The Ubiquitination Cascade: Scoring Touchdowns…Inside Your Body!

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 protein.1 Its signal specificity is conveyed through the inherent dynamics and the multivalency of ubiquitin.1 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 and more,2 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.3 Efforts to drug the ubiquitination pathway have been primarily focused on E1 and E3 enzymes and the 26S proteasome.3 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.4 Previously, we reported the development of novel dual-mode Ube2D1 inhibitors that block both E1 and E3 interactions simultaneously to prevent ubiquitylation in vitro. Currently, we have built upon and improved our initial designs by utilizing a phage-display optimized ubiquitin variant (Ubv) molecule that is capable of selectively binding the backside of Ube2D1.5 Preliminary ubiquitination assays have shown the new design has significantly improved the potency of our inhibitor. Therefore, we are now looking to investigate its potency in vivo as well. 1. Randles, L., & Walters, K. J. (2012). Ubiquitin and its binding domains. Frontiers in Bioscience (Landmark Edition), 17, 2140–2157. https://doi.org/10.2741/4042 2. Park, C.-W., & Ryu, K.-Y. (2014). Cellular ubiquitin pool dynamics and homeostasis. BMB Reports, 47(9), 475–482. https://doi.org/10.5483/bmbrep.2014.47.9.128 3. Petroski, M. D. (2008). The ubiquitin system, disease, and drug discovery. BMC Biochemistry, 9 Suppl 1(Suppl 1), S7–S7. https://doi.org/10.1186/1471-2091-9-S1-S7 4. Stewart, M. D., Ritterhoff, T., Klevit, R. E., & Brzovic, P. S. (2016). E2 enzymes: more than just middle men. Cell Research, 26(4), 423–440. https://doi.org/10.1038/cr.2016.35 5. Garg, P., Ceccarelli, D. F., Keszei, A. F. A., Kurinov, I., Sicheri, F., & Sidhu, S. S. (2020). Structural and Functional Analysis of Ubiquitin-based Inhibitors That Target the Backsides of E2 Enzymes. Journal of Molecular Biology, 432(4), 952–966. https://doi.org/10.1016/j.jmb.2019.09.02