Identification of Cryptosporidium Kinases and Epigenetic Modifiers as Potential Drug Targets Using a Chemical Genetics Approach

Background: Cryptosporidiosis causes diarrhea in infants and immunocompromised people. Nitazoxanide, the best available treatment, is not effective in AIDS patients.  Cryptosporidium species and malaria parasites are related.  Therefore, we hypothesized that well-characterized small molecule kinase inhibitors and epigenetic modifiers with possible anti-malaria activity can be used to identify potential new Cryptosporidium drug targets.

Methods: HCT-8 colon cancer cells grown in 384-well clear-bottomed plates were infected with C. parvum (Iowa strain) 3 hours prior to addition of experimental compounds, and incubation for another 45 hours. Parasites and host cells were then fluorescently stained, and imaged with an automated microscope. The parasites and host cells were then enumerated using NIH ImageJ.

Results: 38 well-characterized small molecule kinase inhibitors and epigenetic modifiers were screened (3 concentrations per compound, n=9 per condition), and results were compared to a DMSO control.  Follow-up dose-response testing on potential inhibitors confirmed in vitro growth inhibition by 13 compounds, including 12 kinase inhibitors and 1 epigenetic modifier.  In vitro IC50 values of the inhibitors ranged from 0.92 µM to 16 µM.  None was toxic to the HCT-8 cell line at concentrations less than 10 µM.  Putative mechanisms-of-action against C. parvum were identified using the Cryptosporidium genome databases and knowledge of the compound targets in other systems.

Conclusion: Because of the well characterized nature of the compounds tested in this study, our data increase knowledge about Cryptosporidium biology and can be exploited for drug development. The results indicate putative new drug targets, and the inhibitors that showed selective toxicity to C. parvum in vitro may themselves be promising chemical scaffolds for drug development. Critical next steps will include confirmation of the targets using in vitro enzyme activity assays and transgenic methods with related model parasite Toxoplasma gondii.  This will be followed by in vivo testing for target validation.