Proteolysis-Targeting Chimeras (PROTAC)

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The Ubiquitin-Proteasome System (UPS)

Compared with traditional protein inhibitors which simply inhibiting the activities of the target proteins, the Protac® technology can knock out functional target proteins with small molecules. It utilizes the UPS in mammalian cells to degrade unnecessary or inappropriate proteins to some extent. The UPS is the main intracellular mechanism that destroys damaged proteins or proteins no longer needed. The ubiquitin comprised of 76 residues attaches to targeted proteins through a lysine isopeptide bond as post-translational modification (PTM) via a cascade of three enzymes: the E1 activating enzyme, the E2 conjugating enzyme, and the E3 ligase[1]. First, free ubiquitin is activated by E1 enzyme in an ATP-dependent manner, during which it is converted to C-terminal thioester. Then trans-thioesterification transfers ubiquitin from E1 to E2. Finally, the E3 ligase catalyzes the direct or indirect transfer of ubiquitin to a lysine residue on the substrate protein.

The composition of Protac®

Protac® is a bifunctional small molecule that can eliminate target proteins from cells. Protac® can link the target protein with E3 ubiquitin ligase to form a ternary complex, one end is the ligand of the protein of interest (POI), the other end is the ligand that can bind to protein degradation systems such as E3 ligase, and the linker connecting the two ends is in the middle. The two proteins bind to Protac® at the same time, so that POI and E3 ligase are spatially closer. Ubiquitin is transferred from the E3 ligase to the target protein, thereby marking the POI as a degrading protein. Then the POI is polyubiquitinated by the E2 enzyme related to the E3 ligase. By "hijacking" the UPS, it promotes the proteasome to recognize the degradation signal and ultimately inhibits tumor cell proliferation[2].


Application of Protac®

Compared with traditional inhibition strategies, targeted degradation has more advantages, including the increased probability of protein removal, a longer-lasting reduction in downstream signals, maintaining reaction time and the enhanced elimination of all related functions of the target protein. Therefore, Protac® technology has been wildly applied in biology discovery and drug development.

In many ways, Protac® represents the chemical equivalent of small interfering RNA (siRNA), although it allows the removal of proteins at the post-translational level, rather than protein silence at the post-transcriptional level. Therefore, they have become useful tools in laboratory for studying the role of proteins in biological systems. In addition, the small molecule nature of Protac® avoids problems related to delivery and biodistribution, which hinder the clinical application of siRNA, and has aroused great interest in the pharmaceutical industry. Protacs are dramatically changing the comprehension of "druggable" criterion. By designing small molecules that can bind to the cavity or pocket of the target protein, the drugability of the target protein usually depend on the inhibition of its activity, thereby obtaining therapeutic benefits. So far, thousands of protein-protein interactions are known, without deep pockets, without clear binding sites and flat protein interfaces, making them challenging targets for small molecules. On the other hand, Protacs have been proven to be suitable for targeting transcription factors lacking active binding sites or membrane-bound proteins. Their efficacy is not limited by equilibrium occupancy. Compared with classical high receptor occupancy dependent drugs, Protac®'s lasting biological effects and different downstream signaling show significant advantages. The unique properties of Protac® provide opportunities for differentiation therapy, and may be helpful to solve pathological problems driven by proteins that were previously considered untreatable by small molecule intervention proteins.

Research Progress

In 2001, Crew and Deshaies first proposed the concept of proteolysis-targeting chimeras (Protac®)[3]. The E3 ligase ligands of Protac®, originally designed by Crew et al., are all derived from peptides, resulting in large relative molecular mass, poor cell permeability, unstable structure, and high hydrolysis[4]. Then in the follow-up research, small-molecule E3 ligase ligands such as: Cereblon (CRBN), mouse double minute 2 homolog (MDM2), IAP, Von Hippel–Lindau (VHL) and cellular inhibitor of apoptosis protein 1 (cIAP1), were discovered. The E3 ligase ligand Protac® designed based on small molecules has the advantages of low relative molecular weight and strong cell permeability. More and more targeted proteins have been studied.

The proteolysis-targeting chimera must have sufficient steric structure so that it can contain two different binding groups in the same molecule. However, it is unbelievable that in the past, many drugs were approved by regulatory authorities despite the lack of understanding of their functionary mechanisms and direct molecular targets[5].

Because of its small molecule nature, Protac® technology is entering clinical research and has many indications. Initial research focused on the degradation of hormone receptors, especially androgen receptors (AR) and estrogen receptors (ER).

  • Protacs Targeting AR
  • Androgen is a type of steroid hormone, which mainly regulates gene transcription through androgen receptor (AR) to affect gene expression level. AR is the main therapeutic target for prostate cancer. Studies have shown that the development of prostate cancer is related to the high expression of AR in malignant tumor cells. AR antagonists, such as Enzalutamide (Rathkopf and Scher, 2013), have been used for prostate cancer treatment and have generated great benefits for the patients. However, there is often resistance. Schneekloth et al. used DHT and an E3 ligase ligand containing arginine to obtain the first compound that can degrade AR[6]. Han et al. synthesized ARD-69 with AR antagonist and small molecule ligand VHL. ARD-69 can inhibit the cell growth of positive prostate cancer cell lines and reduce the level of AR protein by more than 95%[7]. AR Protac® ARV-110 is currently in clinical trials for metastatic castration-resistant prostate cancer.

  • Protacs Targeting ER
  • ER is an important target for the treatment of estrogen receptor-positive breast cancer. Currently, Fulvestrant is the only estrogen receptor degrading agent that has been approved for the treatment of estrogen-positive breast cancer. Clinical experiments have shown that Fulvestrant is helpful for the treatment of breast cancer, but cannot be taken orally because of its poor solubility. In clinical practice, it can only be administered by intramuscular injection.

    Induction of estrogen receptor (ER) degradation is a new treatment strategy. ER Protacs can induce more effective degradation than Fulvestrant and have improved pharmacological properties. Hu et al. connected Fulvestrant and E3 ligase ligand VHL with multiple carbon atoms to obtain ERD-308[8]. In addition, ER Protac® ARV-471 is currently in clinical trials for advanced or metastatic ER+/Her2- breast cancer.

  • Protacs Targeting Aggregate Proteins
  • A challenging class of protein degradation targets are proteins that are prone to agglutinate, such as α-synuclein, transthyretin and tau. It is an attractive method to treat protein aggregation diseases by directly recruiting E3 ligase through Protac® to degrade these proteins while they are in monomer state or aggregate state. Some Protac® studies have begun to meet this challenge by inducing tau degradation, which is closely related to Alzheimer’s disease (AD)[9].

  • Protacs Targeting ALK
  • ALK is a tyrosine kinase receptor, which is related to the occurrence and development of many types of tumors. Studies have shown that cancer cells carrying ALK fusion proteins had a higher ALK activity, so inhibiting ALK activity can inhibit the cancer cells proliferation. Currently, there are 4 ALK inhibitors on the market that have been approved by the FDA for the effective treatment of ALK-positive NSCLC patients, but after these ALK inhibitors are used for a period of time, cancer cells will become resistant. Therefore, new treatment methods need to be developed to delay or overcome drug resistance. Traditional enzyme inhibitors only inhibit the enzyme activities. Protac® degrades the enzyme through the ubiquitin-protease system, which not only serves the same treatment, but also does not produce drug resistance. Zhang et al. obtained two kinds of Protac®, the MS4077 and MS4078, by combining the ALK inhibitor ceritinib and pomalidomide with different linking groups[10]. Kang et al. used ALK inhibitor ceritinib and E3 ligase ligand VHL to design and synthesize TD-004, which not only effectively reduced the level of intracellular ALK fusion protein, but also selectively inhibited the proliferation of fused ALK-positive cancer cells[11].

  • Protacs Targeting CDK8
  • CDKs include more than 13 serine-threonine kinases. Among them, CDK1-4, CDK6 and CDK11 mainly regulate the cell cycle, while CDK7-9 mainly regulate gene transcription and translation. CDK 8 plays an important role in oncogenic signaling pathways such as TGF-β pathway and p53 pathway. Hatcher et al. synthesized JH-VIII-49 on the basis of Cortistatin A, which has better inhibitory effect on CDK8. By connecting JH-VIII-49 and the E3 ligase ligand CRBN via PEG, JH-XI-10-02 was obtained to show a better performance[12].

    In addition, Buhimschi et al. designed and synthesized MT-802, which can targetedly degrade the BTK protein related to chronic lymphocytic leukemia (CLL) and other B cell malignancies[13]. Zengerle et al. connected the small molecule inhibitor JQ1 with E3 ligase ligand VHL with linker to obtain MZ1, which can degrade the BRD protein, an important target for the treatment of acute myeloid leukemia and ovarian cancer[14].


In recent years, targeted drugs have gradually become the research hotspot of anti-tumor drugs. Targeted drugs can specifically act on the lesions, which not only improves the efficacy of the drug but also reduces the damage to normal human tissues. Using the ubiquitin-protease system to specifically degrade target proteins, Protac® has also become a method of targeted therapy. Theoretically, as long as the target protein has a specific ligand, it can form a POI-Protac®-E3 ubiquitin ligase ternary complex with different linkers and E3 ligase ligands, and then degraded by the ubiquitin-protease pathway. This technology has the potential to use a variety of proteomes that were once ineffective with drugs for treatment. Overall, the application prospects of Protac® therapy will be immeasurable.


  1. Burslem, G. M. & Crews, C. M. (2020) Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery, Cell. 181, 102-114.
  2. Wang, P. & Zhou, J. (2018) Proteolysis Targeting Chimera (Protac®): A Paradigm-Shifting Approach in Small Molecule Drug Discovery, Current topics in medicinal chemistry. 18, 1354-1356.
  3. Myung, J., Kim, K. B. & Crews, C. M. (2001) The ubiquitin-proteasome pathway and proteasome inhibitors, Medicinal research reviews. 21, 245-73.
  4. Lai, A. C. & Crews, C. M. (2017) Induced protein degradation: an emerging drug discovery paradigm, Nature reviews Drug discovery. 16, 101-114.
  5. Chamberlain, P. P. & Hamann, L. G. (2019) Development of targeted protein degradation therapeutics, Nature Chemical Biology. 15, 937-944.
  6. Schneekloth, J. S., Jr., Fonseca, F. N., Koldobskiy, M., Mandal, A., Deshaies, R., Sakamoto, K. & Crews, C. M. (2004) Chemical genetic control of protein levels: selective in vivo targeted degradation, J Am Chem Soc. 126, 3748-54.
  7. Han, X., Wang, C., Qin, C., Xiang, W., Fernandez-Salas, E., Yang, C. Y., Wang, M., Zhao, L., Xu, T., Chinnaswamy, K., Delproposto, J., Stuckey, J. & Wang, S. (2019) Discovery of ARD-69 as a Highly Potent Proteolysis Targeting Chimera (Protac®) Degrader of Androgen Receptor (AR) for the Treatment of Prostate Cancer, Journal of medicinal chemistry. 62, 941-964.
  8. Hu, J., Hu, B., Wang, M., Xu, F., Miao, B., Yang, C. Y., Wang, M., Liu, Z., Hayes, D. F., Chinnaswamy, K., Delproposto, J., Stuckey, J. & Wang, S. (2019) Discovery of ERD-308 as a Highly Potent Proteolysis Targeting Chimera (Protac®) Degrader of Estrogen Receptor (ER), Journal of medicinal chemistry. 62, 1420-1442.
  9. Lu, M., Liu, T., Jiao, Q., Ji, J., Tao, M., Liu, Y., You, Q. & Jiang, Z. (2018) Discovery of a Keap1-dependent peptide Protac® to knockdown Tau by ubiquitination-proteasome degradation pathway, European journal of medicinal chemistry. 146, 251-259.
  10. Zhang, C., Han, X. R., Yang, X., Jiang, B., Liu, J., Xiong, Y. & Jin, J. (2018) Proteolysis Targeting Chimeras (Protacs) of Anaplastic Lymphoma Kinase (ALK), European journal of medicinal chemistry. 151, 304-314.
  11. Kang, C. H., Lee, D. H., Lee, C. O., Du Ha, J., Park, C. H. & Hwang, J. Y. (2018) Induced protein degradation of anaplastic lymphoma kinase (ALK) by proteolysis targeting chimera (Protac®), Biochemical and biophysical research communications. 505, 542-547.
  12. Hatcher, J. M., Wang, E. S., Johannessen, L., Kwiatkowski, N., Sim, T. & Gray, N. S. (2018) Development of Highly Potent and Selective Steroidal Inhibitors and Degraders of CDK8, ACS medicinal chemistry letters. 9, 540-545.
  13. Buhimschi, A. D., Armstrong, H. A., Toure, M., Jaime-Figueroa, S., Chen, T. L., Lehman, A. M., Woyach, J. A., Johnson, A. J., Byrd, J. C. & Crews, C. M. (2018) Targeting the C481S Ibrutinib-Resistance Mutation in Bruton's Tyrosine Kinase Using Protac®-Mediated Degradation, Biochemistry. 57, 3564-3575.
  14. Zengerle, M., Chan, K. H. & Ciulli, A. (2015) Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4, ACS chemical biology. 10, 1770-7.

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