Structure-based design, synthesis and biological characterization of isoform-selective covalent-allosteric Akt inhibitors

Abstract

Akt is a serine/threonine kinase that serves as a central regulator of the PI3K/mTORC signaling pathway and plays a critical role in cell survival and metabolism. Dysregulation of Akt is frequently associated with cancer, making it an attractive therapeutic target. However, the three highly homologous Akt isoforms (Akt1, Akt2 and Akt3) perform distinct biological functions, while currently available inhibitors lack isoform selectivity, resulting in off-target effects and limited clinical success. This dissertation aimed to develop isoform-selective covalent-allosteric Akt inhibitors to enable more precise therapeutic modulation. To address the limited structural information available for Akt2 and Akt3, homology modeling, sequence analysis and molecular docking were integrated into a structure-guided drug design strategy. A modular synthetic approach enabled the preparation of a focused library of new inhibitors designed to exploit subtle structural differences within the allosteric pockets of the Akt isoforms. Comprehensive biochemical characterization using HTRF assays, together with NanoBRET target engagement studies, identified potent Akt2-, Akt3- and dual Akt2/Akt3-selective inhibitors. Functional selectivity was further confirmed by CTG cell viability assays in an Akt1-sensitive cellular model and Western blot analyses, while protein mass spectrometry demonstrated covalent target engagement. Moreover, co-crystal structures obtained using an engineered Akt1 construct mimicking the Akt2 allosteric pocket provided detailed structural insights into ligand binding and the molecular basis of isoform selectivity. Collectively, this work establishes covalent-allosteric inhibition as a viable strategy for achieving Akt isoform selectivity and provides a structural and medicinal chemistry framework for the rational development of next-generation precision kinase inhibitors.