ANNEROOS EMMELY NEDERSTIGT

The Development of Novel Protein Inhibitors of the E2-Conjugating Enzyme Ube2D1

Ubiquitin (Ub) is a 76 residue, 8.6 kDa protein that is used as a post-translational modification. Most commonly it is transferred onto the ε-amino group of a lysine in a target substrate. Its signal specificity is conveyed through the inherent dynamics and the multivalency of ubiquitin. Substrate ubiquitination is achieved through a cascade that is mainly controlled by 3 types of enzymes in the human body: 1 E1 activating, 38 E2 conjugating and over 600 E3 ligating enzymes. Since the ubiquitination cascade is involved in many processes such as protein degradation, cell cycle regulation, DNA repair, lysosomal targeting and more, changes in the activity of its participating enzymes are potentially detrimental to some of these processes. As such, a plethora of diseases including cervical cancer, Huntington’s and Parkinson’s disease have been associated with the ubiquitination cascade. Efforts to drug the ubiquitination pathway have been primarily focused on E1 and E3 enzymes and the 26S proteasome. However, few efforts have been focused on finding small molecules that target E2 conjugating enzymes specifically, even though they play a critical role in determining where and how a target is modified by Ub.
Previously, we reported the development of novel linked domain Ube2D protein inhibitors that block both E1 and E3 interactions simultaneously to prevent Ub transfer in vitro. Since then, we have built upon and improved our initial designs by utilizing a phage-display optimized ubiquitin variant (Ubv) molecule in lieu of the Ubl domain originally present in our inhibitors. The Ubv is capable of selectively binding the backside of Ube2D1. Preliminary ubiquitylation assays testing E3 Ligase auto-ubiquitination activity in vitro, show a significantly enhanced potency of the new inhibitor compared to first generation designs. This data is also corroborated by the Kd, which was found to be approximately 5 nM using isothermal titration calorimetry. We are currently looking to investigate the potency of our inhibitors in HeLa cells, in an attempt to establish whether the E2-conjugating enzyme is a potential therapeutic target for treatments for diseases such as cancer.