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Thalidomide and its derivatives exert not only therapeutic effects as immunomodulatory drugs (IMiDs) but also adverse effects such as teratogenicity, which are due in part to different C2H2 zinc-finger (ZF) transcription factors, IKZF1 (or IKZF3) and SALL4, respectively. Here, we report the structural bases for the SALL4-specific proteasomal degradation induced by 5-hydroxythalidomide, a primary thalidomide metabolite generated by the enzymatic activity of cytochrome P450 isozymes, through the interaction with cereblon (CRBN). The crystal structure of the metabolite-mediated human SALL4-CRBN complex and mutagenesis studies elucidate the complex formation enhanced by the interaction between CRBN and an additional hydroxy group of (S)-5-hydroxythalidomide and the variation in the second residue of β-hairpin structure that underlies the C2H2 ZF-type neo-morphic substrate (neosubstrate) selectivity of 5-hydroxythalidomide. These findings deepen our understanding of the pharmaceutical action of IMiDs and provide structural evidence that the glue-type E3 ligase modulators cause altered neosubstrate specificities through their metabolism.

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Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as first-line therapy in many haematological cancer diseases, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4CRBN 1–4. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins5,6. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy2–4,7. However, current IMiDs and IMiD-based PROTACs also unexpectedly break down neosubstrates involved in embryonic development and disease progression2–4,8–10. Here, we showed that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IZKF3, and CK1α, which are involved in anti-haematological cancer activity2–4, and showed stronger antiproliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using the new lenalidomide derivatives for BET proteins induced the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exerted antiproliferative effects in all cell lines examined, including MM and neuroblastoma cell lines. Thus, 6-position-modified lenalidomide is a key strategy for selective TPD using thalidomide derivatives and PROTACs.