Live-cell NanoBRET assay to measure AKT inhibitor binding to conformational states of AKT

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 multi-faceted 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, this assay is useful for screening new AKT inhibitors, and these findings represent important considerations in developing the next generation of AKT inhibitors.