Chemical genetics-guided discovery and modification of anti-mitotic and anti-inflammatory bioactive natural products

Cancer is an intricate disease that has been studied extensively and remains a high priority in medical research. One of the hallmarks of cancer is dysregulated proliferation of cells, which treatments and medications aim to combat.¹ An area of current research is the study of anti-mitotic drugs, which stop cells from dividing and usually interfere with a cell’s microtubules. Chapters 2-4 focus on the discovery of anti-mitotic natural products, their synthetic modification, and their mechanisms of action. Sepsis is a dangerous inflammatory reaction to infection, whereby the body’s natural defense systems overreact, potentially leading to tissue damage, organ failure, or death.² Chapter 5 investigates the regulation of the PI3K signalling pathway, focusing on SHIP1 activation. In Chapter 2 pulchelloid A was isolated and chemically modified to confirm the necessity of Michael acceptors for bioactivity. Pull-down experiments suggested interactions with ubiquitin-related proteins, while a haploid assay identified strong genetic interactions with kinetochore/spindle assembly checkpoint genes, supporting a unique mechanism distinct from traditional microtubule inhibitors like colchicine. A series of alkynyl chemical probes were synthesized for use in pull-down experiments. A previously undescribed rearrangement was observed, which readily occurred under both basic and acidic conditions and yielded the known compound isopulchelloid A. Chapter 3 used bioassay-guided fractionation to identify anemonin from Pulsatilla nuttalliana as an anti-mitotic compound. The first ever resolution of trans-anemonin enantiomers was achieved and confirmed through CD spectroscopy and X-ray crystallography. Cis-anemonin was isolated and characterized for the first time, and a dynamic equilibrium between trans- and cis-anemonin, and protoanemonin was shown to occur, depending on solvent and temperature. Chapter 4 reports the first mitotic arrest activity of (+)-6-Tuliposide A, which was isolated using bioassay-guided fractionation from Erythronium grandiflorum. HT-29 cell assays revealed a distinct mitotic arrest phenotype, differing from tubulin-binding agents like colchicine. In Chapter 5 turnagainolides A and B were confirmed as SHIP1 activators in whole-cell and zebrafish assays. Solubility challenges led to the synthesis of a more polar pyridine-containing macrolide analog, which could not be purified. Unexpectedly, two linear methyl ester analogs exhibited promising anti-inflammatory activity, highlighting the value of structural modifications.