<div>Ebolavirus, a genus of filoviruses, are responsible for outbreaks that cause up to 90% fatality, including the recent outbreak in West Africa that has resulted in over 28,603 reported cases and 11,301 deaths according to the WHO. Inhibitors of Vacuolar-ATPase (V-ATPase), a key protein complex that is responsible for endosomal acidification and represents a unique method to block this common viral pathway. V-ATPase inhibitors have previously been explored as therapies for many diseases but have failed due to high toxicity. Diphyllin is a natural, arylnaphthalene lignan that represents a novel structural class of V-ATPase inhibitors with a greater selectivity index than previous V-ATPase inhibitors. Diphyllin has shown promising anti-tumor and anti-osteoclast activity, as well as strong anti-viral activity against Influenza and Dengue viruses. </div><div>Herein, novel modifications of the lactone and phenol functional groups of diphyllin were explored for the ability to enhance the potency or therapeutic selectivity of the diphyllin core. Four initial sets of derivatives were synthesized and assayed for activity against ebolavirus infection, inhibition of cellular endosomal acidification, cytotoxicity and biochemical inhibition of isolated V-ATPase. Modification of diphyllin’s lactone functional group reduced both activity and selectivity, while alkylation of the phenol groups significantly enhanced activity. The incorporation of basic heterocycles to the alkyl group created an alkylamino series of derivatives that exhibited significantly improved therapeutic selectivity compared to diphyllin. Further investigation of the alkylamino class indicated that they retained activity against Marburgvirus infection, a filovirus related to Ebolavirus. Alkylamino derivatives inhibited ebolavirus infection of human macrophages at low micromolar levels with no apparent cytotoxicity.</div><div>Further investigation of the alkylamino class of diphyllin derivatives was conducted to determine if potency and/or therapeutic selectivity could be optimized. The addition of a 1-methylpiperazine moiety to the end of the alkyl chain improved potency 1260-fold over diphyllin, though therapeutic selectivity was not improved. The modification of the alkylamino linker to an acetamide eliminated cytotoxicity but decreased derivative activity against V-ATPase activity. To evaluate if the cytotoxicity evidenced by the alkylamino derivatives was evidenced in organisms, the derivative toxicity was assessed in zebrafish and mouse models. Derivatives displayed toxicity in a zebrafish developmental model but were all at least 10-fold less toxic than the known V-ATPase inhibitor bafilomycin A1. Three derivatives were well tolerated in CD-1 mice when administered at therapeutically relevant concentrations and caused no abnormal changes in their blood chemistry. Overall, these results demonstrate that the alkylamino and acetamide diphyllin phenol derivatives should be further studied as therapies for ebolavirus infection in addition to other V-ATPase mediated diseases.</div><div><br></div>

Anthracycline-based chemotherapy is the mainstay neoadjuvant therapy for breast cancer. However, it is efficacious in only 60% of patients while carrying substantial toxicity. The application of a predictive marker of response may spare predicted ‘poor responders’ from the toxicity. Previously, we demonstrated a gene expression signature that predicts chemotherapy resistance which is linked to TP53 integrity. Further investigation showed that p53 signatures predict response in only ER+ tumors. We hypothesized that the loss of p53 confers an elevated chemotherapy sensitivity in ER+ breast tumors. We engineered isogenic p53 mutant ER+ breast cancer cell lines and assayed their cell cycle kinetics and chemotherapy sensitivity. Our results demonstrated that the loss of p53 is necessary to abrogate p53-mediated cell cycle arrest and produce an increase in apoptosis. Therefore, p53 signatures may be utilized as a predictive marker of response for patients with ER+ breast tumor and spare ‘poor responders’ from toxicity. Since ER+ p53 wild-type breast tumors are associated with anthracycline resistance, new anticancer drugs against that subgroup of tumors are needed. Phenotypic drug screening approach, which do not focus on isolated targets but instead classify compounds by their impact on cell physiology, is highly suitable for this purpose. Current cell-based phenotypic assays require fixation and staining for phenotypic markers, which reduce screen throughput and introduce potential variations and artifacts. Here we describe a high-content live-cell phenotypic assay, which streamlines the process of cytological profiling and provides a consistent platform for empirically evaluating drug action. Importantly, when combined with chemical similarity clustering, the phenotypic assay provided an inference of structure-activity relationships. Finally, a small-scale phenotypic screen of natural products enabled classification of unknown compounds against the cytological profiles of commercial compounds. Hence, the phenotypic screen provides a new and robust opportunity for accelerating the evaluation of compound activity during high-throughput drug screens. / Thesis / Master of Science (MSc)