PROTACs, also named as Proteolysis-Targeting Chimeras, is a new type of drug that is different from antibodies and traditional small molecular inhibitors. It consists of target proteins ligand, linker, and E3 ubiquitin ligase ligand. One end of the PROTAC molecule binds to the target protein and the other end binds to E3 ubiquitin ligase that can mark a small protein called ubiquitin as defective or damaged by attaching it to the target protein. After that, the cell’s protein shredder (proteasome) recognizes and degrades the labeled target protein.
In recent years, as a new way to regulate protein homeostasis, PROTAC has been widely concerned by academia and industry, and many teams are actively developing protein degradants based on PROTAC technology. In terms of target proteins, according to statistics, there are more than 100 hot targets, including AR-PROTAC, ER-PROTAC, and BTK-PROTAC. In terms of E3 ubiquitin ligase, two kinds of ubiquitin ligases are most commonly used in PROTAC development, namely Von Hippel Lindau (VHL) and cereblon (CRBN), among which CRBN is also another kind of protein degradation agent-molecular gel verified target.
CRBN is a part of CRL4 E3 ubiquitin ligase, which acts as a substrate receptor (SR) to recognize substrate proteins, thus initiating the degradation process. CRLs is the largest family of E3 ubiquitin ligases, with more than 250 kinds formed by assembling substrate receptors and adapter proteins around different cullin skeletons.
To put it simply, molecular gel degradants are a kind of small molecules that can induce a new interaction between E3 ubiquitin ligase substrate receptors (such as CRBN) and target proteins, resulting in the degradation of target proteins. Small molecular immunomodulators such as thalidomide lenalidomide and pomalidomide are a significant example of molecular gel which can redirect CRBN resulting in polyubiquitin and degradation of transcription factors IKZF1 and IKZF3. Similarly, the anticancer sulfonamide drug indisulam can also guide the interaction between E3 ubiquitin ligase DCAF15 and RBM39 to promote the degradation of RBM39. In short, the development of molecular gel and PROTAC technology provides a new strategy for targeting disease-causing proteins, including many proteins that are difficult to target by traditional methods.
A team of scientists from the University of Wisconsin-Madison unveiled a new MDM2 degrader they developed in a paper published in the European Journal of Medicinal Chemistry.
MDM2 (Murine double minute 2) is a key negative regulator of p53 (a powerful tumor suppressor and the most frequently mutated gene in human cancer) that is highly expressed in tumors and plays an important role in the occurrence and development of tumors. Previous studies showed that MDM2 can not only bind to p53 to block its tumor inhibitory transactivation domain, the protein itself is also an E3 ligase that can label p53 to be degraded by proteasomes. Since the wild-type p53 gene is retained in about 50% of human cancers, but the tumor inhibitory function of these p53 is weakened by signal molecules such as MDM2, the researchers hoped that patients carrying wild-type p53 can restore the anti-tumor activity of p53 by developing small molecular inhibitors or degradants to block the interaction between p53 and MDM2.
In August 2019, the team of scientists reported that they had developed an efficient MDM2-PROTAC—WB156 consisting of nutlin derivatives and CRBN ligand lenalidomide. In leukemic cells, WB156 can effectively deplete MDM2, activate wild-type p53, and then induce apoptosis, which, however, can only play a role in a limited number of leukemic cell lines.
To overcome this bottleneck, scientists hypothesized that the fusion of different MDM2 ligands might enable MDM2 degradants to work in a wider range of cancers. In the newly published study, they first used “MDM2 ligand 1” and “MDM2 ligand 2” to prepare ligands for the development of MDM2 degradation agents by four-component Ugi reaction, and then used these ligands as binder of MDM2 to construct active MDM2 degradation agents. After extensive optimization, WB214 has been identified as the most effective antiproliferative drug in various leukemic cell lines.
Surprisingly, mechanism studies have shown that this new WB214 degrader does not activate p53 but induces the degradation of p53, which is completely contrary to the MDM2 degrader WB156 previously reported by the team.
Scientists have conducted a series of experiments to investigate the underlying mechanism behind this effect. The results show that:
- WB214-mediated degradation of MDM2-p53 complex is achieved through ubiquitin-proteasome system.
- p53 is a bystander in the process of MDM2 degradation because it is directly related to MDM2, resulting in “bystander degradation effect”.
- WB214-CRBN complex does not bind to MDM2 at p53 binding site (in contrast, MDM2 ligand 1 or WB13 binds MDM2 at p53 binding site).
The researchers further analyzed the formation of ternary complex induced by WB214, and confirmed that WB214 can effectively induce the formation of ternary complex in a dose-dependent manner, and induced stronger CRBN-MDM2 interaction than WB156 and WB214. The data show that, unlike WB156 (real MDM2 PROTAC), WB214 does not degrade MDM2 through the classical PROTAC mechanism. The action mechanism of WB214 is more consistent with that of molecular gel. In other words, WB214 binds to MDM2 simply by interacting with its CRBN. According to the authors, this is the first time that MDM2 can be used as a neo-substrate for CRBN.