An Overview of Researches on Targeted Protein Degradation (II)


Posted on: 19 November 2020 by chen shanshan

An Overview of Researches on Targeted Protein Degradation (II)

3. 2 Small molecule PROTAC

The use of small molecules as part of the E3 ubiquitin ligase has led to the rapid development of small molecule-based PROTAC technology. PROTAC based on small molecules has many advantages over peptide-based PROTAC. Most importantly, PROTAC based on small molecules is more promising to be develop into drugs, because small molecules are more easily absorbed by the body. At present, more than 600 E3 ligases have been found in the human body. Currently, due to the lack of small molecule ligands for E3 ligase with high affinity and specificity, the only E3 ligases that have been reported to be involved in PROTAC are mainly mouse double minute 2 homologue (MDM2), cellular inhibitor of apoptosis protein 1 (cIAP1), Cereblon (CRBN), Von Hippel-Lindau (VHL), etc.

3. 2. 1 Recruit E3 ubiquitin ligase MDM2

Discovered in 2008, Nutlin-3a is used as a ligand for E3 ubiquitin ligase MDM2. The PROTAC connects Nutlin-3a and the non-steroidal androgen receptor (AR) ligand through a polyethylene glycol chain. In the test of prostate cancer cells, the small molecule PROTAC can degrade AR at 10 μmol/L. Although the activity of this molecule is lower than that of its peptide analogs, the research results have confirmed the feasibility of using the small molecule PROTAC to degrade the target protein.

3. 2. 2 Recruit E3 ubiquitin ligase cIAP1

cIAP1 is an intrinsic inhibitor of apoptosis. Using cIAP1 inhibitor bestatin b (Bestatin) as the recognition group of E3 ligase, a small molecule PROTAC targeting the degradation of intracellular retinoic acid binding protein II (CABP-II) was designed and synthesized. The molecule can induce 75% of CABP-II degradation in cancer cells at a concentration of 10 μmol/L. Studies have shown that this kind of small molecule PROTAC has the following limitations: (1) Since Bestatin is an aminopeptidase inhibitor, it is often accompanied by off-target effects and cause adverse reactions. (2) Due to its low activity in degrading proteins, a higher concentration is required to exert pharmacological effects. (3) IAP ligands often induce IAP self-ubiquitination and degradation. These limitations restrict the further development of Bestatin as a ligand for E3 ubiquitin ligase.

3. 2. 3 Recruiting E3 ubiquitin ligase CRBN

Thalidomide and its derivatives Lenalidomide and Pomalidomide are potent immunomodulators. Studies have found that the combination of thalidomide and E3 ligase CRBN can lead to the degradation of IKZF1 and IKZF3, indicating that thalidomide and its derivatives can be introduced into the design of PROTAC as recognition part of E3 ligase. Bromodomain-containing protein 4 (BRD4) is a member of the BET family. BET bromodomain (bromodomain and extra-terminal domain) contains 4 kinds of proteins, namely BRD2, BRD3, BRD4 and BRDT. Among them, BRD4 is a very popular anti-tumor target in the field of epigenetics.

The targeted drug candidate OTX-015 is linked to pomalidomide through a polyethylene glycol chain to obtain the small molecule PROTAC ARV-825. ARV-825 can almost completely degrade BRD4 protein within 10 nmol/L and 6 hours, and its duration of action is longer than 24 hours. Further testing confirmed that ARV-825 has the dual advantages of anti-tumor cell proliferation and inducing apoptosis compared with BRD4 inhibitors JQ1 and OTX-015, indicating that PROTAC can inhibit the target more thoroughly and long-term than small molecule inhibitors.

BET inhibitor JQ1 and thalidomide derivatives were linked to obtain dBET1. dBET1 can induce 87% degradation of BRD4 protein in human leukemia cell line AML at a concentration of 100 nmol/L, while inactive dBET1 (R) does not degrade protein effect. Due to the lack of binding specificity of JQ1, dBET1 can also cause the degradation of other BET family members (BRD2, BRD3). The pharmacological effects of dBET1 were further evaluated in the anti-tumor cell proliferation test, and the results showed that by inducing the degradation of BRD4, it can significantly inhibit the growth of the lymphoma cells tested, and its activity is better than JQ1. In addition, in a xenograft mouse model of human mv4-11 leukemia cells, continuous administration of dBET for 14 days can make the tumor regression, and 24h after stopping the administration, BRD4 protein level begins to recover. Interestingly, although the structure of ARV-825 is similar to dBET1, its effect in inducing the degradation of BRD4 is 10 times stronger than that of dBET1, which may be mainly due to the difference in the linking chain of the two molecules, indicating the composition and sum length of the linking chain are the key variables that determine the activity of PROTAC. The linking chain will also affect the lipid solubility and cell membrane permeability of PROTAC, which is an important module for PROTAC design and optimization. A small molecule PROTAC ZBC260 can be obtained by linking the highly active BRD4 inhibitor HJB97 with the lenalidomide analog via aliphatic chain, which can degrade the BRD4 protein of the leukemia cell line at at a rate of 30 pmol/L and inhibit the proliferation of leukemia cell line. Intravenous injection of the small molecule PROTAC ZBC260 can make tumor regression in the transplanted tumor mice.

Studies have shown that many kinase target inhibitors and thalidomide and its derivatives are connected by suitable linking chains to obtain small molecule PROTAC, which can also successfully degrade the target proteins. These targets mainly include anaplastic lymphoma kinase (ALK), cyclin-dependent kinase CDK8 and cyclin-dependent kinase CDK9, etc.

3. 2. 4 Recruit E3 ubiquitin ligase VHL

VHL is another E3 ubiquitin ligase widely used in PROTAC design. Recent studies have shown that some small molecules can competitively bind to the main HIF-binding site on VHL. The Crews team developed a small molecule PROTAC for targeted degradation of serine/threonine kinase RIPK2. They linked the VHL ligand and the RIPK2 inhibitor through a polyethylene glycol chain to obtain a PROTAC-RIPK2 molecule that degrades the RIPK2 protein. The value of DC50 (the chimera dose required to reduce the protein level by 50%) is 1.4 nmol/L, and the value of DC95 is as low as 10 nmol/L.

Since the VHL ligand has strict spatial conformational restrictions, its epimer PROTAC-RIPK2-epi has no degradation activity. The sub-stoichiometric catalytic properties of PROTAC-RIPK2 were confirmed by in vitro reconstruction ubiquitination experiments. The results showed that PROTAC-RIPK2 at 1 pmol/L can induce degradation of RIPK2 at 3.4 pmol/L, proving that PROTAC can circulate in cells.

Small molecule VHL ligands have also been used in the design of PROTAC targeting BRD4 protein. Connecting the BRD4 inhibitor JQ1 with the VHL ligand to obtain two small molecules, PROTAC MZ1 and PROTAC MZ2. Both of these two small molecules can induce the degradation of BRD4 within 24h, while the MZ1 has a shorter polyethylene glycol link chain, showing a higher degradation effect. This discovery once again proved that the composition and length of the connection chain are key variables that determine the activity of PROTAC, which are also important modules for the design and optimization of PROTAC. Partial degradation of BRD2 and BRD3 can be observed after 24 hours of use of these compounds, while the degradation of BRD2 and BRD3 can be observed within 24 hours of use of PROTAC ARV-825 and dBET1 based on E3 ligase CRBN targeting BRD4. The dual function of PROTAC should satisfy the matching of the target protein and E3 ligase, thereby effectively ubiquitinating and degrading the target protein, indicating that the type of E3 ligase can affect the sensitivity and selectivity of the target protein to the degradation induced by PROTAC.

To be continued in Part III…

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