Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders

Abstract

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined "functional hotspots". Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here, we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

Constitutive protein degradation induces acute cell death via proteolysis products

“…Taken together, these results suggest that MZ1 resistance may arise from combinatorial effects of attenuated target degradation, lower production of IBM peptides, and dysfunctional cell death signaling. Previous reports indicate that the mechanisms of acquired 33,34 or engineered 35 resistance to targeted BRD4 degradation impact ligase machinery; our collective findings emphasize how these differences converge on the reduced capacity to produce IBM (which was not certified by peer review) is the author/funder. All rights reserved.…”

E3-Specific Degrader Discovery by Dynamic Tracing of Substrate Receptor Abundance

“…This allows selection of therapeutically enticing CRL E3 ligases, taking into account their characteristics such as disease relevance, expression pattern, and target complementarity. In fact, recently, we have shown that essentiality of an E3 ligase can have a profound impact on the emergence of resistance to degrader modalities, further highlighting the need to expand the targetable E3 ligase space . In principle, ligase tracing assays capture POI recruitment on a proteome-wide scale.…”

“…In fact, recently, we have shown that essentiality of an E3 ligase can have a profound impact on the emergence of resistance to degrader modalities, further highlighting the need to expand the targetable E3 ligase space. 48 In principle, ligase tracing assays capture POI recruitment on a proteome-wide scale. Future research will be required to determine the threshold of neo-substrate abundance required for this assay.…”

Advancing Targeted Protein Degradation via Multiomics Profiling and Artificial Intelligence

“…To this goal, base-editor screens, deep mutational scanning (DMS), and CRISPR tiling screens can play an important role. For instance, both DMS and CRISPR tiling have recently highlighted hotspots in ligase and POI that are functionally relevant for degrader activity. , …”

“…For instance, both DMS and CRISPR tiling have recently highlighted hotspots in ligase and POI that are functionally relevant for degrader activity. 237,238…”