Abstract
Akt plays a major role in tumorigenesis and the development of specific Akt inhibitors as effective cancer therapeutics has been challenging. Here, we report the identification of a highly specific allosteric inhibitor of Akt through a FRET-based high-throughput screening, and characterization of its inhibitory mechanism. Out of 373,868 compounds screened, 4-phenylquinolin-2(1H)-one specifically decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC50 = 6 µM) and downstream signaling. 4-Phenylquinolin-2(1H)-one did not alter the activity of upstream kinases including PI3K, PDK1, and mTORC2 as well as closely related kinases that affect cell proliferation and survival such as SGK1, PKA, PKC, or ERK1/2. This compound inhibited the proliferation of cancer cells but displayed less toxicity compared to inhibitors of PI3K or mTOR. Kinase profiling efforts revealed that 4-phenylquinolin-2(1H)-one does not bind to the kinase active site of over 380 human kinases including Akt. However, 4-phenylquinolin-2(1H)-one interacted with the PH domain of Akt, apparently inducing a conformation that hinders S473 and T308 phosphorylation by mTORC2 and PDK1. In conclusion, we demonstrate that 4-phenylquinolin-2(1H)-one is an exquisitely selective Akt inhibitor with a distinctive molecular mechanism, and a promising lead compound for further optimization toward the development of novel cancer therapeutics.
Highlights
Akt is a serine/threonine protein kinase that belongs to the AGC group within the eukaryotic protein kinase superfamily
We devised an HTRF assay using fluorophore d2 conjugated to anti-Akt monoclonal antibody (Mab) and europium cryptate conjugated to anti-pS473-Akt Mab as the acceptor and donor fluorophores
To test the suitability of the HTRF assay, Neuro 2A cells were treated with insulin-like growth factor (IGF) which is known to phosphorylate Akt and the cell lysates were incubated with the fluorophore-conjugated antibodies
Summary
When activated Akt was incubated with ATP and a substrate peptide in the presence of 20 μM G7 and the phosphorylation of the substrate peptide was evaluated by an ELISA-based assay (Fig. 6a), only negligible change of the substrate phosphorylation was observed, indicating that G7 did not interfere with the ATP or substrate binding. Allosteric inhibitors including MK-2206 and Akt Inhibitor VIII (EMD Millipore) are thought to interact with both PH and the kinase domains promoting the formation of inactive conformation and impeding membrane translocation of Akt[23, 52] Unlike these cases, G7 did not appear to affect the Akt-membrane interaction and the resulting interdomain conformational changes detected by the chemical cross-linking approach (Fig. 7c). We have identified an allosteric Akt inhibitor with excellent selectivity and less toxicity, presenting a promising lead compound for further optimization and development of novel therapeutic agents against cancers
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