Introduction
Proteolysis targeting chimeras (PROTACs) are an interesting type of hetero-bivalent compounds that attach and bring together the target and the E3 enzyme concurrently to enhance the ubiquitination of a target protein. These compounds comprise three structural elements: a linker that joins the two ligands, one of which binds the protein of interest (POI) and the other of which binds an E3 ubiquitin ligase to facilitate POI ubiquitination. Current advancements in the field demonstrate how important linker length and composition are to obtaining the best possible PROTAC characteristics, as well as how they affect binding kinetics and have a significant impact on potency and selectivity (1,2).
PROTACs are typically small molecules since the molecular weight and characteristics of the linker and ligands determine their stability, bioavailability, and capacity to pass across cell membranes. The optimization of linker properties, including chemical nature, length, hydrophilicity, and rigidity, is necessary for each PROTAC molecule to attain optimal cell permeability and appropriate spatial alignment between the E3 ubiquitin ligase and the target POI (3).
PEG-based linkers
PROTAC linker plays a vital role in efficient ubiquitination of the target protein and its ultimate degradation. To date, several kinds of linkers have been reported and applied in PROTAC ternary complexes formation, such as PEG linker, Alkyl linker, alkyne linkers, and click chemistry linker, etc.
The stability, bioavailability, and permeability of PROTAC molecules are all influenced by PROTAC linkers, which are essential to their performance. To improve cell permeability and spatial orientation, recent research has highlighted the various types of linkers that are frequently utilized in PROTAC design. Particular attention has been paid to improving features such as chemical nature, length, hydrophilicity, and rigidity (4).
Polyethylene glycol (PEG), different-length alkyl chains, alkynes, triazoles, and saturated heterocycles like piperazine and piperidine are examples of frequently used PROTAC linkers. Because they offer the required flexibility, synthetic accessibility, and compositional and length tunability, these linkers are essential for the formation of effective PROTAC compounds. The development of powerful PROTACs requires the proper mix of linker properties (5).
When designing PROTACs (Proteolysis Targeting Chimeras), polyethylene glycol (PEG) is a flexible and popular linker that is essential to the effectiveness and operation of these targeted protein degraders(6). PEG is a biocompatible synthetic polymer made of ethylene glycol units repeated. Because of its distinctive qualities—namely, its hydrophilicity, biocompatibility, and flexibility—it is the perfect option for linkers in PROTAC molecules. PEG offers various benefits as a linker in PROTAC design. Because of their flexibility, PROTAC molecules can take on a variety of conformations that let them interact with the target protein and E3 ligase to effectively ubiquitinate and subsequently degrade the target protein (7). PEG’s hydrophilic qualities help PROTAC chemicals become more soluble and stable, which improves their pharmacokinetic profile and bioavailability. Additionally, PEG linkers can influence the permeability and oral bioavailability of PROTAC by shielding specific functional groups within the molecule. PEG linkers can increase PROTACs’ total cellular absorption and membrane permeability by decreasing the compound’s effective polar surface area. This characteristic is very beneficial for the creation of drugs, as maximizing oral bioavailability is essential for effective treatment (8).
Linker Design Strategy for PEG linker
Simplifying the analytical characterization of PEGylated proteins is fundamental to the design of PEG linkers. The key component of this strategy is the addition of cleavable linkers, which enable the identification of PEG attachment sites by being enzymatically cleaved during standard peptide mapping sample preparation procedures (9). The design makes use of transglutaminase chemistry to include a cleavable moiety that consists of a glutamine residue that is protected by cbz, followed by glycine residues, and either an arginine or glutamic acid residue. By separating the enzyme cleavage site from the conjugation site and PEG chain spatially, this configuration avoids interference and guarantees proper analysis. Verifying the efficacy of the PEG linker requires experimental confirmation, namely by transglutaminase-mediated conjugation of proteins such as interferon α-2b (IFN) and interleukin-2 (IL-2) (9,10).
Specific PEG attachment sites can be identified with the use of peptide mapping analysis, whereas conjugation products are analyzed using analytical techniques including LC-MS and MALDI-MS. Careful examination of the experimental data informs the interpretation of the results, enabling researchers to optimize the linker design in light of analytical and empirical findings. By balancing simplicity, dependability, and analytical robustness, this iterative procedure makes sure that the developed linkers satisfy the strict requirements for characterizing PEG-protein conjugates (7).
Optimization strategy
Optimizing PROTAC characteristics for targeted protein breakdown necessitates a strategic approach in PEG linker design. The spatial orientation of the ligand binding domains with respect to the target protein and E3 ligase is largely dependent on the length of the PEG linker. Longer PEG linkers may be more flexible, enabling the ligands to be positioned optimally for effective protein breakdown. The hydrophilic property of PEG improves PROTAC molecules’ solubility and stability, which is essential for their pharmacokinetic profile and bioavailability. For optimal cellular absorption and target engagement, hydrophilicity must be balanced with other characteristics (11).
The PEG linker’s interactions with the target protein and E3 ligase can be altered by adding particular functional groups to it. The potency, selectivity, and general effectiveness of PROTAC molecules can all be improved by functionalized PEG linkers. Effective protein degradation can result from the regulated release of ligands following target contact, which can be facilitated by adding cleavable links to the PEG linker. A flexible method for controlling PROTAC activity and reducing off-target effects is provided by cleavable PEG linkers. To keep the PROTAC molecule stable and active, the right conjugation chemistry must be chosen when adding ligands to the PEG linker. A successful PROTAC design requires compatibility with the linker chemistry as well as the ligands (12,13).
Conclusion
To sum up, the optimization of targeted protein degradation is greatly influenced by the strategic design of PROTACs, especially when it comes to utilizing polyethylene glycol (PEG) linkers. By carefully evaluating linker length, hydrophilicity, and functionalization, scientists can improve PROTAC properties like stability, selectivity, and solubility. A versatile way to control PROTAC activity that minimizes off-target effects and maximizes therapeutic efficacy is provided by cleavable PEG linkers. Thus, PEG linker design’s iterative optimization highlights its significant advancement in the realm of targeted protein breakdown for medicinal applications (9).
References
1. Troup RI, Fallan C, Baud MG. Current strategies for the design of PROTAC linkers: a critical review. Exploration of Targeted Anti-tumor Therapy. 2020;1(5):273.
2. Zagidullin A, Milyukov V, Rizvanov A, Bulatov E. Novel approaches for the rational design of PROTAC linkers. Exploration of targeted anti-tumor therapy. 2020;1(5):381.
3. Hu Z, Crews CM. Recent Developments in PROTAC‐Mediated Protein Degradation: From Bench to Clinic. ChemBioChem. 2022 Jan 19;23(2):e202100270.
4. Li K, Crews CM. PROTACs: past, present and future. Chemical Society Reviews. 2022;51(12):5214–36.
5. Bemis TA, La Clair JJ, Burkart MD. Unraveling the Role of Linker Design in Proteolysis Targeting Chimeras: Miniperspective. J Med Chem. 2021 Jun 24;64(12):8042–52.
6. Testa A, Hughes SJ, Lucas X, Wright JE, Ciulli A. Structure‐Based Design of a Macrocyclic PROTAC. Angewandte Chemie. 2020 Jan 20;132(4):1744–51.
7. Filpula D, Zhao H. Releasable PEGylation of proteins with customized linkers. Advanced drug delivery reviews. 2008;60(1):29–49.
8. Riley T, Riggs-Sauthier J. The benefits and challenges of PEGylating small molecules. Pharmaceutical Technology [Internet]. 2008 [cited 2024 Mar 18];32(7). Available from: https://www.pharmtech.com/view/benefits-and-challenges-pegylating-small-molecules
9. Saha-Shah A, Sun S, Kong J, Zhong W, Mann BF. Design and Study of PEG Linkers That Enable Robust Characterization of PEGylated Proteins. ACS Pharmacol Transl Sci. 2021 Aug 13;4(4):1280–6.
10. Li Q, Li W, Xu K, Xing Y, Shi H, Jing Z, et al. PEG linker improves antitumor efficacy and safety of affibody-based drug conjugates. International journal of molecular sciences. 2021;22(4):1540.
11. Stefanick JF, Kiziltepe T, Bilgicer B. Improved peptide-targeted liposome design through optimized peptide hydrophilicity, ethylene glycol linker length, and peptide density. Journal of biomedical nanotechnology. 2015;11(8):1418–30.
12. He S, Dong G, Cheng J, Wu Y, Sheng C. Strategies for designing proteolysis targeting chimaeras (PROTACs). Medicinal Research Reviews. 2022 May;42(3):1280–342. 13. Burke PJ, Hamilton JZ, Jeffrey SC, Hunter JH, Doronina SO, Okeley NM, et al. Optimization of a PEGylated glucuronide-monomethylauristatin E linker for antibody–drug conjugates. Molecular Cancer Therapeutics. 2017;16(1):116–23.
