Product News | E3 Ubiquitin Ligase Ligands in PROTACs Technology

Mar.16.2023

Induced protein degradation is emerging as a powerful strategy for regulating protein function and studying the corresponding cellular signalling pathways, including the use of proteolytic targeted chimeras (PROTACs) and molecular gel degraders for target protein degradation [1]. PROTACs use chemically synthesised bifunctional molecules to induce target proteins into close proximity to E3 ubiquitin ligases and use the ubiquitin-proteasome system to degrade specific proteins. As different E3 ligases have specific expression in certain tissue types, cell types or cellular compartment regions, their selective and specific ligands are important building blocks for designing and expanding the chemical space of current PROTACs, which can effectively expand the range of target proteins that can be applied to aid the research and treatment of related diseases [2]. However, compared to the more than 600 E3 ligases encoded in the human genome, the number of E3 ligands identified and applied to PROTACs is limited (about 10 E3 ligases exist with corresponding ligands), and further research and expansion is still needed [3-5].

Figure 1: PROTACs degradation mechanism and design strategy

The Ubiquitin-proteasome system (UPS) is an important pathway for selective protein degradation. PROTACs consist of protein targeting ligands, linkers and E3 ubiquitin ligase ligands, which can induce E3 ligases to They form a ternary complex that spatially brings the target protein and E3 ligase into close proximity, thereby mediating the ubiquitination of the target protein and its degradation by the proteasome [6, 7].

VHL ligands

In 2001, Deshaies Raymond's team introduced the concept of PROTACs and reported the first PROTAC molecule based on the Skp1-Cullin-F protein complex peptide ligand I kappa B alpha phosphopeptide for the degradation of methionine aminopeptidase-2 (MetAP-2). [8] The restricted cellular permeability, ease of synthesis and poor stability of PROTACs molecules due to polypeptide molecules prompted efforts to develop additional non-peptide E3 ligase ligands. In the early 2000s, the central hydroxyproline residue in the ALAPYIP recognition sequence of hypoxia-inducible factor-1 (HIF-1α) was identified as being required for the formation of the HIF-1α/VHL complex via a hydrogen bond between the hydroxyproline of HIF-1α and the hydroxyproline pocket on VHL. in 2004, Schneekloth and Crews et al. used the ALAPYIP recognition sequence as the first cell-permeable VHL recruiter for PROTAC to target and degrade the FK506 binding protein (FKBP12). The VHL ligand was then further optimised to obtain its small molecule analogue. This provided a strong basis for the widespread application of PROTACs technology that interferes with proteins at the post-translational level, offering the opportunity to create chemical knockdowns to study protein function without genetic modification [9].

CRBN ligands

Over the next decade or so, other E3 ligase ligands continued to be discovered. Thalidomide, a drug originally developed in the 1950s to treat morning sickness in pregnant women, was found to have a severe congenital defect known as seal limb syndrome, which is characterised by short limbs. Recent studies have found that thalidomide or other analogues of immunomodulatory drugs (e.g. lenalidomide, pomalidomide, etc.) are able to induce substrate proteins to bind to CRBN to form a complex that is recognised by Cullin-RING E3 ubiquitin ligase 4 (CRL4) and then degraded via the ubiquitin-proteasome route. As a result, compounds such as thalidomide became new E3 ubiquitin ligase ligands that could be designed and synthesised for PROTACs.10 In 2019, ARV-110 and ARV-471, designed and synthesised based on CRBN ligands, became the first PROTACs molecules to enter clinical trials.In 2020, Arvinas announced the role of ARV-110 in metastatic desmoplastic The pharmacological efficacy of PROTACs in the clinic was validated, setting a new milestone in the development of the PROTACs class of drugs [11-13].

Figure 3: Common CRBN ligands and PROTACs molecules entering clinical studies

Other ligands

In addition to VHL and CRBN ligands, new E3 ligase ligands have been disclosed and successfully used in the design and synthesis of PROTACs molecules: mouse double microsomal 2 protein (MDM2), inhibitor of apoptosis protein (cIAP), ring finger protein 4/114 (RNF4/114), DCAF 11/15/16 (DDB1- and CUL4-associated factor 11/15/16), FEN1B and other E3-ligase corresponding ligands [14, 15].

Figure 4: Other reported successful applications of E3 ligase ligands for PROTACs

Current E3 ligase recruiting molecules may not be sufficient for the selective degradation of any target protein of interest, but PROTACs have been extensively demonstrated to efficiently induce degradation of a wide range of proteins [16].

Thus, for more than 600 E3 ligases, only a few E3 ligases exist with corresponding ligands and their research prospects and application potential are enormous. The application of new E3 ligase ligands to PROTACs technology could not only expand the range of target proteins that can be degraded, but also provide new chemical approaches for the treatment of corresponding clinical diseases [17].

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