Live-Cell NanoBRET Assay to Measure AKT Inhibitor Binding to Conformational States of AKT
AKT kinase plays a pivotal role in the PI3K-AKT signaling pathway, critical for cell growth, survival, and proliferation, particularly in cancer cells. Despite significant interest, existing ATP-competitive and allosteric inhibitors targeting AKT have faced challenges in clinical efficacy, likely due to complex interactions affecting AKT’s conformation and phosphorylation states.
In this study, Harris and colleagues developed a live-cell NanoBRET assay to precisely evaluate how AKT inhibitors interact with different isoforms (AKT1, AKT2, AKT3) and conformational states of AKT within a cellular environment. Their findings indicate that ATP-competitive inhibitors exhibit uniform binding across all isoforms, while allosteric inhibitors show variability in isoform selectivity.
A significant discovery was the enhanced binding affinity of ATP-competitive inhibitors upon phosphorylation of AKT at residue T308. This phosphorylation state notably improved drug binding and correlated strongly with increased sensitivity of triple-negative breast cancer cells to these inhibitors.
The researchers further explored pathogenic AKT mutants and found improved binding to ATP-competitive inhibitors in these activated states, underscoring the importance of conformational and phosphorylation contexts in drug targeting. Conversely, allosteric inhibitors generally displayed reduced binding efficacy in these mutant forms.
These findings provide a valuable platform for screening and developing AKT inhibitors, particularly emphasizing the importance of T308 phosphorylation as a biomarker for inhibitor efficacy. This approach could significantly impact the strategic design of next-generation targeted therapies for cancer treatment.
Keywords: AKT, NanoBRET, kinase inhibitors, phosphorylation, ATP-competitive inhibitors, allosteric inhibitors, PI3K-AKT pathway, triple-negative breast cancer, target engagement assay, bioluminescence imaging.
