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

Abstract

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.

An expanded lexicon for the ubiquitin code

“…In addition to their high specificity, these ‘molecular degraders’ surpass classical inhibitors by being active at substoichiometric concentrations, as they can be recycled after the target protein has been ubiquitylated. However, drug resistance and on-target toxicities remain a problem 197 , 198 . Several PROTACs, including ARV-110 and ARV-471, which target the androgen receptor and oestrogen receptor, displayed promising results in cell lines and mouse models for prostate cancer and breast cancer, respectively, and have thus entered clinical trials 199 .…”

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2

“…PROTACs have provided a new pharmacological paradigm of targeted protein degradation, with over a dozen compounds today in clinical trials, and many more in pre-clinical development, that degrade a variety of different targets against different diseases 59 . Resistance mechanisms already observed for VHL and CRBN, the most widely hijacked E3 ligases with PROTAC degraders, converge in the loss or missense mutations in E3 ligase components, including the substrate adaptors [60][61] . These observations motivate the search for novel E3 ligase recruiters, as expanding the reach of degraders to new E3 ligases could circumvent such innate or acquired resistance.…”

Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

“…Though first studies focussing on BET degrading PROTACs disclosed both acquired and intrinsic resistance mechanisms towards both CRBN-and VHL-recruiting PROTACs, 214,215 the essential role of VHL for survival of many cancerous cell lines results in a lower fraction of disruptive mutations on the substrate receptor VHL compared to CRBN. 216 Instead, Cul2 loss has been identified as primary resistance mechanism induced by MZ1 as exemplary VHL-recruiting degrader. 214,215,217 Interestingly, MZ1-resistant cells have shown to be still sensitive towards mM concentrations of the more potent VHL-based BET protein degrader ARV-771, 214 and to exhibit undiminished sensitivity towards CRBN-based BET degraders.…”