Degrader Building Blocks for Targeted Protein Degradation
What is targeted protein degradation?
TPD generally refers to the strategy of targeted protein degradation by the endogenous protein degradation mechanism of cells. One important way TPD works is through the use of heterobifunctional molecules, often called degraders or PROTAC degraders, that bind both E3 ligase and target protein (POI). A terpolymer complex is formed between E3 ligase, degrader and POI to polyubiquitinate POI, and the ubiquitinated protein is recognized and degraded by the proteasome 26S in the cell. Utilizing small molecule degradation, rather than simply inhibiting target proteins, has a number of significant advantages.
Design and synthesis of protein degraders
How to design and find efficient degraders through effective evaluation system is the focus of the research and development of degraders. Based on the structural characteristics of the degraders, the modular process design of E3 ligase ligand, linker and POI ligand can be carried out.
Most of the inhibitors published to date have primarily targeted E3 ligases including von hippel lindau (VHL), cereblon (CRBN), and apoptosis protein inhibitors (cIAP). Among them, CRBN and VHL ligands have been the main ligands of PROTAC.
When it comes to the design of linker, it is crucial to select suitable linker binding sites for POI and E3 ligases to ensure high binding affinity. The link sites are typically amide-coupled, click chemistry, nucleophilic substitution, or reductive amination. In addition, the length of the linker can also affect the degradation activity, and the optimal Linker length can be determined using a simple alkyl chain and PEG chain between the two ligands, after which the optimization of linker stability can be focused on improving the PK/PD characteristics of the degrader.
POI ligands generally select inhibitors with certain activity that are listed or reported in the literature. Generally, some structural derivative optimization of the inhibitors is performed first, and then they are used as POI ligands.
A considerable bottleneck in the development of TPD is the synthesis and screening of a large number of candidate degraders, and the use of commercially available degrader building blocks can reduce time and economic costs. In addition, our screening library can help you select an E3 ligand targeting cereblon, VHL or IAP, an alkyl or PEG Linker, and a functional group coupled to your POI ligand to quickly generate the right customized solution.
Cereblon-based (CRBN) degrader
Immunomodulatory IMiDs, such as pomalidomide, thalidomide, and lenalidomide, target the CRBN substrate receptor CUL4-RBX1-DDB1-CRBN of E3 ubiquitin ligase. These CRBN ligands are widely used in the development of heterobifunctional degraders, including the clinical candidates ARV-110 and ARV-471 for Arvinas.
Studies have shown that proteins containing zinc finger (ZF) motifs are susceptible to IMIDS-induced CRBN ubiquitination and are degraded by proteasomes, known as "new substrates". Typical examples are the transcription factors IKZF1 and IKZF3, which have important biological functions, so off-target degradation can have long-term effects. By improving junction sites and linkers containing IMiDs, it is possible to reduce or even avoid degradation of new substrates. The 5' -phthalimide modification has been shown to reduce ZF protein degradation more than the 4' -phthalimide modification, while the incorporation of fluorogroups into the 6' position of IMiDs, such as ARV-110, further reduces off-target fall.
A) Schematic diagram of the ternary complex of PROTAC degrader based on CRBN. B) CRBN ligand used in clinical drug candidate ARV-471. C, D) Powerful and stable phenyl-glutarimide CRBN ligands. (Min, J., 2021)
IMiDs contain phthalimide functional groups, which have been reported to be unstable in hydrolysis and may reduce the half-life of the degrader in cells. To solve this problem, researchers from St. Jude Children's Research Hospital synthesized a novel cereblon ligand that does not contain phthalimide, called phenyl-glutarimide (PG). Compared to IMiDs, PG derivatives retain the affinity of CRBN while exhibiting significantly improved chemical stability. For example, analogues of dBET1 (JQ-1 based BET-bromine domain degraders), whose IMiDs are replaced by PG ligands, show a 6-fold higher degradation titer and increase degradation efficiency by 15 times after 24 hours of incubation. Other studies have shown that baricitinib-based JAK2 degraders can off-target the new substrate GSPT1 (translation termination factor), but the PG derivatives of the degraders can not only maintain the degradation efficiency of the target protein, but also effectively reduce the off-target degradation of GSPT1. These in vitro results indicate that PG ligands are expected to be more suitable and efficient CRBN ligands.
CRBN ligands at BOC Sciences
VHL-based degrader
In 2012, Alessio Ciulli et al. collaborated at JACs to announce the first small ligand molecule targeting the E3 ligase VHL, which can disrupt the interaction of VHL and HIF-1α. This work laid the foundation for the later development of high-performance and highly selective VHL ligands. VHL-associated small molecule inhibitors such as VH 032 and VH 101 are introduced to PROTAC as E3 ligase recruiters. These commonly used VHL ligands have higher molecular weights than IMIDs-based CRBN ligands, resulting in substantial differences in the physicochemical properties of the final degraders. This is also one of the key reasons for limiting the entry of VHL-based inhibitors into the clinic.
Using the benzyl site of VH 101 as the Linker junction site, researchers from the University of Dundee and Boehringer Ingelheim University selectively targeted SMARCA2 (BAF chromatin remodeling complex ATPase) by carefully adjusting the exit vector, Linker length and type. To demonstrate that bioavailable VHL-based degraders can be developed for export.
VHL ligands at BOC Sciences
IAP based degrader
IAP plays a crucial role in inhibiting apoptosis pathways by regulating caspase activity. Five of the eight IAPs, particularly apoptosis suppressor 1 (cIAP1) and X-linked apoptosis suppressor protein (XIAP), containing the RING domain of the E3 ligase complex, have been used for TPD by small molecule methods.
The first generation of heterobifunctional degraders uses the aminopeptidase inhibitor Bestatin to recruit IAP. These compounds, known as SNIPERs (specific and non-hereditary IAP-dependent protein eliminators), are capable of degrading many targets, such as estrogen receptor-α (ERα), androgen receptors, and BCR-ABL, but are only able to induce degradation when applied in concentrations greater than 10 μM. Bestatin was then optimized to produce potent and highly selective IAP ligands, such as LCL 161 and a 410099.1. LCL 161-based snipers were able to degrade ERα at low nanomolar DC50 (< 3 nM) and showed in vivo activity in mouse xenograft models.
IAP ligands at BOC Sciences
KEAP1-based degrader
KEAP1, a member of the Kelch-like protein family, is a substrate adaptor for REDOX regulation of the CUL3-dependent ubiquitin E3 ligase complex. KEAP1 is expressed higher than VHL or CRBN in human cancers and has a unique tissue distribution, being highly expressed in lung, kidney, breast, and prostate cancers. Therefore, the recruitment of KEAP1 protein to achieve the degradation of key targets is a potential strategy to treat these malignancies.
Researchers at Massachusetts General Hospital used the CoraFluor assay platform to detect a range of electrophilic reversible covalent inhibitors on wild-type, full-length KEAP1: CDDO-Me, DMF, and obtusaquinone, as well as the non-covalent inhibitor KI-696.
Ligand-linker conjugates simplify the synthesis of heterobifunctional degraders
Conventional heterobifunctional protein degraders usually consist of a small molecular ligand that binds to a target protein at one end and an E3 ubiquitin ligase at the other end, linked by a suppurably designed linker. The complexity of such structures often results in cumbersome and inefficient synthesis processes. Our ligand-linker conjugate eliminates the prior synthesis step and requires only a chemical reaction to attach the target ligand to the end functional group.
Thalidomide-CH2CONH-C2-COOH is a synthesized E3 ligase ligand-linker conjugate.
E3 ligase ligand-linker conjugates at BOC Sciences
The diversity of ligand-linker conjugates
Common E3 ligands (VHL ligands, MDM2 ligands, CRBN ligands, and cIAP1 ligands) can be coupled to different ligands (PEG, alkyl chain, alkyl and ether, etc.).
E3 ligases ligands: While an increasing number of E3 ligases are being used to target protein degradation, a few are most commonly used to develop protein degraders. Our conjugates include ligands for the proven E3 ligases CRBN, VHL, IAP and MDM2.
Linkers: Alkyl and PEG linkers are excellent starting points for sampling a wide range of hydrophobicity, flexibility and length. In addition, we offer many "hybrid" and rigid joints to diversify the joint features in your library.
Terminal chemistry: Various popular functional groups can be used to connect target warheads.
Advantage of ligand-linker conjugates
Save synthesis time: ligand-junction conjugates simplify the synthesis of single degraders and parallel synthesis of library construction
Molecular design: The arrangement of the most interesting E3 ligands, exit vectors, and splices in the complexes simplifies the previous combinational library design
Compatibility: The joint binds to common functional groups present on the target ligand
Scalable and high-throughput screening: Multiple ligand-linker combinations can be synthesized and tested simultaneously, accelerating the process of drug development and saving significant time and resources.
BOC Sciences provides a complete set of customized solutions for PROTACs research, including the building blocks of linkers, ligand for E3 ligase and ligand for target protein, etc. Our excellent product quality, flexible customization services and professional technical team have greatly promoted innovation and progress in the field of protein degraders.
References:
- Sievers, Q. L., et al. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science. 2018, 362(6414): eaat0572.
- Min, J., et al. Phenyl‐glutarimides: alternative cereblon binders for the design of PROTACs. Angewandte Chemie International Edition. 2021, 60(51): 26663-26670.
