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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Crystallographic approach to identification of cyclin-dependent kinase 4 (CDK4)-specific inhibitors by using CDK4
M Ikuta1, K Kamata, K Fukasawa
1Banyu Tsukuba Research Institute/ Merck Research Laboratories, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan.
Abstract:
Genetic alteration of one or more components of the p16(INK4A)-CDK4,6/cyclin D-retinoblastoma pathway is found in more than half of all human cancers. Therefore, CDK4 is an attractive target for the development of a novel anticancer agent. However, it is difficult to make CDK4-specific inhibitors that do not possess activity for other kinases, especially CDK2, because the CDK family has high structural homology. The three-dimensional structure of CDK2, particularly that bound with the inhibitor, has provided useful information for the synthesis of CDK2-specific inhibitors. The same approach used to make CDK4-specific inhibitors was hindered by the failure to obtain a crystal structure of CDK4. To overcome this problem, we synthesized a CDK4 mimic CDK2 protein in which the ATP binding pocket of CDK2 was replaced with that of CDK4. This CDK4 mimic CDK2 was crystallized both in the free and inhibitor-bound form. The structural information thus obtained was found to be useful for synthesis of a CDK4-specific inhibitor that does not have substantial CDK2 activity. Namely, the data suggest that CDK4 has additional space that will accommodate a large substituent such as the CDK4 selective inhibitor. Inhibitors designed to bind into this large cavity should be selective for CDK4 without having substantial CDK2 activity. This design principle was confirmed in the x-ray crystal structure of the CDK4 mimic CDK2 with a new CDK4 selective inhibitor bound.
Insights
Developing specific CDK4 inhibitors for cancer therapy is challenging due to structural similarities with CDK2. A novel CDK4 mimic protein enabled the design of selective CDK4 inhibitors, overcoming this challenge.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- The p16(INK4A)-CDK4,6/cyclin D-retinoblastoma pathway is frequently altered in human cancers, making CDK4 a key therapeutic target.
- Developing selective CDK4 inhibitors is difficult due to high structural homology with other kinases, particularly CDK2.
Purpose of the Study:
- To overcome the challenge of synthesizing CDK4-specific inhibitors by developing a novel strategy based on structural mimicry.
- To obtain structural information of CDK4 in complex with inhibitors to guide the design of selective anticancer agents.
Main Methods:
- Synthesis of a CDK4 mimic CDK2 protein by replacing the ATP binding pocket of CDK2 with that of CDK4.
- Crystallization of the CDK4 mimic CDK2 protein in both free and inhibitor-bound forms.
- X-ray crystallography to determine the three-dimensional structure of the CDK4 mimic CDK2-inhibitor complex.
Main Results:
- The CDK4 mimic CDK2 protein was successfully synthesized and crystallized, providing valuable structural insights.
- Structural data revealed an additional binding space in CDK4, absent in CDK2, suitable for larger substituents.
- This information facilitated the design and confirmation of a novel CDK4-selective inhibitor with minimal CDK2 activity.
Conclusions:
- A CDK4 mimic protein strategy is effective for obtaining structural information and designing selective kinase inhibitors.
- The identified structural feature in CDK4 allows for the development of potent and specific anticancer agents targeting this kinase.
- This approach advances the development of targeted cancer therapies by enabling the creation of highly selective kinase inhibitors.
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