Nat Chem Biol | Rao Yu/Ying Meidan team designed CDK2 degradation agent and applied it to AML differentiation treatment​

Cyclin-dependent kinases (CDK) are a group of serine/threonine protein kinases with a shorter N-terminus (β-sheet) and a longer C-terminus (a-helix).

CDK interacts with the corresponding cyclin and cyclin-dependent kinase activated kinase (CAK), plays a role in all stages of the cell cycle, participates in the physiological processes of cell growth, differentiation, and proliferation, and mediates the orderly cell cycle get on.

CDK2 is a member of the CDK family and is widely expressed in mammalian cells, but its function can be compensated by other CDK family members, so CDK2 is not necessary for most normal cells and tissues.

However, CDK2 plays an important role in tumorigenesis, cell differentiation, meiosis, and hearing damage repair.
Recent studies have found that CDK2 knockdown can induce differentiation of AML cells.

However, traditional small molecule inhibitors are difficult to achieve selective CDK2 inhibition, nor can they eliminate the non-enzymatic function of CDK2, with insufficient efficacy and serious side effects.

Gene editing technology has many shortcomings in application efficiency and clinical use.

Although it is considered that CDK2 is a good drug target, it is difficult for existing tools to achieve efficient and selective regulation of it.

PROTAC technology is a technology that uses small molecules to induce ubiquitination of target proteins and uses the ubiquitinated proteasome pathway to achieve targeted protein knockdown.

PROTAC is composed of target protein ligand, E3 ubiquitin ligase ligand and linker (Figure 1).

The small molecule can induce the target protein to interact with the E3 ubiquitin ligase, thereby realizing the non-natural ubiquitination of the target protein, and the ubiquitinated target protein is further recognized and degraded by the proteasome.

The degradation achieved by PROTAC technology not only depends on the affinity of small molecules, but also requires the specific spatial interaction between the target protein and the E3 ubiquitin ligase.

Therefore, under reasonable design and fully optimized conditions, PROTAC technology can theoretically achieve better selectivity compared with traditional small molecule inhibitors.

At present, there is no literature report on the highly selective CDK2 knockdown PROTAC molecule.

Figure 1.
The principle of PROTAC technology.
On March 4, 2021, the Rao Yu team of the School of Pharmacy of Tsinghua University and the Ying Meidan team of the School of Pharmacy of Zhejiang University published a titled Discovery of a first-in-class CDK2 in Nature Chemical Biology.
A research paper on selective degrader for AML differentiation therapy.
This work reports a new type of CDK2 selective degrading agent that achieves high-efficiency and low-toxicity in AML cell differentiation therapy, and provides a potential differentiation therapy for AML therapy.

How to achieve selective knockdown of CDK2 is the core technical problem that the author solves in this work.

The affinity level is the basis for the function of PROTAC.
In order to maintain the affinity with the CDK2 protein, the researchers have fully simulated the molecular structure.
The structural units that have important interactions with CDK2 need to be retained and the sites exposed to the solvent region are used for Linker extension, this design can retain the level of affinity with CDK2 to the greatest extent, while reducing the affinity with non-targeted proteins.

After multiple rounds of iterative design and molecular structure optimization, finally based on the selected ligands, the CDK2 selective degradation agent CPS2 with J2 as the ligand was successfully developed.

The new type of CDK2 degradation agent developed by researchers has the following characteristics.

Wide range of applications: In this work, the author tried to test more than 10 cell lines, CPS2 can achieve effective knockdown of CDK2 at nanomolar concentrations.

Good selectivity: The selectivity of CPS2 is significantly improved with smaller molecular inhibitors.

The author used kinomics, kinase activity test, proteomics, and western blot experiments to prove that when CPS2 is used to treat cells, the degradation of CDK2 is the most significant within the observable protein range, while it has no significant effect on other proteins.
(figure 2).

Figure 2.
CPS2 induces efficient and selective degradation of CDK2 in a variety of cell lines.
CPS2 solves the problem of the selectivity of CDK2 inhibitors and is less toxic to normal cells (Beas2b, 293T cell lines), but CPS2 can inhibit tumor cells The way of proliferation produces anti-tumor effect (Figure 3).

The author also conducted acute toxicity experiments in animals, and the results showed that the safety of CPS2 is much higher than that of its parent nucleus inhibitor J2 (Figure 4).

Figure 3.
CPS2 has little effect on normal cells, but it can block the rapid proliferation of tumor cells.
Figure 4.
CPS2 is safer in vivo.
Researchers have further applied CPS2 in the differentiation therapy of AML.

Under CPS2 treatment, the differentiation index of AML cells was significantly increased, and the cell morphology was more mature (Figure 5).

At the same time, the authors conducted multiple sets of Rescue experiments, which proved that CPS2 performs the differentiation-inducing function by causing the degradation of CDK2 in cells.

In order to fully demonstrate the importance of CPS2 as a selective degradation agent of CDK2 in the clinical treatment of AML, the researchers collected multiple primary cells from AML patients and added CPS2 for treatment.

The results show that the primary cells can also be significantly differentiated under the treatment of drugs, which fully proves the potential clinical application significance of CPS2.

Figure 5.
Under CPS2 treatment, AML cells differentiated significantly.
In this work, the researchers developed a class of CDK2 selective degradation agents and initially studied the safety and effectiveness of the molecule.

The researchers applied this new discovery to the field of AML differentiation therapy, providing a potentially high-efficiency, low-toxicity new treatment plan for AML treatment (Figure 6).

In addition to the treatment of AML, this type of degrading agent may also play an important role in the treatment of other diseases related to CDK2.

Figure 6.
The functional characteristics of CPS2 Tsinghua University doctoral student Wang Liguo, Zhejiang University postdoctoral student Xuejing Shao, Tsinghua University doctoral student Zhong Tianbai, Tsinghua University doctoral student Wu Yue and Zhejiang University doctoral student Xu Aixiao are the co-first authors of this work, and Tsinghua University Rao Yue The professor and Professor Ying Meidan of Zhejiang University are the co-corresponding authors.

This research was greatly helped by Professor Wu Wei, Professor Tao Qinghua, Professor Li Haitao, and Professor Wang Qianfei from the Beijing Institute of Genomics, Chinese Academy of Sciences.

Link to the original text: https://doi.
org/10.
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