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Published on: March 13, 2018
Enhancing Akt imaging through targeted reporter expression
Limin Zhang1, Mahaveer S Bhojani, Brian D Ross
1Department of Radiation Oncology, Center for Molecular Imaging, University of Michigan, Ann Arbor, MI 48109, USA.
Abstract:
The serine/threonine kinase PKB/Akt is a key mediator of survival and resistance to cancer therapy. Pharmacologic inhibition of Akt and its biologic sequelae may significantly impact the treatment of cancer. The use of molecular imaging technologies has contributed significantly to drug discovery research with an emphasis on drug efficacy, the mechanism of action, and target validation studies. We constructed a genetically engineered hybrid bioluminescent Akt reporter (BAR) molecule that reports on Akt serine/threonine kinase activity. Based on the fact that Akt is recruited to the plasma membrane on activation, we here describe a modified version of this reporter molecule (myristoylated and palmitoylated bioluminescent Akt reporter [MyrPalm-BAR]), which is membrane bound and whose bioluminescence activity can be used to monitor Akt activity at the cell membrane. Using changes in Akt activation status with small molecule inhibitors of Akt, we demonstrated that the membrane-targeted Akt reporter was more sensitive and quantitative. In addition, inhibition of upstream signaling kinases such as epidermal growth factor receptor and phosphatidylinositol 3-kinase activity resulted in changes in Akt activity that were quantitatively monitored by bioluminescence imaging. Based on these results, we propose that the membrane-associated Akt reporter may be better suited for identification of novel compounds that modulate the Akt pathway by high-throughput screening.
Insights
A novel membrane-bound bioluminescent Akt reporter (MyrPalm-BAR) offers a sensitive and quantitative method for monitoring Akt kinase activity. This tool aids in discovering new cancer drugs targeting the Akt pathway.
Area of Science:
- Molecular Biology
- Cancer Research
- Biotechnology
Background:
- The serine/threonine kinase Akt (Protein Kinase B) is crucial for cancer cell survival and therapy resistance.
- Targeting Akt is a significant strategy in cancer treatment.
- Molecular imaging aids in drug discovery, efficacy assessment, and target validation.
Purpose of the Study:
- To develop a genetically engineered bioluminescent reporter for Akt kinase activity.
- To create a membrane-localized version (MyrPalm-BAR) for enhanced sensitivity and quantification of Akt activity.
- To validate the reporter's utility in monitoring Akt pathway modulation by small molecule inhibitors.
Main Methods:
- Construction of a hybrid bioluminescent Akt reporter (BAR) molecule.
- Modification to create a membrane-bound version (myristoylated and palmitoylated bioluminescent Akt reporter [MyrPalm-BAR]).
- Utilized bioluminescence imaging to monitor Akt activity in response to small molecule inhibitors targeting Akt and upstream kinases (EGFR, PI3K).
Main Results:
- The membrane-targeted MyrPalm-BAR reporter demonstrated higher sensitivity and quantitative accuracy compared to non-membrane-bound reporters.
- Bioluminescence imaging effectively monitored changes in Akt activity upon inhibition of Akt itself or upstream signaling pathways.
- The reporter quantitatively tracked Akt activation status changes induced by small molecule inhibitors.
Conclusions:
- The membrane-associated Akt reporter (MyrPalm-BAR) is a sensitive and quantitative tool for assessing Akt kinase activity.
- This reporter is well-suited for high-throughput screening to identify novel compounds that modulate the Akt pathway.
- The MyrPalm-BAR reporter facilitates drug discovery and validation in cancer therapy research.
