Eisert, J. | bioRxiv Science

bioRxiv2025

Multi-scale classification decodes the complexity of the human E3 ligome

E3 ubiquitin ligases are key regulators of protein homeostasis, targeting specific proteins for degradation via the ubiquitin-proteasome system (UPS). They provide crucial substrate specificity, making them promising candidates for the design of novel therapeutics. This work presents a comprehensive, annotated dataset of high-confidence catalytic human E3 ligases, termed the "E3 ligome". Integrating disparate data from various granularity layers, including protein sequence, domain architecture, 3D structure, function, localization, and expression, we learn an emergent distance metric, capturing authentic relationships within this heterogeneous group. A weakly-supervised hierarchical classification framework identifies conserved features of E3 families and subfamilies, consistent with RING, HECT, and RBR classes. This classification explains functional segregation, identifies multi-subunit and standalone enzymes, and integrates substrate and small molecule interaction networks. Our analysis provides a global view of E3 biology, opening new strategies for drugging E3-substrate networks, including drug re-purposing and designing new E3 handles.

bioinformatics↗

bioRxiv2024

Engineering CRBN for rapid identification of next generation binders

The majority of clinical degrader candidates utilize an immunomodulatory imide drug (IMiD)-based derivative, that directs their target substrate to the E3 ligase receptor Cereblon (CRBN), however, concomitant neo-substrate degradation by IMiDs often results in severe off-target effects. Further biophysical screening is needed to discover CRBN binders that might overcome these safety concerns, but the previously reported CRBN protein constructs suffer significant limitations that reduce their applicability as tools for biophysical assays making large-scale screening efforts a costly endeavor. This is due to the requirement of CRBN co-expression with DDB1 in insect cells to produce soluble protein that contains all the reported structural features necessary for proper compound interaction. Here, a near full-length human CRBN construct was designed that retains these required features, but for the first time allows the generation of highly homogenous and cost-efficient expression in E.coli, eliminating the need for DDB1 co-expression. We have extensively profiled the new construct against the existing CRBN constructs in binary and ternary degrader complexes, showing it to be the "best of both worlds" in terms of binding characteristics and ease of protein production. We then designed the Enamine CRBN focused IMiD library of 4480-compounds and demonstrated applicability of the new construct to high throughput screening by identifying novel CRBN binders with high potency and ligand efficiency. The new methods described here should serve as valuable tools for the development of the next generation IMiD-based degraders.

biophysics↗