iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells

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

“…IMiDbound CRBN recognizes a small b-turn motif often found in zinc finger (ZF) transcription factors. Appending this small b-turn motif, commonly called a degron, to other proteins makes the resulting fusion protein susceptible to IMiD-induced degradation (9,(14)(15)(16).…”

Orthogonal IMiD-Degron Pairs Induce Selective Protein Degradation in Cells

“…At least ten CRL4 CRBN neosubstrates are known, including Ikaros family zinc finger protein 1 (IKZF1; Ikaros) and Ikaros family zinc finger protein 3 (IKZF3; Aiolos), ZFP91 zinc finger protein (ZFP91), G1 to S phase transition 1 protein (GSPT1), casein kinase 1 alpha 1 (CK1α), and spalt-like transcription factor 4 (SALL4); the latter of which is thought to be the effector of the teratogenic properties of thalidomide ( 13–23 ). Although these neosubstrates do not possess a specific consensus sequence, they all share a β-hairpin motif with a conserved glycine residue which binds at the CRBN-IMiD interface ( 6 , 24 ), and is frequently part of a C2H2 zinc finger (ZF). This motif is an example of a degron, broadly defined as a targeting signal that confers metabolic instability on some, or all, of the peptide bonds in a protein ( 25–27 ).…”

“…A combination of IMiD small molecules and ZF-based degrons offer an attractive alternative as inducible degrons; the low molecular weight inducers employed can readily enter cells and do not display a hook effect. In addition, the ZF degron motif can be as small as 23 amino acid residues, but works optimally at 60 amino acids ( 6 , 24 ), minimally perturbing the POI (degron = 7.0 kDa). Existing implementations of such systems have used degron motifs derived from IKZF1/3, SALL4, and hybrid sequences comprising halves of two different neosubstrate ZF degron sequences, such as Superdegron and iTAG ( 24 , 42–45 ), These IMiD-degron systems have been used to degrade chimeric antigen receptors (CARs) in T-cells, and modulate CRISPR Cas9 genetic editing ( 44–47 ).…”

“…In addition, the ZF degron motif can be as small as 23 amino acid residues, but works optimally at 60 amino acids ( 6 , 24 ), minimally perturbing the POI (degron = 7.0 kDa). Existing implementations of such systems have used degron motifs derived from IKZF1/3, SALL4, and hybrid sequences comprising halves of two different neosubstrate ZF degron sequences, such as Superdegron and iTAG ( 24 , 42–45 ), These IMiD-degron systems have been used to degrade chimeric antigen receptors (CARs) in T-cells, and modulate CRISPR Cas9 genetic editing ( 44–47 ). While this elegant approach overcomes some limitations of other inducible degrons, the use of existing small molecule IMiDs presents important selectivity-related limitations.…”

Degron tagging for rapid protein degradation in mice

“…However, the development of potent and selective degraders for new protein targets remains costly and time consuming, particularly for compounds destined for the clinic, which often require extensive optimisation to achieve desirable pharmacokinetic properties ( Pike et al, 2020 ). An alternative approach leverages both genome editing and protein engineering (see poster, ‘Degron tagging via chemically induced E3 ligase proximity’) to fuse additional peptide sequences to target proteins (hereafter referred to as ‘degron tags’) that interact with E3 ubiquitin ligases in a chemically controllable manner ( Bond et al, 2021 ; Bouguenina et al, 2023 ; Carbonneau et al, 2021 ; Nabet et al, 2018 , 2020 ; Nishimura et al, 2009 ; Nowak et al, 2021 ; Veits et al, 2021 ; Yamanaka et al, 2020 ). This renders the degron-tagged protein susceptible to degradation using generic pre-validated degrader molecules, over time scales of minutes to hours, in a manner that is reversible and dosage controllable.…”