Kinase Conformations Reveal Insights into Regulatory Mechanisms and Drug Interactions

Conceitos essenciais

Genetically encoded KinCon reporters enable systematic monitoring of cellular kinase activity states and conformations, providing insights into the impact of protein interactions, mutations, and small molecule inhibitors on kinase regulation.

Resumo

The content discusses the use of the KinCon reporter technology to study the conformational dynamics of various disease-associated kinases, including BRAF, LKB1, RIPK1, and CDK4/6. Key highlights:

The KinCon reporter system is a Renilla luciferase-based protein fragment complementation assay that can track changes in kinase conformations in live cells in response to factors like mutations, protein-protein interactions, post-translational modifications, and small molecule binding.

For the BRAF kinase, the KinCon reporter was used to show that FDA-approved melanoma drugs and a drug candidate can convert the opened, active BRAF-V600E conformation to a more closed, inactive state.

The LKB1-STRADα-MO25 trimeric complex formation was found to promote the closing and activation of the LKB1 KinCon reporter. Certain patient-derived LKB1 mutations disrupted this complex formation and prevented the closing of the LKB1 conformation.

RIPK1 KinCon reporters revealed that both activating and inactivating mutations, as well as allosteric inhibitors, can induce conformational changes in RIPK1, suggesting a role for structural dynamics in regulating its scaffolding and catalytic functions.

For CDK4/6, the KinCon reporters showed that mutations reducing the binding affinity for the inhibitor p16INK4a led to a more opened conformation, but clinically used CDK4/6 inhibitors did not induce significant conformational changes.

The study highlights how the KinCon technology can provide insights into the structural dynamics of kinases and their regulation by diverse cellular factors, which is crucial for designing more effective therapeutic strategies.

Estatísticas

Transient over-expression of BRAF, LKB1, RIPK1, and CDK4/6 KinCon reporters in HEK293T cells showed consistently increasing bioluminescence signals over time (10h, 16h, 24h, 48h).
The BRAF-V600E KinCon reporter exhibited a more opened conformation compared to wild-type BRAF, which was converted to a more closed state upon treatment with the melanoma drug PLX8394.
Co-expression of LKB1, STRADα, and MO25 led to a more than 10-fold increase in the STRADα KinCon reporter signal, indicating a shift to a partially closed conformation.
Mutations in RIPK1 (S14/15/166A, S14/15/166E, K45A, D324A/E/H, C601Y) all resulted in an opened conformation of the RIPK1 KinCon reporter.
The CDK4-R24C and CDK6-R31C mutations, which reduce binding to the inhibitor p16INK4a, led to a more opened conformation of the respective KinCon reporters.

Citações

"The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies."
"Alterations of the ON and OFF modes of regulating kinases are also pertinent to kinase-related disorders. Deregulation of kinase functions is associated with the development of numerous diseases, such as cancer, inflammatory, infectious and degenerative conditions."
"Conventional methods often fall short in capturing the dynamics of kinase phosphotransferase and scaffolding activities within their native cellular environments, making innovative biotech approaches essential for a comprehensive understanding of their roles in the cell."

Principais Insights Extraídos De

Kinases in motion: impact of protein and small molecule interactions on kinase conformations

by Kugler,V., S... às www.biorxiv.org 01-12-2024

https://www.biorxiv.org/content/10.1101/2024.01.11.575270v3

Perguntas Mais Profundas

How could the KinCon reporter technology be further developed or combined with other techniques to provide a more comprehensive understanding of kinase regulation and drug interactions?

The KinCon reporter technology has shown great potential in tracking kinase conformational changes in response to various stimuli, including drug interactions and protein binding. To further enhance its capabilities and provide a more comprehensive understanding of kinase regulation and drug interactions, several strategies could be considered:

Integration with High-Resolution Imaging Techniques: Combining KinCon reporters with high-resolution imaging techniques such as super-resolution microscopy or cryo-electron microscopy can provide detailed insights into the spatial organization of kinases and their interactions with regulatory proteins or inhibitors.

Multiplexing Assays: Developing multiplexing assays that allow the simultaneous monitoring of multiple kinases and their conformational changes can provide a more holistic view of kinase signaling networks and their responses to different stimuli.

Live-Cell Imaging: Incorporating live-cell imaging approaches can enable real-time visualization of kinase dynamics in living cells, allowing for the observation of temporal changes in kinase conformation and activity in response to various stimuli.

Integration with Proteomics: Combining KinCon technology with proteomic approaches such as mass spectrometry can help identify novel kinase substrates, regulatory proteins, and signaling pathways, providing a more comprehensive understanding of kinase regulation.

Development of Kinase-Specific Biosensors: Designing kinase-specific biosensors that can selectively monitor the conformational changes of individual kinases or kinase families can offer a more targeted approach to studying kinase regulation and drug interactions.

What are the potential limitations or caveats of the KinCon reporter approach, and how could they be addressed to improve the reliability and applicability of the system?

While the KinCon reporter approach offers valuable insights into kinase conformational dynamics, there are some limitations and caveats that should be considered:

Overexpression Artifacts: High levels of exogenous expression of KinCon reporters may lead to non-physiological interactions and conformational changes. Optimizing the expression levels of KinCon reporters to mimic endogenous kinase levels can help mitigate this issue.

Cell Line Specificity: The behavior of KinCon reporters may vary across different cell lines, potentially impacting the reliability and reproducibility of the results. Validating the KinCon technology in multiple cell types can help address this limitation.

Limited Spatial Resolution: The KinCon reporter technology provides information on kinase conformational changes at the whole-cell level, lacking spatial resolution. Combining KinCon reporters with spatially resolved techniques can help overcome this limitation.

Inability to Capture Post-Translational Modifications: KinCon reporters may not capture the effects of post-translational modifications on kinase conformation. Integrating KinCon technology with phospho-specific antibodies or mass spectrometry can provide a more comprehensive view of kinase regulation.

Need for Validation: Validating the KinCon reporter results with orthogonal techniques such as biochemical assays or structural studies is essential to ensure the accuracy and reliability of the findings.

Given the diverse roles of kinases in cellular signaling and disease, how could insights from KinCon studies on kinase conformations be leveraged to develop novel therapeutic strategies beyond the examples discussed in the content?

Insights from KinCon studies on kinase conformations can be leveraged to develop novel therapeutic strategies in the following ways:

Identification of Novel Drug Targets: KinCon studies can help identify novel kinase conformations associated with disease states, providing potential new drug targets for therapeutic intervention.

Personalized Medicine: Understanding the conformational dynamics of individual kinases in patient samples can enable the development of personalized treatment strategies tailored to the specific kinase profiles of patients.

Combination Therapies: Insights from KinCon studies can guide the development of combination therapies targeting multiple kinases or signaling pathways simultaneously, potentially overcoming drug resistance and improving treatment outcomes.

Drug Repurposing: KinCon studies can reveal new conformational states of kinases that may be targeted by existing drugs, leading to the repurposing of approved drugs for novel therapeutic indications.

Development of Allosteric Inhibitors: KinCon studies can inform the design of allosteric inhibitors that target specific kinase conformations, offering a new approach to modulating kinase activity with increased selectivity and efficacy.

By leveraging the insights gained from KinCon studies on kinase conformations, researchers and clinicians can advance the development of innovative therapeutic strategies for a wide range of diseases beyond the examples discussed in the content.