MDM2 degraders turned out to degrade p53?

PROTACs, fully known as Proteolysis-Targeting Chimeras, or proteolytically targeted chimeras, are a new drug type different from antibodies and traditional small molecule inhibitors and consist of three parts: target protein binder, linker, and E3 ubiquitin ligase binder. That is, one end of the PROTAC molecule binds to the target protein and the other end binds to the E3 ubiquitin ligase. E3 ubiquitin ligases, on the other hand, can label small proteins called ubiquitin as defective or damaged proteins by attaching them to target proteins. Afterwards, the cell's protein crusher (i.e., proteasome) recognizes and degrades labeled target proteins.

In recent years, as a new way to regulate proteostasis, PROTAC has received much attention from academia and industry, and many teams are actively developing protein-degrading agents based on PROTAC technology. In terms of target proteins, it is statistically stated that there are currently more than 100 popular targets targeted by PROTAC, and the fastest progressing ones include AR-PROTAC, ER-PROTAC, BTK-PROTAC, etc.; in terms of E3 ubiquitin ligases, there are currently two most commonly used for PROTAC development, which are von Hippel Lindau (VHL) and cereblon (CRBN).

CRBN is part of the CRL4 E3 ubiquitin ligase that recognizes substrate proteins as substrate receptors (SRs), thereby initiating the degradation process. CRLs, the largest family of E3 ubiquitin ligases, form over 250 CRLs by assembling substrate acceptor and adapter proteins around different cullin backbones.

Simply put, molecular gel-degrading agents are a class of small molecules that can induce novel interactions between E3 ubiquitin ligase substrate receptors (e.g., CRBN) and target proteins, which lead to target protein degradation. Thalidomide, lenalidomide, and pondolamine small molecule immunomodulators are a remarkable example of molecular glues that redirect CRBN, thereby polyubiquitinating transcription factors IKZF1 and IKZF3, resulting in IKZF1 and IKZF3 degradation by the proteasome. Analogously, the anticancer sulfonamide indisulam also guides the interaction between the E3 ubiquitin ligase DCAF15 and RBM39 and promotes the degradation of RBM39. In conclusion, the development of molecular glue and PROTAC technology provides a new strategy for targeting pathogenic proteins, including many proteins targeted using traditional methods.

A team of scientists from the University of Wisconsin-Madison announced their development of a new type of MDM2 degradant in a paper published in the European Journal of Medicinal Chemistry.

MDM2, fully known as mouse double minute 2, is a key negative regulator of p53 (a powerful tumor suppressor, the most frequently mutated gene in human cancer), which is highly expressed in tumors and plays an important role in tumor development and progression. Previous studies have shown that MDM2 can not only bind to p53 to block its tumor suppressor transactivation domain, the protein itself is an E3 ligase that labels p53 for degradation by the proteasome. Since the wild-type p53 gene is retained in about 50% of human cancers, but the tumor suppressor function of these p53 is weakened by signaling molecules such as MDM2, the researchers hope that patients carrying wild-type p53 can restore the tumor suppressor activity of p53 by developing small molecule inhibitors or degradation agents to block the interaction between p53 and MDM2.

In August 2019, the team of scientists reported that they had developed a highly effective MDM2-PROTAC, WB156, which consists of a nutlin derivative linked to the CRBN ligand lenalidomide. In leukemia cells, WB156 is able to efficiently deplete MDM2 and activate wild-type p53, which in turn induces apoptosis. However, although WB156 is able to effectively degrade MDM2, induce p53 activation, and show anti-proliferative effects, this molecule can only act in a limited number of leukemia cell lines.

To overcome the bottleneck, scientists envision that fusion of different MDM2 ligands may enable MDM2 degraders to act in a wider range of cancers. In this newly published new study, they first prepared ligands for the development of MDM2 degraders by performing a four-component Ugi reaction using "MDM2 ligand 1" and "MDM2 ligand 2", and then used these ligands as binders of MDM2 to construct active MDM2 degraders. After extensive optimization, WB214 was identified as the most effective antiproliferative agent in various leukemia cell lines.

Surprisingly, mechanistic studies showed that this novel WB214 degrader did not activate p53, and conversely, WB214 induced the degradation of p53, which is completely opposite to the MDM2 degrader WB156 previously reported by the team.

Scientists have conducted a series of experiments to investigate the potential mechanisms of action behind this effect. The results showed that WB214-mediated degradation of the MDM2-p53 complex was achieved through the ubiquitin-proteasome system; p53 was a bystander in the MDM2 degradation process because it was directly associated with MDM2, thus producing a "bystander degradation effect"; and the WB214-CRBN complex did not bind MDM2 at the p53-binding site (in contrast, either MDM2 ligand 1 or WB13 bound MDM2 at the p53-binding site).

Since ternary complex formation is a prerequisite for proteasomal degradation, the researchers further analyzed WB214-induced ternary complex formation and confirmed that WB214 was able to effectively induce ternary complex formation in a dose-dependent manner and that WB214 induced a stronger CRBN-MDM2 interaction than WB156. These data suggest that unlike WB156 (bona fide MDM2 PROTAC), WB214 does not degrade MDM2 via the classical PROTAC mechanism. The mechanism of action of WB214 is more consistent with molecular glue. In other words, WB214 simply leads to binding to MDM2 by interacting with its CRBN. The authors state that MDM2 was first discovered to act as a new substrate (neo-substrate) for CRBN.