PROTAC (Proteolysis Targeting Chimera) is an innovative technology that induces targeted protein degradation via the ubiquitin-proteasome system. A PROTAC molecule consists of three parts: a ligand that binds to the target protein, a ligand that recruits the E3 ubiquitin ligase, and a linker that connects the two.
Figure 1. PROTAC-induced targeted degradation of protein.
The design and selection of the linker are crucial for the drug-like properties of PROTAC molecules. The type, length, and linkage site of the linker can affect the structural rigidity, hydrophobicity, and solubility of the PROTAC, thereby influencing the formation of the ternary complex and the eventual degradation activity.
Here, we will primarily analyze and discuss two aspects: the structural types and characteristics of PROTAC linkers, and the impact of linker properties on the biodegradation efficiency of PROTACs.
The Structural Types And Characteristics Of PROTAC Linkers
PROTAC linkers can be categorized into two broad groups, according to their structural types: flexible linkers and relatively rigid linkers.
Flexible linkers are the most commonly used in PROTACs, accounting for 67.66% of the total. These linkers include alkyl-based and polyethylene glycol (PEG)-based linkers, allowing for systematic variations in length and rapid synthesis of molecules with distinct linkers.
Alkyl-based linkers in peptide PROTACs use amino acids or short peptides like aminohexanoic acid (Ahx), glycine, serine, and GSGS. While effective in degrading targets, these PROTACs often have poor cell permeability and are easily cleavage by proteases. To overcome these issues, cell-penetrating amino acids or peptides are added to improve flexibility and cell uptake.
PEG-based linkers, consisting of multiple ethylene glycol units, increase the water solubility and adaptability of PROTACs in the body. They allow for easy modification and attachment of other chemical groups, and are biocompatible, reducing the risk of immune response or toxicity. However, compared to alkyl-based linkers, PEG linkers can be less stable, more complex to synthesize, and more expensive. They are also more prone to oxidative metabolism in vivo.
In contrast, due to the limitations of synthesis technology in the PROTAC field, relatively rigid linkers are used less frequently, comprising a total of eight types. Among these, cycloalkanes, particularly those containing piperazine and piperidine components, are often used because they enhance the solubility and stability of the ternary complex. Triazole-based linkers are another commonly used rigid linker. With the advent of click chemistry, copper-catalyzed azide‒alkyne cycloaddition (CuAAC) reaction, they are highly compatible with different functional groups and maintain consistent reaction rates. Another type of linker, the photo-controlled PROTAC linker, such as photo-switchable PROTAC linkers, uses azo fragments instead of alkyl or polyether fragments. When exposed to specific wavelengths of light, these linkers cause the resulting PROTAC to undergo reversible photoisomerization. This enables precise and adjustable regulation of PROTAC biodegradation rates (Figure 2).
Figure 2. Structural types and characteristics of PROTAC linkers.
The Impact Of Linker Properties On The Biodegradation Efficiency Of PROTACs.
Firstly, the length of the linker significantly affects the formation of the POI-PROTAC-E3 ternary complex. The optimal linker length depends on the interaction pattern, distance, and spatial structure of the ternary complex. If the linker is too long, it may weaken the interaction between the POI and the E3 ligase, reducing the likelihood of forming a stable ternary complex. Conversely, a shorter linker may introduce steric hindrance, disrupting the formation of the ternary complex and lowering the biodegradation efficiency of the PROTAC.
Figure 3. The impact of linker length on the formation of the ternary complex between POI, PROTAC, and E3 is significant.
Secondly, the chemical composition of the linker directly affects the physicochemical properties of the PROTAC, which in turn influences its cell permeability. This significantly impacts the biodegradation efficiency of the PROTAC.
Figure 4. The linker group type significantly impacts the cell permeability of PROTAC.
Thirdly, the flexibility of the linker is a key factor in determining the biodegradation efficiency of PROTACs. Linkers with significant conformational flexibility can enhance interactions between the PROTAC, POI, and E3 proteins, thereby preventing their stable binding at fixed interfaces. Conversely, introducing rigid groups into a flexible linker can enhance stiffness and replicate the original geometry of the PROTAC, leading to new interactions and increased stability of the ternary complex.
Figure 5. Linker flexibility has an impact on PROTAC stability.
Finally, the linkage sites of the linker with the POI and E3 influence the interaction between the POI and E3. Optimizing the PROTAC linker typically involves determining the most favorable structural derivatization sites to ensure maximum binding affinity. This selection process often includes analyzing the solvent-exposed areas of the POI-ligand or E3-ligand interaction interfaces. By introducing the optimal linker in these solvent-exposed regions, protein-protein interactions can be maximized while retaining the original interactions between the ligand and the POI or E3.
Figure 6. The linkage site of the linker is essential for protein‒protein interaction (PPI).
Given these intricate relationships, it is clear that the linker is a key factor in ensuring higher specificity and targeting efficiency of PROTACs. While optimizing the length, functional group types, flexibility, and linkage sites of the linker can enhance the efficacy of PROTACs, several challenges must be overcome.
1) The complexity and diversity of PROTAC structures hinder the establishment of clear structure-activity relationships. To address this, exploring optimal linker properties for specific biological degradation systems can facilitate the rapid identification of more effective PROTACs.
2) The physicochemical properties of PROTACs often deviate from classical “rule of five”, necessitating the investigation of drug-likeness rules tailored to PROTACs to minimize the potential for designing PROTACs with poor pharmacokinetic characteristics.
3) Challenges in purification and low yields due to the large molecular weight and complex structure pose difficulties in the synthesis and optimization of PROTACs. Therefore, the development of advanced synthesis techniques tailored to PROTACs is crucial for obtaining a wider range of structural types.
4) The large size of ternary complexes composed of POI-PROTAC-E3 makes crystal structure determination challenging. To address this issue, it is necessary to develop advanced crystallographic techniques or more precise computational simulation methods to gain further insights.
We believe that addressing these challenges will greatly facilitate progress in PROTAC research. Biopharma PEG is dedicated to the R&D of PROTAC Linker, providing high purity PEG linkers with various reactive groups to continuously assist your project development.
References:
[1] Yawen Dong, Tingting Ma, Ting Xu, Zhangyan Feng, Yonggui Li, Lingling Song, Xiaojun Yao, Charles R. Ashby, Ge-Fei Hao, Characteristic roadmap of linker governs the rational design of PROTACs, Acta Pharmaceutica Sinica B, 2024, ISSN 2211-3835, https://doi.org/10.1016/j.apsb.2024.04.007.
[2] Cyrus K, Wehenkel M, Choi EY, Han HJ, Lee H, Swanson H, Kim KB. Impact of linker length on the activity of PROTACs. Mol Biosyst. 2011 Feb;7(2):359-64. doi: 10.1039/c0mb00074d. Epub 2010 Oct 4. PMID: 20922213; PMCID: PMC3835402.