Continuous evolution of compact protein degradation tags regulated by selective molecular glues

Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36–amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo–electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40’s activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons. Editor’s summary Degron tags enable rapid and tunable control of target protein levels using small molecules. The ability to develop tags with desirable properties could expand their use in research and biotechnology. Mercer et al. report a continuous evolution platform for generating high-affinity molecular glue complexes. Using this approach, the authors evolved a compact zinc-finger degron that engages the protein cereblon in the presence of thalidomide derivatives that avoid endogenous proteins, unlike the immunomodulatory drugs commonly used to trigger protein degradation. This work provides a compact orthogonal degron tag and a powerful system with which to engineer molecular glue interactions using diverse small molecules. —Di Jiang Rapid evolution of molecular glue interfaces yields a compact small molecule–triggered degron with high target protein specificity. INTRODUCTION Small molecule–triggered protein degradation tags (degrons) are powerful tools to study the effects of the loss of a protein of interest and for target validation. In the presence of a small molecule in mammalian cells, degron tags form a complex with an E3 ubiquitin ligase effector such as cereblon (CRBN). Proteins recruited to this degron•small molecule•E3 ligase ternary complex by fusion to a degron tag are degraded by the ubiquitin-proteasome system. RATIONALE Most known degron tags are large protein domains that cannot be easily or cleanly installed in endogenous protein-coding genes. Although zinc finger (ZF) degrons are theoretically small enough for facile endogenous tagging, their degradation is triggered by thalidomide, pomalidomide, and lenalidomide (immunomodulatory drugs, or IMiDs) with substantial off-target degradation effects, precluding their use as specific modulators of target protein levels. Recently, we reported the development of new IMiD derivatives such as PT-179 that bind CRBN but do not induce degradation of off-target proteins. In this study, we developed a phage-assisted continuous evolution (PACE) platform for generating engineered molecular glue interactions and applied it to evolve ZF degrons that bind CRBN complexed with the otherwise-inactive IMiD derivative PT-179. RESULTS To apply continuous evolution to molecular glue complexes, we developed PACE selections that link phage propagation to molecular glue ternary complex formation (MG-PACE). Over 250 hours of continuous evolution requiring survival through an overall dilution of 10271-fold yielded SD40, a 36–amino acid ZF protein that tightly binds CRBN in complex with PT-179. In mammalian cells, PT-179 potently degrades proteins fused to SD40 at either the N or C terminus. SD40 is small enough for efficient and precise in-frame insertion in target protein-coding genes in human cells using prime editing, enabling minimally perturbative studies of endogenous proteins generated from genes that maintain their native regulatory contexts. To reveal mechanistic insights into the evolution and molecular basis of SD40’s activity and specificity, we determined high-resolution cryo–electron microscopy (cryo-EM) structures of SD40 in complex with CRBN bound to PT-179 or pomalidomide. SD40 interacts with CRBN’s IMiD-binding C-terminal domain while also forming an extensive interface with its N-terminal domain. We also used MG-PACE to evolve a second ZF degron, SD56, that is functional in mouse cells. Compared with human CRBN, mouse CRBN contains an amino acid substitution that prevents binding of endogenous mouse ZF proteins upon IMiD treatment. MG-PACE evolved SD56 in the presence of mouse CRBN and PT-179. We showed that PT-179 potently degrades SD56-tagged proteins in cultured mouse cells and that SD56 can respond to canonical and next-generation IMiDs, including pomalidomide and mezigdomide. CONCLUSION We developed a continuous evolution system for generating new molecular glue complexes and applied it to remodel the CRBN•IMiD•ZF degron ternary complex. MG-PACE yielded SD40, a compact ZF that mediates potent degradation of tagged proteins in human cells in response to an IMiD-derived small molecule with no detected off-target neosubstrates. SD40 evolved to engage both the N- and C-terminal domains of CRBN, driving CRBN to its putatively active closed conformation. SD40’s small size facilitates efficient installation in endogenous protein-coding genes with prime editing, enabling tunable and selective modulation of endogenous proteins. We also used MG-PACE to develop SD56, a ZF degron that is compatible with PT-179 and other IMiDs in mouse cells. Collectively, our efforts provide a platform for the rapid evolution of tailor-made molecular glue complexes, including compact ZF tags that overcome shortcomings associated with existing degrons. An evolved zinc finger mediates specific degradation of tagged endogenous proteins. (A) We developed a selection system for phage-assisted continuous evolution of molecular glue complexes (MG-PACE). We applied MG-PACE to evolve ZF degrons that engage the E3 ubiquitin ligase substrate receptor CRBN only when it is bound to PT-179, a thalidomide derivative that does not exhibit off-target degradation of endogenous human proteins. Assembly of the CRBN•PT-179•ZF ternary complex in host Escherichia coli promotes transcription of the essential phage gene gIII. (B) An ~2.4-Å resolution cryo-EM structure revealed that the evolved ZF protein, SD40 (magenta), engages both CRBN’s IMiD-binding C-terminal domain (CRBNCTD, cyan) and N-terminal domain (CRBNNTD, green) through contacts not previously observed between CRBN and a ZF. (C) SD40 is only 36 amino acids long, enabling clean installation of SD40 in-frame into endogenous genomic protein-coding genes in mammalian cell culture with prime editing. SD40-tagged endogenous proteins in human cells are potently, rapidly, and selectively degraded in response to treatment with PT-179.