E3 Ligase Ligands for PROTACs: How They Were Found and How to Discover New Ones

Abstract

Bifunctional degrader molecules, also called proteolysis-targeting chimeras (PROTACs), are a new modality of chemical tools and potential therapeutics to understand and treat human disease. A required PROTAC component is a ligand binding to an E3 ubiquitin ligase, which is then joined to another ligand binding to a protein to be degraded via the ubiquitin-proteasome system. The advent of nonpeptidic small-molecule E3 ligase ligands, notably for von Hippel-Lindau (VHL) and cereblon (CRBN), revolutionized the field and ushered in the design of drug-like PROTACs with potent and selective degradation activity. A first wave of PROTAC drugs are now undergoing clinical development in cancer, and the field is seeking to extend the repertoire of chemistries that allow hijacking new E3 ligases to improve the scope of targeted protein degradation.Here, we briefly review how traditional E3 ligase ligands were discovered, and then outline approaches and ligands that have been recently used to discover new E3 ligases for PROTACs. We will then take an outlook at current and future strategies undertaken that invoke either target-based screening or phenotypic-based approaches, including the use of DNA-encoded libraries (DELs), display technologies and cyclic peptides, smaller molecular glue degraders, and covalent warhead ligands. These approaches are ripe for expanding the chemical space of PROTACs and usher in the advent of other emerging bifunctional modalities of proximity-based pharmacology.

Keywords: E3 ubiquitin ligase; PROTACs; binding ligands; molecular glues; targeted protein degradation.

Figure 1.

Graphical representation of the degradation mechanism of proteolysis-targeting chimeras (PROTACs).

Figure 2.

Von Hippel–Lindau (VHL)-based and cereblon (CRBN)-based small-molecule proteolysis-targeting chimeras (PROTACs). (A) Exemplified structures of VHL ligands and the PROTACs composed of them: VH032,^, VH101, VH298,^, MZ1, PROTAC_ RIPK2, PROTAC_ ERRα, ARD-266, VZ185, and ACBI1. (B) Exemplified structures of thalidomide and PROTACs composed of CRBN ligands: thalidomide, dBET1, dFKBP12, QCA570, PROTAC6, ZNL-02-096, and d9A-2.

Figure 3.

Representative structures and biological data of recently disclosed patents: 406, I-685, Compound 1, Example 9, and STEAP1-L1BC8.

Figure 4.

Inhibitor of apoptosis protein (IAP)-based and mouse double minute 2 homolog (MDM2)-based proteolysis-targeting chimeras (PROTACs), and von Hippel–Lindau (VHL)-based and cereblon (CRBN)-based homo-PROTACs. (A) Exemplified structures of methyl bestatin (MetBS) and Specific and Nongenetic Inhibitors of Apoptosis Protein (IAP)-Dependent Protein Eraser (SNIPERs): MetBS, SNIPER-2 (R = O) and SNIPER-4 (R = NH), SNIPER(ER)-87, SNIPER(BRD4)-1, and SNIPER(ABL)-62. (B) Structures of MDM2 inhibitors and PROTACs incorporating with them: nutlin, idasanutlin, PROTAC 14, A1874, Compound 3, and MD-224. (C) Structures of homo-PROTACs and related PROTACs composed with CRBN and VHL ligands: CM11, Compound 15a, CRBN-6-5-5-VHL, Compound 14a, and TD-165.

Figure 5.

Proteolysis-targeting chimeras (PROTACs) using nonconventional E3 ligase ligands. (A) Structures of CDDO-Me^, and CDDO-JQ1. (B) Structures of sulfonamide derivatives, indisulam, E7820, CQS, and DP1. (C) Structure of CCW 28-3. (D) Structures of nimbolide and XH2. (E) Structure of KB02-SLF. (F) Structure of β-NF-ATRA.

Figure 6.

Noncovalent and covalent fragment-based approaches for hit finding and ligand development for E3 ligases. (A) Initial hit compound to the final clinical compound, ASTX660.^– (B–F) Examples of specific residue-selective covalent warheads: (B) cysteine, (D) lysine, and (F) methionine; and (C,E,G) the reaction mechanisms.