File(s) under embargo
Reason: One of the chapters in this thesis contains unpublished data.
until file(s) become available
DEVELOPMENT AND CHARACTERIZATION OF NOVEL PROBES TO ELUCIDATE THE ROLE OF UBIQUITIN C-TERMINAL HYDROLASE L1 IN CANCER BIOLOGY
Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme (DUB) that is expressed in the central nervous system under normal physiological conditions, comprising 1-5% of the total soluble tissue in the brain. Ectopic expression of UCHL1 is observed in a number of aggressive forms cancers, where increased protein levels correlate with an increased invasive phenotype, supporting UCHL1 as an oncogene. Despite this, there is a significant lack of useful probes to validate targeting UCHL1 in vivo. In this work, both small molecule inhibitors and macromolecular inhibitors of UCHL1 were designed and characterized in an effort to alleviate this issue.
Few small molecule inhibitors of UCHL1 are available, with the current gold standard LDN- 57444 suffering for poor chemical stability as well as an inability to engage UCHL1 in cells. Two previously published inhibitors, MT-19 and VAEFMK, represent viable alternatives to LDN-57444. However, it was imperative to characterize these molecules in detail to confirm their utility. Using a number of approaches, it was determined that while MT-19 is a potent inhibitor of UCHL1, it suffers from non-specific toxicity and engages other proteins in the cell, making it unsuitable for use as a probe. On the other hand, a similar approach validates VAEFMK as a UCHL1-selective probe, though it suffers from low potency and requires high concentrations in cells to fully engage UCHL1.
Alternatively, the high intrinsic affinity of UCHL1 for ubiquitin (Ub) provides a starting point for macromolecular inhibitor design. However, Ub interacts with many proteins, including the nearly 100 DUBs expressed in the human genome. Using the co-crystal structures of UCHL1 and UCHL3 with Ub, a number of Ub variants (UbVs) were designed in silico to leverage the difference in the Ub-interfaces of UCHL1 and UCHL3. Biochemical testing shows that selectivity towards UCHL1 was achieved, though this selectivity was lost upon conversion of the UbV into an activity- based probe (ABP). Molecular dynamics (MD) simulations provide data to rationalize why this occurs.
Although much work remains to identify an inhibitor that displays suitable potency and selectivity towards UCHL1 in cells, this work sheds light the mechanistic rationale for a variety of inhibitors and will serve as a useful reference for the design of future UCHL1-selective probes.