Targeted protein degradation as a powerful research tool in basic biology and drug target discovery

Abstract

Controlled perturbation of protein activity is essential to study protein function in cells and living organisms. Small molecules that hijack the cellular protein ubiquitination machinery to selectively degrade proteins of interest, so-called degraders, have recently emerged as alternatives to selective chemical inhibitors, both as therapeutic modalities and as powerful research tools. These systems offer unprecedented temporal and spatial control over protein function. Here, we review recent developments in this field, with a particular focus on the use of degraders as research tools to interrogate complex biological problems.

Fig. 1.

Schematic representations of key concepts in protein degradation. a, Bifunctional degrader (PROTAC). Model of CRL2^VHL E3 ligase in complex with MZ-1 (PROTAC) and BRD4_BD2 (as a model POI) PDB: 5T35 and 5N4W (chain A and R). b, ‘Molecular glue’ degrader. Model of CRL4^DCAF15 E3 ligase in complex with indisulam and RBM39 (POI). PDB: 6Q0R (chain B, C and D), PDB: 4A0C (chain C and D) and PDB: 4A0K (chain C). c, dTAG system as an example for a tagging strategy. Model of CRL4^CRBN E3 ligase in complex with dTAG-13 recruiting FKBP12^F36V-BRD4_BD1 (POI). PDB models as in B, FKBP12 PDB: 1FKJ. Structure visualization created with the Illustrate program

Fig. 2.

An overview of degradation tag fusion strategies for targeted protein degradation. Please refer to descriptions in the main text. POI based on BRD4 as an example, PDB: 6BOY; Auxin-inducible Degron based on TIR/IAA structure, PDB: 2P1Q (chain C), TIR structure, PDB: 2P1Q (chain B); Destabilzing Domain FKBP12 structure, PDB: 1FKJ; dTAG CRBN structure, PDB: 6BOY; IKZF3 tag based on structure of IKZF1 ZF2, PDB: 6H0F (chain C); HaloTag based apo HaloTag structure, PDB: 5UY1; VHL structure, PDB: 5T35 (chain D); NS3 protease structure in SMASh, PDB: 4WF8; NS4A degron based structure of NS4A peptide (shorter than the length of degron), PDB: 2OBO (chainB); Structure visualizations created with theIllustrate program.

Fig. 3.

Schematic representations of targeted protein degradation strategies mediated by macromolecular conjugates. a, Trim-away. b, Lysosome targeting chimera. c, Peptide-directed lysosomal degradation.

Fig. 4.

Validation methods for targeted protein degradation at different stages of the ubiquitin-proteasome pathway. P97 PDB: 5FTJ; model of CRL4^CRBN E3 ligase apo structure and structure with BRD4 created with PDB: 4A0C (chain C and D), PDB: 4A0K (chain C) and 6BOY (chain B and C); Human 26S proteasome PDB: 5GJR;Ubiquitin PDB: 1UBQ;POI BRD4 PDB: 6BOY (chain C). Structure visualizations created with the Illustrate program.