Date of Award

2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Christopher D. Huston

Abstract

Cryptosporidiosis is a common diarrheal disease caused by intestinal infection with the apicomplexan parasite Cryptosporidium, in humans usually either with C. hominis or C. parvum. Unfortunately, given a large burden of disease in children and immunocompromised people like AIDS patients, the only currently approved treatment, nitazoxanide, is unreliable for these patient populations. To address the urgent need for new drugs for the most vulnerable populations, large phenotypic screening efforts have been established to identify anti-Cryptosporidium growth inhibitors in vitro (hits). However, in the absence of a gold standard drug, the in vitro and in vivo characteristics that should be used to prioritize screening hits are not known. This thesis is focused on identifying promising anti-Cryptosporidium hits and drug leads, and using them to establish validated methods to guide hit-to-lead studies for anti-Cryptosporidium drug development.

A re-analysis of our phenotypic screen of the Medicines for Malaria Venture Open Access Malaria Box identified a promising C. parvum growth inhibitor, MMV665917. It had similar in vitro activity against C. hominis, C. parvum Iowa, and C. parvum field strains, and it was amenable to preliminary structural activity relationship studies using commercially available variants, with one variant demonstrating nanomolar potency. Furthermore, MMV665917 was effective in vivo in an acute interferon-γ mouse model of cryptosporidiosis; and it appeared to cure an established infection in the chronic NOD SCID gamma (NSG) mouse model, unlike nitazoxanide, paromomycin, and clofazimine. We hypothesized that anti-Cryptosporidium activity in the highly immunocompromised chronic NSG mouse model might relate to compounds being capable of killing and eliminating parasites (cidal), rather than only preventing growth (static). To test this, we developed a novel in vitro parasite persistence assay that showed that MMV665917 was potentially cidal, whereas nitazoxanide, paromomycin and clofazimine appeared static. This pharmacodynamic assay also provided the concentration of compound required to maximize rate of parasite elimination, which could help design in vivo experiments.

To further characterize compounds based on mechanism of action, we developed a range of in vitro medium-throughput life-stage assays. To validate and gain value from the assays, a “learner set” of compounds from our in-house screens and collaborations were tested in all of the in vitro assays and in the in vivo NSG mouse model. Using these assays, it was possible to group molecules based on chemical class/mechanism of action. Because compounds from distinct groups showed activity in the NSG mouse model, these methods could be used to obtain a diverse set of early-stage Cryptosporidium inhibitors for prioritization. Furthermore, compounds that appeared static in the in vitro parasite persistence assay did not have activity in the NSG mouse model. In summary, we report the identification and development of a highly promising initial lead, MMV665917, and report a range of in vitro assays that can be used to prioritize anti-Cryptosporidium hits and leads.

Language

en

Number of Pages

201 p.

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