Proteolysis-targeting chimeras (PROTACs) are a novel class of heterobifunctional molecules that harness the cell’s ubiquitin-proteasome systems (UPS) to selectively degrade disease-related proteins. A typical PROTAC consists of three key elements: a ligand that binds to the protein of interest (POI), a ligand that recruits an E3 ubiquitin ligase, and a chemical linker that connects the two moieties. Upon formation of a ternary complex involving the POI, PROTAC, and E3 ligase, the target protein is polyubiquitinated and subsequently degraded by the proteasome.
As PROTACs gain increasing traction in drug discovery, it is now widely recognized that the linker is far more than a passive spacer. Linker length, composition, flexibility, polarity, and attachment points can all influence the formation and stability of the ternary complex, cellular permeability, pharmacokinetics, and ultimately the degradation efficiency of the molecule.
Common types of linkers: Flexible vs. Rigid
In PROTAC design, the linker plays a critical role in connecting the warhead and E3 ligase ligand. Its structural properties significantly influence molecular stability, conformational flexibility, solubility, and bioactivity. Based on structural characteristics, linkers are generally classified into flexible and rigid types.
Flexible linkers, widely used in early and classical PROTACs, typically include alkyl-based and PEG-based structures.
Alkyl-Based Linkers
Alkyl linkers consist of saturated or unsaturated alkyl chains, often incorporating functional groups such as ether, amine, carbonyl, or amide to tune polarity and rigidity. These linkers are synthetically accessible and chemically stable, but tend to be hydrophobic, which may limit aqueous solubility and cellular uptake. Polar modifications can help address this.
PEG-based Linkers
PEG linkers are composed of consecutive ethylene glycol units, which impart excellent hydrophilicity and improve the water solubility of PROTACs, enhancing their compatibility with physiological environments. These linkers offer good biocompatibility and allow for versatile chemical modifications, aiding in fine-tuning molecular properties. However, compared to alkyl-based linkers, PEG linkers may have reduced metabolic stability in vivo and can be more challenging and costly to synthesize.
Rigid linkers, in contrast, contain cyclic or planar elements that restrict molecular conformation and improve metabolic stability. Key categories include:
Triazole-Based Linkers
Triazole-containing linkers are among the most widely used rigid linkers in PROTAC development. They are typically synthesized through the copper-catalyzed azide-alkyne cycloaddition. The resulting triazole moiety is metabolically stable and helps to reduce oxidative degradation in vivo, making it ideal for therapeutic applications.
Cycloalkane-Based Linkers
Linkers containing cycloalkane structures such as piperazine, piperidine, or cyclohexane are another commonly used class in PROTACs. These fragments introduce rigidity while also enhancing water solubility and metabolic stability. PROTACs using these linkers tend to maintain their structural integrity in biological systems, translating to improved pharmacokinetics and bioavailability.
Aromatic-based linkers
Aromatic linkers, typically based on phenyl rings, provide planarity and rigidity while maintaining chemical simplicity. Their delocalized π-electrons not only improve linker stability but also enhance the non-covalent interactions, such as π- π stacking, help stabilize the ternary complex between the PROTAC, E3 ligase and target protein. Aromatic linkers are often introduced to improve binding geometry or fine-tune the overall length of the PROTAC molecule.
Spiro-Based Linkers
Spirocyclic structures offer a unique 3D rigid geometry that locks molecular conformation and reduces rotational freedom. Their use in PROTAC linkers can enhance selectivity by pre-organizing the molecule into an active conformation that favors ternary complex formation.
Fused-Heterocyclic Linkers
Fused heterocycles, such as benzoimidazole, thienopyrimidine, or cycloheptapyridazine, are gaining attention as rigid linkers due to their ability to introduce directional rigidity and enhance metabolic resistance. These bicyclic or tricyclic fragments often mimic natural ligand scaffolds, supporting better protein binding and biological compatibility. Their inclusion can also affect the solubility and lipophilicity of the PROTAC molecules.
Photocaged Linkers
Photocaged linkers integrate light-sensitive groups that can be removed through exposure to specific wavelengths, often UV light. This design allows PROTAC activity to be switched on only in targeted regions, helping to minimize systemic side effects.
Photoswitchable Linkers
Photoswitchable linkers take light-responsiveness a step further by enabling reversible control. These linkers typically contain azobenzene unites, which can switch between trans and cis isomers under different light wavelengths. The structural change alters the spatial configuration of the PROTAC molecule, allowing researchers to precisely modulate degradation activity in real time.
Macrocyclic Linkers
Macrocycles in PROTACs are relatively novel but offer compelling benefits. By forming large ring structures, typically 14 atoms or more, macrocyclic linkers impose a highly constrained conformation that can significantly enhance selectivity and reduce entropy penalties during binding. These linkers are often designed via computer-aided drug design (CADD) approaches to maintain an optimal distance and orientation between the E3 ligase and the target ligand. Though challenging to synthesize, they represent a promising direction in the design of next generation PROTACs.
Conclusion
The choice of linker remains one of the most critical determinants of PROTAC performance, influencing not just degradation efficiency but also pharmacokinetics, cellular uptake, and target selectivity. At PrecisePEG, we support PROTAC innovation by providing high-quality linker building blocks and ligand-linker conjugates and work closely with clients to accelerate PROTAC discovery and optimization.
Reference
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