Protein kinases are key cancer therapy targets. But kinase studies are hindered by lack of powerful in vivo analysis methods. We address this challenge with Akt1 and Akt2 as initial model. Though sharing high sequence homology, they display functional different in metastasis. In colon cancer, Akt2 is a strong promoter of metastasis, but Akt1 is an inhibitor. Understanding the difference begs for better analysis methods. Previously, we have combined the chemical genetic method developed by Dr. Kevan Shokat and nanoparticle intracellular delivery of the bulky ATP analog N6-Benzyl-ATP-gamma-S (A*TP-gamma-S). According to the Shokat method, we mutated the gate-keeper Methionine to Glycine, creat...
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Protein kinases are key cancer therapy targets. But kinase studies are hindered by lack of powerful in vivo analysis methods. We address this challenge with Akt1 and Akt2 as initial model. Though sharing high sequence homology, they display functional different in metastasis. In colon cancer, Akt2 is a strong promoter of metastasis, but Akt1 is an inhibitor. Understanding the difference begs for better analysis methods. Previously, we have combined the chemical genetic method developed by Dr. Kevan Shokat and nanoparticle intracellular delivery of the bulky ATP analog N6-Benzyl-ATP-gamma-S (A*TP-gamma-S). According to the Shokat method, we mutated the gate-keeper Methionine to Glycine, creating Akt(m-g) with enlarged ATP binding pocket. Nanoparticle-delivered A*TP-gamma-S was used exclusively by Aktm-g in kinase reactions in live cells. Thus, we created an in vivo version of the Shokat method, i.e., Akt-substrate relationship determination in live cells. This project has two goals. First, we are creating fluorogenic version of A*TP (MANT- and TNP-A*TP). Nanoparticle-delivered MANT- or TNP-A*TP, fluorescent only upon Akt(M-G) binding, serve as fluorescent probes for in vivo Akt activity quantification. This method will enable previously impossible fluorescent library screening for Akt1 or Akt2 modulators in live cells. Second, we are generating the Akt(M-G) with CRISPR/Cas9-genomic-editing, so that Akt(M-G) stays in the native genomic context. Then, a comparative mass-spectrometry Akt1 and Akt2 substrate analysis based on live cell substrate thio-phosphorylation via nanoparticle-delivered A*TP-gamma-S will be performed. Given the Akt1 and Akt2 functional difference in cancer, the former will lead to valuable therapeutic agents, and the latter to downstream therapeutic targets. The strategy should be widely applicable, setting a new paradigm for studying oncogenic and tumor suppressive protein kinases as well as targeted therapeutic development.
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