Release date:2020/9/5 7:55:12

PROteolysis TArgeting Chimera (PROTAC) is a new type of treatment that can induce the dynamic degradation of intracellular or nuclear protein of interest (POI), and and it plays a significant role in solving drug resistance through the degradation of the entire pathogenic proteins without compensatory increase or mutation. The core concept of PROTAC is that the bifunctional molecule binds to POI at one end and E3 ligase at the other end to form a ternary complex that hijacks the cellular ubiquitin-proteasome system (UPS) for proteasomal degradation of POI.

At present, most current PROTACs use small molecules as targeting warheads,  which rely heavily on POI binding pockets. With the rapid development of structural biology, it is becoming more and more convenient to obtain peptides with high affinity to POI epitopes. Therefore, the design of PROTAC based on specific peptides (p-PROTAC) is an emerging method that can achieve the specific and effective degradation of POI, and expand the scope in regards to “undruggable” proteins targeting, while avoiding the restriction of shallow binding pockets through large interacting surfaces between POI and peptides.

P-PROTAC consists of the following components, a peptide-based targeting warhead, a chemical linker, and a recruitment ligand for E3 ubiquitin ligase.

Design of p-PROTAC

The schematic diagram of p-PROTAC
The schematic diagram of p-PROTAC. Image source: Theranostics 2020; 10(22):10141-10153. doi:10.7150/thno.46985

Design of peptide targeting warhead

The appropriate choice of the peptide targeting warheads has a significant impact on the binding affinity and spatial orientation of POI and E3 ligase, which will affect the ubiquitination efficiency. The acquisition of targeting peptides is not a tedious process of trial and error like small molecules that rely heavily on databases and virtual screening. Generally, based on the crystal structure of the endogenous complex of POI and binding protein, which reveals key protein-protein interaction (PPI) motifs and residues, peptides targeting warheads, binding to the POI selectively, can be designed to disrupt the PPI. Then, the obtained sequence of protein epitope mimetic can be used as the main candidate for the synthesis of PROTAC warheads. In order to maximize the efficacy of protein epitope mimetics, the point mutations on non-critical interacting residues can be further used to optimize peptides targeting warheads that have high affinity for specific POIs.

Exploitation of E3 ubiquitin ligase-recruiting ligand

Since the ubiquitination tags guide the degradation caused by proteasomes which are hijacked by p-PROTAC to promote the degradation of POIs, so it is very important to select and design the recruiting moiety reasonably. There are many E3 ubiquitin ligases coded in our bodies, which have specific degron recognition motifs, providing huge theoretical possibilities for the development of PROTAC drugs. However, PROTAC can only hijack less than 1% of E3 ligases in the body, including Von Hippel-Lindau (VHL), Cereblon (CRBN), IAP, Keap1, RNF4, RNF114 and MDM2. So far, most of the reported PROTACs have selected VHL or CRBN as the E3 ligase due to the presence of their specific and high-affinity ligands.

Choice of linking moiety

Linking moiety has a major influence on the stability of the ternary complex and subsequent function. In order to maintain the delicate balance between affinity and spatial effect, the linker should have an appropriate stereochemical structure and provide an appropriate solvent exposure position to connect the peptide targeting warhead with the E3 ubiquitin ligase-recruiting ligand. So far, amino acids are commonly used linkers in p-PROTAC, including aminohexanoic acid (Ahx), glycine and serine. Polyethylene glycol (PEG) is another linker commonly used to improve the hydrophilicity of p-PROTAC.

Application of p-PROTAC 

The interface of most protein-protein complexes is usually hydrophobic, relatively flat, and lacking a deep interface pocket. Compared with small molecules, peptide-based modulators have greater potential to modulate PPI due to their larger contact surface area and ease of modification. Therefore, p-PROTAC is a more attractive strategy to eliminate intracellular pathogenic proteins through the UPS-dependent degradation cascade. Several p-PROTACs have been successfully used in certain cancers (such as ovarian cancer, breast cancer, pancreatic cancer, neuroma, etc) and neurodegenerative diseases with specific POIs, including ERa, PI3K, AKT, CREPT, X protein, Tau protein and FRS2α, as summarized in the following table.

Name Target Warhead E3 ligase Linker Indication
PROTACAKT Akt tri_a VHL PEG5 Ovarian cancer
TD-PROTAC Erα PERMs VHL Ahx Breast cancer
ErbB2PPPI3K  PI3K ErbB2 peptide VHL PEG3 Breast cancer; Ovarian cancer
PRTC CREPT CREPT ligand VHL Ahx Pancreatic cancer
PROTACX-protein X-protein oligomerization peptide VHL HBV-induced HCC
TrKAPPFRS2α FRS2α TrKA phosphorylation site VHL Ahx2 Neuroma (PC12)
PROTAC Tau-protein Tau-protein Sequence targeting Tau Keap1 GSGS Neurodegenerative disease

Successful application of p-PROTACs

Although PROTAC technology has made significant progress in drug discovery, the future development of PROTAC still faces many challenges, including off-target toxicity caused by non-specific degradation of POI in normal cells, major obstacle of pharmacokinetic (PK) evaluation for PROTAC, limited choices of E3 ligases for PROTAC design, and restricted applications for only intracellular proteins but not extracellular proteins (including membrane proteins and secreted proteins). In addition, as an important form of PROTAC, p-PROTAC further broadens the applicable targets in " undruggable proteins" with potentially high affinity. However, p-PROTAC also has its own limitations, including poor structural stability, easy degradation and poor transmembrane ability.

Overall, p-PROTAC technology provides an attractive platform that can provide leading drug candidates for potential translation studies. We expect that emerging p-PRTOACs will become the leading form of PROTAC modality in new drug discovery.

Biochempeg, as a professional PEG derivatives supplier, offers our clients with high purity PEG linkers for PROTAC development.  For more details regarding Biochempeg's PROTAC service, please feel free to contact us to assist you.

​The peptide PROTAC modality: a novel strategy for targeted protein ubiquitination, Theranostics 2020; 10(22):10141-10153. doi:10.7150/thno.46985

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PROTACs and Targeted Protein Degradation

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