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Live-Cell NanoBRET Assay to Measure AKT Inhibitor Binding to Conformational States of AKT
Jeremy W Harris1, Flávio Antônio de Oliveira Simões1, Erin N Ryerson1
1Department of Genetics, The University of Alabama at Birmingham, Birmingham, Alabama 35294, United States.
Abstract:
AKT is the main protein kinase of the PI3K-AKT pathway, interacting with over one hundred protein partners to facilitate cellular processes that allow cancer cells to survive and proliferate. It is an attractive target due to its control over many cellular outputs. However, ATP-competitive and allosteric AKT inhibitors have performed poorly in clinical trials. AKT inhibitor interactions with AKT are multifaceted and influence the catalytic activity of AKT, its conformation, its ability to interact with binding partners, and its phosphorylation state. Therefore, a better understanding of how these inhibitors influence these parameters is needed, especially in a cellular context. Using a live-cell NanoBRET target engagement assay to query the binding of AKT inhibitors to all isoforms of AKT, we found that ATP-competitive inhibitors bind similarly across all three isoforms and allosteric inhibitors bind more heterogeneously. Further, assaying gain-of-function pathological mutants and myristoylated active versions of all AKT isoforms revealed that T308 phosphorylation enhances the binding of ATP-competitive inhibitors. We found that this phosphorylation is a good indicator of cell viability sensitivity to ATP-competitive inhibitors when comparing effects on known resistant and sensitive triple-negative breast cancer cell lines. Taken together, these findings are useful for screening new AKT inhibitors, and these findings represent important considerations in developing the next generation of AKT inhibitors.
Insights
Understanding how AKT inhibitors interact with AKT is crucial for cancer therapy. This study reveals phosphorylation at T308 enhances ATP-competitive inhibitor binding, aiding the development of next-generation AKT inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The PI3K-AKT pathway is central to cancer cell survival and proliferation.
- AKT protein kinase is a key regulator, making it an attractive therapeutic target.
- Current ATP-competitive and allosteric AKT inhibitors show limited clinical efficacy.
Purpose of the Study:
- To investigate the multifaceted interactions between AKT inhibitors and AKT isoforms within a cellular context.
- To understand how inhibitor binding affects AKT's catalytic activity, conformation, partner interactions, and phosphorylation.
- To guide the development of more effective next-generation AKT inhibitors.
Main Methods:
- Utilized a live-cell NanoBRET target engagement assay to assess AKT inhibitor binding across all AKT isoforms.
- Examined inhibitor interactions with pathological mutants and myristoylated active AKT variants.
- Correlated T308 phosphorylation status with cell viability in response to ATP-competitive inhibitors.
Main Results:
- ATP-competitive inhibitors demonstrated similar binding across all AKT isoforms.
- Allosteric inhibitors exhibited heterogeneous binding patterns among AKT isoforms.
- T308 phosphorylation was found to enhance ATP-competitive inhibitor binding.
- T308 phosphorylation served as a reliable indicator of cell sensitivity to ATP-competitive inhibitors.
Conclusions:
- The binding characteristics of AKT inhibitors vary significantly among isoforms and are influenced by phosphorylation.
- T308 phosphorylation is a critical factor modulating the efficacy of ATP-competitive AKT inhibitors.
- These findings provide valuable insights for screening new AKT inhibitors and designing improved therapeutic strategies.
