ORCID
0009-0002-1361-9999
Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
Christopher D. Huston
Second Advisor
Jonathan E. Boyson
Abstract
Cryptosporidiosis is a prevalent diarrheal disease characterized by infection of the small intestine by apicomplexan Cryptosporidium parasites. These intestinal parasites effectuate life-threatening diarrhea in young children and immunocompromised patients such as those on long-term immunosuppressants, or people living with HIV/AIDS. There are presently no vaccines to prevent cryptosporidiosis in humans. Unfortunately, the only FDA-approved treatment, nitazoxanide, lacks efficacy in immunocompromised patients but shows moderate efficacy in children, populations in which cryptosporidiosis is most severe and persistent. To engage the obvious paucity in the availability of novel anti-Cryptosporidium therapeutics, large-scale phenotypic screenings of compounds made available by Merck KGaA, Darmstadt, Germany, led to the identification of pyrazolopyrimidine human phosphodiesterase (PDE)-V (hsPDE-V) and 1,5-naphthyridine phosphatidylinositol-4 kinase (PI4K) inhibitors with potent in vitro anticryptosporidial characteristics and in vivo efficacy following oral administration in C. parvum-infected immunocompromised mouse models of cryptosporidiosis. The lead phosphodiesterase inhibitor (PDEi) and phosphatidylinositol-4-kinase inhibitor (PI4Ki) series showed comparable anti-C. parvum and C. hominis potency, are fast-acting in tissue culture, and have minimal off-target effects in a preliminary safety screening assay panel. Our main objective was to validate the molecular targets of the novel PDEis and PI4Kis lead series in Cryptosporidium. By extension, we sought to uncover the mode-of-action of these novel lead series in C. parvum and highlight important target-based strategies that can be exploited for drug target identification in anticryptosporidial drug discovery. We demonstrated the mode-of-action of the lead series by employing life-cycle phenotypic assays which identified the parasite egress stage as the key life stage blocked by the PDEi and PI4Ki series. Subsequently, we utilized in vitro enzyme assays to confirm on-target engagement of our lead PDEis and PI4Kis against recombinant CpPDE1/CpPDE3 and CpPI4K enzymes, respectively. Guided by in silico analyses, we identified two residues (Val900 and His884) in the CpPDE1 active site predicted to be important for pyrazolopyrimidine PDEi binding. We produced a CRISPR-engineered C. parvum CpPDE1-V900A transgenic strain which exhibited altered susceptibility to our lead PDEi series, providing genetic support for CpPDE1-pyrazolopyrimidine PDEi interaction. Our findings suggest that CpPDE1, a validated pyrazolopyrimidine molecular target, can be exploited for target-based lead optimization in our anticryptosporidial drug development scheme. Furthermore, to genetically validate CpPI4K as a molecular target of the novel naphthyridine PI4Ki lead series, we overexpressed the wild-type CpPI4K gene in wild-type C. parvum to confirm the development of a resistance phenotype in the CpPI4K over-expressing transgenic strain. The expression of an additional copy of the wild-type CpPI4K gene conferred a moderate resistance phenotype in the presence of a naphthyridine PI4Ki and a separate imidazopyrazine PI4Ki by about 3-fold. These results provide compelling evidence that CpPI4K is a molecular target of the imidazopyrazine and novel naphthyridine PI4Ki lead series. In summary, we have identified and validated CpPI4K and CpPDE1 as molecular targets of our PI4Ki and PDEi lead series, respectively. Our target identification efforts on CpPDE1 marks the first characterization of the CpPDE1 as a druggable target in C. parvum. Meanwhile, our genetic validation of CpPI4K druggability will build on existing research in the anti-Cryptosporidium drug discovery field. Collectively, the results from this work will inform medicinal chemistry lead optimization efforts to advance anticryptosporidial drug development.
Language
en
Number of Pages
291 p.
Recommended Citation
Ajiboye, Jubilee, "Combinative Strategy To Advance Target-Based Anticryptosporidial Drug Discovery." (2025). Graduate College Dissertations and Theses. 2008.
https://scholarworks.uvm.edu/graddis/2008
