Charting functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders

Targeted protein degradation is a new pharmacologic paradigm established by drugs that recruit target proteins to E3 ubiquitin ligases via a ternary ligase-degrader-target complex. Based on the structure of the degrader and the neosubstrate, different E3 ligase interfaces are critically involved in this process, thus forming defined functional hotspots. Understanding disruptive mutations in functional hotspots informs on the architecture of the underlying assembly, and highlights residues prone to cause drug resistance. Until now, their identification was driven by structural methods with limited scalability. Here, we employ haploid genetics to show that hotspot mutations cluster in the substrate receptors of the hijacked ligases and find that type and frequency of mutations are shaped by the essentiality of the harnessed ligase. Intersection with deep mutational scanning data revealed hotspots that are either conserved, or specific for chemically distinct degraders or recruited neosubstrates. Biophysical and structural validation suggest that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we identified and validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and experimentally widely accessible methodology that empowers the characterization of small-molecule degraders and informs on associated resistance mechanisms. ### Competing Interest Statement S.B. is an employee at Proxygen, a company that is developing molecular glue degraders. M.B. is scientific founder, shareholder, and employee at Proxygen. G.E.W. is scientific founder and shareholder at Proxygen and Solgate and coordinates a Research Collaboration between CeMM and Pfizer. A.C. is a scientific founder, shareholder, and advisor of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms. S.J.H. and A.T. are currently employees of Amphista Therapeutics. The Ciulli laboratory receives or has received sponsored research support from Almirall, Amgen, Amphista Therapeutics, Boehringer Ingelheim, Eisai, Nurix Therapeutics, and Ono Pharmaceutical. The other authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this work.