DEVELOPMENT OF E2 ENZYME MODULATORS USING PHAGE DISPLAYED UBIQUITIN VARIANTS

Therapeutic advancements in targeting the ubiquitin (Ub) system for cancer treatment are currently on the rise. This system can be perturbed in a variety of ways, which include targeting the enzymes that add Ub to proteins, the enzymes that remove Ub from proteins, and components that recycle cellular Ub. Although a large number of small molecules have been developed towards enzymes within this network, these constitute a small fraction of the total number of targets in this system. A significant barrier to developing new drugs for better treatments is target validation. Phage display, a technique that connects proteins with the genes that encode them, allows us to rapidly develop highly selective target validation tools to modulate large superfamilies of enzymes in the human genome in a reversible and dose-dependent manner. This method is an alternate approach to small molecules as it involves protein engineering to design Ub variant (UbV) libraries that streamline target validation. We have previously applied this method to develop a Ub-specific protease (USP)-interface library and used it for the high throughput screening of modulators of enzymes within the two protein families, E3s and deubiquitinases (DUBs). In this thesis, I describe the application of this method for the high throughput screening of modulators of E2 enzymes. To this end, structures of E2 enzymes with Ub bound to their backside were explored. Structural data was used to design a phage displayed library with 3 x 1010 UbVs with amino acid variations at twenty-one positions of Ub that constitute the surface binds to E2 backsides. This E2-interface library was screened alongside the USP-interface library against 15 different E2 enzymes. UbVs were developed against 10 different E2s. For Ube2D1, Ube2V2, Ube2G1 and Ube2G2, we substituted the residue at position 22 (typically Ser) at their backside surface with Arg. Substitution with Arg at this site abolished UbV binding. Thus, it was demonstrated that the UbVs raised against these E2s bind specifically to the backside surface. In addition to the backside surface, data presented in this thesis shows the successful development of binders to other sites (active site and C-terminal extension site) on E2s. The backside binding UbVs where then tested in vitro for binding affinities, specificities and inhibition activities. The UbVs were shown to bind to their cognate E2 with high affinity and specificity. Furthermore, in enzymatic assays, the backside surface binding UbVs against Ube2D1 and Ube2V1 were shown to inhibit chain elongation, whereas the backside surface binding UbVs against Ube2G1 and Ube2G2 were shown to modulate E2 charging. Crystal structures of the UbVs in complex with their cognate E2 proteins revealed distinctive molecular interactions in each case, but they also highlighted a common backside pocket that all three UbVs utilized for enhanced affinity and specificity. Therefore, we identify the backside surface on E2s as being a multifunctional regulatory region, which can regulate these enzymes in ways outside the scope of what can be achieved by active site inhibitors. Not only can we completely inhibit the enzymatic activity of these enzymes, but we can also rewire their activity towards different modification lengths. These findings validate the E2 backside as a target for small-molecule inhibitors and provide structural insights to aid inhibitor design and screening efforts.

Keywords: Cancer biology
E2 backside inhibitors
E2 Ub-conjugating enzymes
Phage display
Protein Engineering
Ubiquitin
Advisor: Sidhu, Sachdev