Date of Award
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
Christopher D. Huston
Abstract
The intestinal protozoan parasite Cryptosporidium is a leading cause of diarrheal disease and is a significant burden worldwide. For healthy adults, disease is mild and self-limiting, however young children and the immunocompromised are those most heavily impacted. Unfortunately, the only FDA approved treatment is ineffective. Over the last decade and a half, many anticryptosporidial leads have been identified and studied extensively, however some have unknown drug targets. Surprisingly knowing a drug target isn’t required for development, but understanding mechanism does provide benefits, one being a means to screen patients for potential drug-resistant parasites. Given the history of antimalarials, there is cause for concern for the rapid emergence of drug-resistant Cryptosporidium.
One lead, MMV665917, was identified by screening the Medicines for Malaria Venture “Malaria Box” library of compounds. MMV665917 has been shown to be active against both C. parvum and C. hominis in vitro, and in various animal models providing ample information on dosing and toxicity. Development of this compound has led to a drug series with improved anticryptosporidial activity as well as mitigating toxicity and off target effects. Phenotypic assays have described the timing of MMV665917’s activity in the parasite lifecycle and its impact on sexual differentiation. However, its target remains unknown, making it an ideal candidate for drug target identification studies. The focus of this work was on identifying the molecular target of MMV665917 utilizing a combination of forward and reverse genetics, transcriptomics, and proteomics.
Drug resistance is an imperfect outcome for drug development, but also serves as a powerful tool for identifying drug targets. Research methods for Cryptosporidium are limited and in their infancy, requiring us to develop strategies to study drug resistance. Here, we have established a protocol for generating drug resistant C. parvum in a chronic mouse model. The method is reliant on a mutator strain of the parasite, CpMut, which is deficient in its proofreading ability. Using CpMut, we independently selected for two strains resistant to MMV665917, and using a backcrossing method show we can mate resistant and susceptible parasites to map mutations relevant to resistance. Whole genome sequencing of these resistant strains has provided several candidates to evaluate as potential MMV665917 targets. Using RNA-seq, we profiled changes in C. parvum’s gene expression in response to exposure to various anticryptosporidials. Treatment with MMV665917, various analogs, and outgroup compounds revealed differential expression in Cryptosporidium’s SUMOylation pathway specific to MMV665917 and its analogs. Using checkerboard assays, and inhibiting SUMOylation as well as treating with MMV665917 showed they act in synergy, suggesting SUMOylation is playing a role in C. parvum’s compensatory response to MMV665917.
These approaches have further characterized MMV665917, uncovered clues about its impact on parasite biology, and have directed us towards a potential drug target. Further evaluation is required to pin-point the mechanism of this compound. All together, we provide a framework for future drug target identification studies in Cryptosporidium, which will be a paramount aspect of future development of anticryptosporidials.
Language
en
Number of Pages
349 p.
Recommended Citation
Mattice, Ethan Benjamin, "Drug Target Identification Methods For Cryptosporidium" (2024). Graduate College Dissertations and Theses. 1977.
https://scholarworks.uvm.edu/graddis/1977