Plasticity in binding confers selectivity in ligand-induced protein degradation

Abstract

SUMMARY Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.

Designing Molecular Spanners to Throw in the Protein Networks

“…In another notable example starting from in vitro work, Nowak et al. showed the possibility of leveraging the plasticity of protein–protein interaction surfaces to develop molecules that selectively stabilize complexes between one partner and different homologues of the second partner . The authors concentrated on cereblon–BRD4 (CRL4 CRBN –BRD4) complexes.…”

“…Celebron (CRL4 CRBN ) is a protein that contains an E3 ligase subunit. The work by Nowak and co‐workers highlights the possibility of redirecting the ubiquitinylation effects of celebron towards different targets through structural understanding of its dynamics, and subsequent rational design of small molecules …”

“…In another notable examples tartingf rom in vitro work, Nowak et al showed the possibility of leveraging the plasticity of protein-protein interaction surfaces to develop molecules that selectively stabilize complexes between one partner and different homologues of the second partner. [60] The authors concentrated on cereblon-BRD4 (CRL4 CRBN -BRD4) complexes. By solving multiple X-ray crystal structures of CRL4 CRBN -BRD4 complexes in the presence of differents mall-molecule stabilizers, they showed that inter-protein contacts are mediated by a number of distinct binding conformations, whichever conformation is present depends directly on which small molecule is bound to the complex.…”

Development of Dual and Selective Degraders of Cyclin‐Dependent Kinases 4 and 6

“…Fore xample,i namouse model of non-small-cell lung carcinoma, genetic ablation of CDK4 but not CDK6 induced senescence in lung cancer cells expressing mutant K-Ras. [10] Thes mall molecule thalidomide,w hich binds to the ubiquitously expressed E3 ligase substrate adapter Cereblon (CRBN), has often been used as the E3 ligase binding moiety in degraders. [6] We also recently demonstrated that CDK6, but not CDK4, phosphorylates NFAT-family transcription factors to promote T-cell activation and enhance the antitumor immune response.…”

“…[7] Since current CDK4/6 inhibitors target their highly conserved ATP-binding pockets and are approximately equipotent inhibitors of both proteins,w eh ypothesized that achieving selectivity would require exploiting selectivity determinants outside of the ATP-binding pocket. [10] Thes mall molecule thalidomide,w hich binds to the ubiquitously expressed E3 ligase substrate adapter Cereblon (CRBN), has often been used as the E3 ligase binding moiety in degraders. [8] This strategy has been deployed to achieve selective degradation by using non-selective ligands.F or example,s elective degradation of CDK9 was achieved by converting the non-selective CDK inhibitor SNS-032 into ad egrader.…”