Thalidomide, lenalidomide, and pomalidomide are clinically approved therapies for the treatment of multiple myeloma and other hematologic malignancies. These drugs induce rapid ubiquitination and proteasomal degradation of two transcription factors, Ikaros (IKZF1) and Aiolos (IKZF3), by recruiting them to the CRL4^CRBN E3 ubiquitin ligase through a Cys2-His2 (C2H2) zinc finger (ZF) domain that is present in both proteins and required for their destruction.

Transcription factors have been challenging drug targets because they lack discrete catalytic domains amenable to small-molecule inhibition. Thalidomide analog–induced degradation of IKZF1 and IKZF3 through a C2H2 ZF domain raised the possibility that the >800 C2H2 ZF–containing proteins encoded by the human genome, many of which are putative transcription factors, could be similarly destabilized. We therefore set out to (i) define the human ZF “degrome” in the context of thalidomide, lenalidomide, and pomalidomide; (ii) characterize the ZF-drug-CRBN interaction structurally and functionally; and (iii) determine whether different thalidomide analogs degrade distinct ZFs.

Using a reporter of substrate degradation, we screened 6572 C2H2 ZFs for degradation in the presence of thalidomide, lenalidomide, and pomalidomide, identifying 11 ZF degrons, motifs that are capable of mediating drug-dependent degradation, of which six were found to mediate degradation of their full-length protein. Surprisingly, the 11 ZF degrons lack an identifiable consensus sequence. Saturation mutagenesis of the IKZF1/3 ZF degron and crystal structures of two ZF degrons bound to pomalidomide-engaged CRBN demonstrate that the drug-CRBN interface accommodates ZF degrons with diverse amino acid sequences. Computational docking in combination with in vitro binding assays revealed that a large number of ZFs are capable of weakly binding the drug-CRBN interface, indicating that this interface may be more permissive than suggested by the 11 ZF degrons identified in the degradation screen. To test this hypothesis, we screened the ZF library against two thalidomide analogs with chemical alterations at the ZF-drug-CRBN interface. The two thalidomide analogs induced degradation of distinct sets of C2H2 ZF degrons, including ZFs that bind the CRBN-pomalidomide complex weakly in vitro, but were not degraded by pomalidomide in cells.

We found that thalidomide analogs mediate CRL4^CRBN-dependent degradation of a larger number of C2H2 ZF proteins than previously anticipated. ZFs compatible with the drug-CRBN interface show little sequence conservation apart from residues that stabilize the ternary ZF fold. In addition to the complex ZF-CRBN side chain interactions, direct contacts between thalidomide analogs and varying ZF residues provide another layer of specificity. Thalidomide analogs with altered chemical scaffolds thus allow selective degradation of distinct ZF targets. Our results provide a structural and functional basis for the chemical modulation of CRL4^CRBN to degrade C2H2 ZF transcription factors. Degradation of C2H2 ZF–containing proteins through derivatized thalidomide analogs may be a general paradigm for therapeutically targeting C2H2 ZF transcription factors, a class of proteins previously perceived to be “undruggable.”

We created a cellular library in which each cell expresses one of 6572 individual C2H2 ZF domains from the human proteome fused to enhanced green fluorescent protein (eGFP). Cells …