasite targets,while mitoxantrone,doxorubicin,vincristine and ivermectin inhibit C.parvum by acting on both parasite and host cell targets.This is for the first time that the on-target action of nitazoxanide(the only FDA-approved drug for treating human cryptosporidiosis)is elucidated.