Protein degradation targeting chimera (PROTAC) is a technology that uses E3 ligase-mediated ubiquitination labeling step in the ubiquitin proteasome pathway to silent target protein to E3 ligase by PROTAC small molecules and increase the chance for target protein to be labeled and then degraded. Since the concept of PROTAC was introduced in 2001, over 10 such drugs have so far advanced to the stage of further clinical development, of which ARV-471 and ARV-110 in clinical phase II have been disclosed ^[1]. We have mentioned the basic principle of PROTAC molecule, the advantages compared with traditional small molecules and the building blocks, target protein ligands, linkers, and E3 ubiquitin ligase ligands, in our previous introduction.

In spite of that PROTAC still has problems such as solubility, membrane permeability and selectivity, but its modular design strategy is a great ' black technology ' in the field of drug development.

As we all know, the protein degradation pathways of eukaryotes are mainly divided into lysosome pathway, ubiquitin proteasome pathway, cytosol protease hydrolysis pathway and mitochondrial protease pathway. Among them, the proteasome pathway is mainly aimed at the degradation of cell cycle proteins, transcription factors, cell surface receptors, intracellular variant proteins and abnormal proteins, which is inferior to the lysosomal pathway in the degradation of surface membrane proteins and endocytosis proteins ^[2]. Therefore, in recent years, molecules similar to PROTAC but functioning through lysosomal pathway, such as: LYTAC, AUTAC, and ATTEC, reported so far ^[3].

At present, there are clinically used PROTACs targeting ER, AR, BTK and IRAK which are all tumor-related proteins. It can be seen in the actual investigation that these proteins have specific characteristics in specific tumor cells or are carcinogenic themselves by nature, which provide targeting strategies for PROTAC molecules.

What if a non-specific protein needs to be developed into a highly targeting PROTAC? We summarized three strategies: light controlled linker, PAC molecule, specific E3 ligase.

Light-controlled linker is mainly divided into two types, one is the photosensitive group shelter, and the other is the photosensitive group isomerism.

Xue et al. shielded the bromodomain-4 (BRD4) ligand JQ1 of the original PROTAC molecule dBET1 with 4,5-dimethoxy-2-nitrobenzene (DMNB), reducing the activity by more than 100 times, and then released dBET1 under 365 nm light. After that, the target protein BRD4 was almost completely degraded at 1 μM concentration (D_max = 93%) ^[4].

Pfaff et al. used the characteristic that the cis-trans distance difference of azobenzene isomers was 3−4 Â, which was similar to the difference between activity and inactivity length of PROTAC linker. The linker of the original PROTAC molecule ARV-771 was modified to contain the structure of cis-azobenzene. Cis-azobenzene could be transformed into the trans-structure under the light of 415 nm, and then active chain length of ARV-771 was restored ^[5].

The PROTAC-Antibody Conjugate (PAC) molecule comprise an antibody conjugated via a linker (L1) to a PROTAC, wherein the PROTAC comprises a ubiquitin E3 ligase binding group (“E3LB”), a linker (“L2”) and a protein binding group (“PB”).

Maneiro et al. connected the above BRD4 degradation agent ARV-771 on trastuzumab through a linker fragment, and the whole molecule only degraded BRD4 in HER2 positive breast cancer cell lines instead of HER2 negative ones ^[6].

Traditional E3 ligase whose ligands can be used for PROTAC nowadays are CRBN, VHL and IAP, among which VHL can be used in receptor tyrosine kinase (RTK) so it may be a choice for degradation of membrane proteins ^[7]. In consideration of the E3 ligands, beside the influence of the ligand itself on the physical properties of PROTAC, special E3 ligands can also be used to achieve tissue targeting. For example, IAP, an inhibitor of apoptosis protein, is highly expressed in cancer cells. Based on this, the corresponding PROTAC molecules were developed, which were also named Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER) ^[8].

Ubiquitin-like PHD and ring finger domain 1 (UHRF1) are over-expressed in various cancers such as breast cancer, which is the formation of the ring finger E3 ubiquitin ligase subfamily and plays a role by forming a complex with H3K9me3. Senisterra et al. screened the small molecules from 44000 compound libraries, which destroyed the interaction between UHRF1 and H3K9me3, and finally found the NV03 with K_d value of 2.4 μM through optimization ^[9]. Unfortunately, no relevant PROTAC molecule has been developed for breast cancer based on this research.

Although PROTAC molecules can modularize the construction of target protein degradation molecules, the problem of target selectivity makes it impossible for some non-special proteins. Fortunately, the existing molecules can be optimized by combining photo-controlled groups or PAC technology to improve their tissue selectivity. However, the final solution of selectivity still needs to fundamentally change the structure itself. Therefore, the research for specific tissue distribution of more than 600 known E3 ligases and their related ligands are undoubtedly another effective way out ^[10].

To build efficient PROTAC molecules, the right components are necessary. MCE is the world's leading supplier of scientific chemicals and bioactive compounds, who can provide PROTAC-related modular products for scientists. We have PROTAC, SNIPER, PROTAC-Linker Conjugate for PAC and their constituent modules online. We also provide integrated synthesis services for PROTAC products.