P27 as a molecular target for cancer therapeutics: Discovering small molecule inhibitors of p27 proteolysis and structure-activity relationship and mechanistic studies of largazole, a potent inhibitor of histone deacetylase

Protein ubiquitination is a dynamic process, depending on a tightly regulated balance between the activity of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). The family of E3 ligases has been studied intensively and it is well established that their deregulation contributes to diverse disease processes, including cancer. The SCFSkp2 ubiquitin ligase is a central regulator of the eukaryotic cell cycle that functions as an E3 ligase to catalyze ubiquitination of a number of cell cycle regulatory proteins, including the well-characterized p27. Since decreased p27 levels in cancer cells have been associated with enhanced ubiquitin- dependent degradation and are linked to poor prognosis, p27 is a promising target for mechanism-driven cancer drug discovery. Therefore, I have taken a forward biochemical approach to identify small molecules that inhibit p27 proteolysis. Using a high throughput in vitro AlphaScreen assay and an in vivo cell-based assay combined with reconstituted biochemical experiments, I identified an asymmetrical disulfide compound that inhibits the ubiquitin E1-E2 pathway and a hydroquinone molecule that perturbs binding between Skp2 and a small adaptor protein Cks1. As the Skp2-Cks1 interaction dramatically increases the affinity of Skp2 to p27, thus facilitating its ubiquitination and subsequent degradation, it is an attractive interaction to target for disruption with small molecules. Both compounds impeded polyubiquitination of p27, a process that has been put forward by many scientists as a valuable target for pharmacological intervention.;With its picomolar inhibition of histone deacetylase (HDAC) activity and remarkable cytotoxicity against a number of solid tumors, the natural marine product largazole has been designated as the most potent and selective HDAC inhibitor to date. In the second part of my thesis work, I describe collaborative efforts to enrich structure-activity relationship studies by assessing antiproliferative effects of 15 largazole analogues (synthesized by Dr. Phillips' group) in colon and breast cancer cell lines. Replacing a valine residue within the macrocyclic backbone with smaller amino acids such as glycine and alanine increased largazole's inhibitory activity by 3 and 2.5 fold, respectively. A further inquiry into the mechanism of largazole revealed that largazole can block substrate ubiquitination by impeding the adenylation step during E1 activation, designating largazole as a dual specifi city inhibitor.