Targeted Chimera Proteolysis (PROTAC) technology represents a breakthrough development in the field of drug discovery, utilizing the ubiquitin proteasome system to specifically degrade disease-associated proteins. PROTACs has a two-part function structure, that is, two functional domains are connected through linkers.
Linker plays a key role in determining the biodegradable efficacy of PROTACs. Although an advanced PROTAC connectivity system is under development. However, the relationship between connectome characteristics and PROTAC efficacy has not been fully understood and studied.
Here, we will conduct a multidisciplinary analysis of the PROTAC linker and its effect on efficacy, thus guiding the rational design of the linker. In this paper, the structural types and characteristics of PROTAC, design and optimization strategies, as well as the influence of different characteristics of connectors on the degradation efficiency of PROTAC were discussed. Hopefully, this article will provide a valuable reference for the further development of the PROTAC field.
Types and features of PROTAC linkers
PROTAC linker can be broadly divided into two categories: flexible linker and relatively rigid linker.
Flexible linker is the most widely used type, mainly including alkyl linker and polyethylene glycol (PEG) linker. Flexible linkers, which are prevalent in the design of PROTAC molecules, typically use long, flexible chain-like structures. But this flexible design increases the body’s sensitivity to oxidative metabolism.
Because PROTAC is subject to certain limitations in synthesis, flexible structures are used less frequently than rigid ones, and there are a total of 8 types at present. Cycloalkanes, especially those containing piperazine and piperidine, are often used because of their ability to improve the solubility and stability of ternary complexes. For example, the triazolyl linker is another rigid linker, which is obtained by the 1,3 dipole cycloaddition reaction catalyzed by copper, and has a fast reaction rate with different functional groups and high compatibility.Another type of linker, the photo controlled PROTAC linker, such as the photo switch PROTAC linker, uses azo fragments instead of alkyl or polyether fragments, which, when exposed to specific wavelengths of light, result in reversible photoisomerization of the resulting PROTACs. This makes it possible to precisely and mutually adjust the biodegradation rate of PROTACs (Fig.1)
Common PROTAC linkers at BOC Sciences
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Design and optimization of PROTAC linker
In general, the design and optimization of PROTAC linker relies on empirical or computer-aided methods. The general steps for designing and optimizing PROTAC linker based on experience are as follows:
First, researchers need to design PROTACs based on previous experience, mainly optimizing the following four aspects:
1. Adjust the linker length to achieve the optimal configuration of a particular PROTAC.
2. Modify the type of linker to balance the hydrophilicity and phobicity of PROTAC.
3. Modify the flexibility of the linker and the junction site to increase the stability of the ternary complex.
4. Design and synthesize multiple PROTACs with multiple different types of linker.
Whereas, the general process based on computer-aided strategies is:
1. Crystallography or molecular docking techniques were used to determine the binding modes of POI to its ligand and E3 to its ligand, respectively.
2. The global protein-protein docking simulation was carried out by using MOE, Rosetta, PatchDock and other calculation software, and the model structure sets of poi-ligand and e3-ligand were obtained.
3. After evaluation using molecular dynamics (MD), analyze protein-protein interactions (PPIs) using a rational model structure and identify proteins that interact with each other at the proximal end of their binding pockets.
4. Designed and generated a series of POI-PROTAC-E3 structures by using different types of linker fragments. Then, the structure-activity relationship and combination mode of PROTAC are analyzed, and the rules are summarized. Finally, the optimal linker is found through analysis.
Although this strategy is widely applicable to the design and optimization process, it also has limitations. This approach often requires the synthesis of multiple PROTAC with various linkers to gain a comprehensive understanding of SAR, which can be time-consuming, laborious, and costly. Another limitation is the accuracy of theoretical predictions, which affects the design of the linker.
In recent years, the application of artificial intelligence in the design of PROTAC joints has significantly improved the accuracy and efficiency of linker optimization. Using the help of artificial intelligence, the researchers were able to gain insight into the structural, physical and chemical properties of PORTAC linker. Some of these conclusions have also been applied, which can help the design of PROTAC more accurately. (Fig.2).
Effect of Linker properties on degradation
PROTAC is a heterobifunctional molecule linked by a target protein ligand and an E3 ligase ligand via different linkers, and this structure contributes to the establishment of a stable ternary complex, resulting in the target protein ubiquitination being recognized and degraded by the 26s proteasome in the organism.
It was found that the properties of linker play a key role in the stable formation of ternary complexes and the physicochemical and pharmacokinetic properties of PROTACs.
Firstly, the length of the linker significantly affects the formation of the POI-PROTAC-E3 ternary complex, and the optimal linker length depends on the interaction mode, distance and spatial structure of the ternary complex. Conversely, a shorter linker may lead to an increase in steric hindrance and affect the formation of complexes, thereby reducing the biodegradation efficiency of PROTACs.
Then, the chemical structure composition of linker would affect the physicochemical properties of PROTACs and the membrane permeability of PROTACs, resulting in changes in the biodegradation efficiency of PROTACs.
Therefore, achieving optimal linker length is critical to generate maximum interaction between POI and E3 ligase, resulting in efficient ubiquitination and biodegradation of POI. On the other hand, the flexibility of the linker is a key factor in determining the biodegradation effect of PROTACs, and the linker with significant conformational flexibility can enhance the interaction between PROTACs, POIs, and E3 proteins, thereby preventing their stable binding at the fixed interface.
On the contrary, the introduction of rigid groups in the flexible linker can improve the rigidity of PROTACs and replicate the original geometry of POI and E3 ligands in PROTACs, resulting in new interactions and improved stability of ternary complexes.
In addition, the linker’s attachment site to the POI and E3 affects the interaction between the POI and E3. Optimization of PROTAC ligands involves selecting the most suitable structure-derived structural sites to preserve the best affinity. This selection process typically involves analyzing the solvent-exposed region of the POI-ligand or E3-ligand interaction interface, and by introducing an optimal linker in the solvent-exposed region, the protein-protein interaction can be maximized while preserving the original ligand interaction with the POI or E3.
Given these intricate relationships, it is clear that linker is a key factor in ensuring greater specificity and targeting efficiency of PROTACs (Fig.3).
While optimizing the length, group, and connection location of linker can help improve the efficiency of PROTACs, several challenges must be overcome.
1. The complex diversity of PROTAC structure affects the analysis of structure-activity relationship. To solve this problem, exploring the optimal linker characteristics of specific biodegradable systems can facilitate the rapid identification of more effective PROTACs.
2. Due to the large molecular weight of PROTACs, its physical and chemical properties often do not meet the 5 principles of class drugs. Therefore, it is necessary for researchers to study drug rules suitable for PROTACs to help the design and development of PROTAC drugs.
3. Due to the complex structure of PROTAC and the low synthetic yield, the design and optimization of PROTACs are greatly challenged. Therefore, the development of advanced PROTACs synthesis technology is also crucial.
4. The large size of the ternary complex composed of POI-PROTAC-E3 makes it difficult to determine the crystal structure. In order to solve this problem, it is necessary to develop advanced crystallographic techniques and computer simulation methods to predict crystal binding patterns.
We believe that addressing these issues will greatly facilitate the progress of PROTAC research.
In conclusion, this article analyzes the structure and properties of PROTAC linkers and their effects on the biodegradation effect of PROTACs from a multidisciplinary perspective. It is hoped that it will provide valuable insights into the rational design of the PROTAC linker.
We analyzed three key areas:
1. Various structural types and characteristics of PROTAC linker reported so far are analyzed.
2. Rational design of the optimization strategy for effective PROTAC joints.
3. The effects of linker length, group type, flexibility, and linker site on ternary complex formation, pharmacokinetics, protein-protein interactions, and ultimately on the biodegradation efficiency of PROTACs.
It is hoped that this article will increase the understanding of the rational design of PROTAC linker, thereby promoting future research in this field.
References
- Dong, Yawen, et al., Characteristic roadmap of linker governs the rational design of PROTACs. Acta Pharmaceutica Sinica B (2024).
- Bemis, Troy A., et al., Unraveling the role of linker design in proteolysis targeting chimeras: Miniperspective. Journal of Medicinal Chemistry 64.12 (2021): 8042-8052.
- Cyrus, Kedra, et al., Impact of linker length on the activity of PROTACs. Molecular BioSystems 7.2 (2011): 359-364.