Host cells with transient overexpression of MDR1 as a novel in vitro model for evaluating on-target effect for activity against the epicellular Cryptosporidium parasite

Abstract

Objectives To rapidly generate host cells with resistance to multiple compounds for differentiating drug action on parasite target or the host cell target (i.e. on-target or off-target effect) against the zoonotic enteric parasite Cryptosporidium parvum. Methods Transient overexpression of a multidrug resistance protein 1 (MDR1) gene in host cells (HCT-8 cell line) was explored to increase drug tolerance of the host cells to selected anti-cryptosporidial leads. In vitro cytotoxicity and anti-cryptosporidial efficacy of selected compounds were evaluated on the parasite grown in WT parental and transiently transfected HCT-8 cells. The approach was based on the theory that, for an epicellular parasite receiving consistent exposure to compounds in culture medium, overexpressing MDR1 in HCT-8 cells would increase drug tolerance of host cells to selected compounds but would not affect the anti-cryptosporidial efficacy if the compounds acted solely on the parasite target and the drug action on host cell target played no role on the antiparasitic efficacy. Results Six known anti-cryptosporidial leads were tested. Transient overexpression of MDR1 increased drug tolerance of HCT-8 cells on paclitaxel, doxorubicin HCl and vincristine sulphate (2.11- to 2.27-fold increase), but not on cyclosporin A, daunorubicin HCl and nitazoxanide. Increased drug tolerance in host cells had no effect on antiparasitic efficacy of paclitaxel, but affected that of doxorubicin HCl. Conclusions Data confirmed that, at efficacious concentrations, paclitaxel acted mainly on the parasite target, while doxorubicin might act on both parasite and host cell targets. This model can be employed for studying the action of additional anti-cryptosporidial leads, and adapted to studying drug action in other epicellular pathogens. The limitation of the model is that the anti-cryptosporidial leads/hits need to be MDR1 substrates.

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

“…In comparison to NC cells, there were >1.5-fold increase of MDR1 protein and >9-fold increases of mRNA in MDR1 cells, respectively (Fig 2C, 2D). The fold increases were lower, but the levels were more consistent over the time, than those in our previously reported transiently transfected cells (i.e., 2.12- to 3.37-fold for protein and >40-fold for mRNA) [15].…”

“…The availability of host cells with >2 to 3-fold increase of drug resistance to NTZ, PTX and four other compounds made it possible to evaluate whether, and how much, the anti-cryptosporidial activities of these compounds were attributed to their actions on the parasite targets. In theory, if a specified inhibitor inhibited the epicellular C. parvum in vitro by solely acting on the parasite target and its action on host cell target made no contribution to the antiparasitic activity, the increase of resistance to the inhibitor in the host cells would not affect the anti-cryptosporidial activity [15]. This could be achieved by comparing the anti-cryptosporidial efficacy (EC 50 values) with cytotoxicity (TC 50 values) of the inhibitor between MDR1(PTX) and NC or between MDR1(NTZ) and NC cells.…”

“…This notion was tested using elacridar, a third generation of MDR1 inhibitor [16,20–22]. The concentration of elacridar at 300 nM was used based on previous studies that elacridar at this concentration could produce strong inhibition on the activity of MDR1 with no significant cytotoxicity to HCT-8 cells [15]. In this study, we also confirmed that elacridar at 300 nM produced no or little effect on the growth of the six cell lines and on the growth of C. parvum cultured with NC, MDR1(PTX) and MDR1(NTZ) cells (Fig 7).…”

“…We have recently developed an HCT-8 cell-based transient MDR1- transfection model for evaluating the routes of drug actions on C. parvum [15]. The model takes advantage of the relatively broad-spectrum substrates of the multidrug resistance-1 transporter (MDR1), allowing rapid development of drug resistance in host cells on selected anti-cryptosporidial compounds.…”

“…The transient MDR1 -transfection model can be quickly established with increased tolerance to multiple drugs. However, the application of the transient transfection model is limited to the intrinsic substrates of MDR1 (e.g., paclitaxel), but inapplicable to non-substrates of MDR1 (e.g., NTZ) [15]. Here we report the development of a new model with the potential to evaluate anti-cryptosporidial on/off-target effects of unrestricted classes of compounds.…”