Molecular glue degraders (MGDs), such as pomalidomide, induce degradation of non-native substrates by the cullin-RING E3 ligase 4 (CRL4) through its substrate receptor cereblon (CRBN). Here, to explore CRBN programmability, we tested whether reported CRBN-MGD substrates are part of a network of latent CRBN interactors, proteins capable of MGD-induced CRBN binding without detectable degradation. Leveraging a highly parallel protein complementation assay (GluePCA) to measure MGD-induced interaction between CRBN and zinc fingers, we identified ~210 zinc fingers bound to CRBN-pomalidomide, where top binders are already reported as degraded by dedicated MGDs. To map latent CRBN-MGD interactions proteome-wide and define the accessible CRBN interaction space, we combined artificial intelligence-derived protein surface queries (MaSIF-mimicry) with GluePCA. This pipeline identified 6 known and 43 novel CRBN-pomalidomide binders, including orthogonally validated hits. We find that these binders provide privileged starting points for MGD development. We expect this binding-focused workflow to be applicable to other MGD-E3 ligase systems, potentially extending the scope of this emerging drug class.
Pius Galli, Shuhao Xiao, Y. Meng et al.· Nature Biotechnology· 0 citations
Protein kinases are central to biological regulation, dysregulated in many diseases, and the targets of a hundred clinically-approved drugs. Structural conservation of kinase active sites makes the development of specific inhibitors challenging. Targeting functional secondary sites can increase specificity, reduce toxicity, overcome resistance mutations, and also activate kinases. However, the functional secondary sites to target in most kinases are unknown, and the conservation of allosteric networks in kinases and other proteins that share the same structural fold is unclear. Here, we quantify the activity and abundance of >160,000 variants to construct complete maps of the energetic and allosteric architectures of five human kinase domains: SRC, FGR, JNK2/MAPK9, ZAK/MAP3K20, and TSSK2. For inhibition, all five kinases have distance-dependent but anisotropic allostery and each kinase has a unique allosteric architecture, surface, and set of pockets to therapeutically target. A set of functional secondary sites is conserved in all five proteins, but other allosteric pockets are protein-specific or switch from inhibitory to activating in different proteins. The differences in the energetic architectures are particularly striking for activation, where the allosteric maps are highly diverged. The allosteric architecture of each kinase is therefore unique, with a distinct set of functional secondary sites to regulate and therapeutically target.
Carla Folgado, Antoni Beltran, Ben Lehner· bioRxiv· 0 citations
Programmed translational readthrough produces C-terminally extended protein isoforms via decoding of stop codons by near-cognate tRNAs. Human genes experimentally validated as readthrough targets share a CUAG motif downstream of a UGA stop codon. However, the full sequence determinants of readthrough efficiency, how they combine, and how generalisable they are across genes remain largely unexplored. Here we use deep mutational scanning to quantify ∼1,400 sequence variants for each of the three examples of human readthrough in the genes AQP4, MAPK10 and OPRK1. In addition to the core CUAG motif, mutations that modulate readthrough elements extend up to +27 nucleotides downstream of the stop codon and across six codons (18 nucleotides) upstream. For the downstream sequence, an additive model with a sigmoidal global epistasis function captures most of the within-gene readthrough variance for double mutants (R²=0.84-0.96), with additional contributions from a small number of strong pairwise interactions. Mutational effects nonetheless generalise poorly between genes: only the immediate -3 to +4 nucleotide window shows consistent behaviour, while mutations in more distal positions have context-dependent effects. Combinatorial assembly of sequence blocks from different genes into chimeras reveals strong interactions (epistasis) between sequences upstream and downstream of the stop codon. This study provides comprehensive quantitative maps of the sequence determinants of human programmed readthrough and suggests that three examples of programmed readthrough are located on distinct local fitness peaks, each defined by different upstream and downstream architectures built around a shared CUAG core motif.
Ignasi Toledano, Fran Supek, Ben Lehner· bioRxiv· 0 citations
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