ATP-competitive and allosteric inhibitors induce differential conformational changes at the autoinhibitory interface of Akt1

Akt is a master regulator of pro-growth signaling in the cell. Akt is activated by phosphoinositides that disrupt the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains and then is phosphorylated at T308 and S473. Akt hyperactivation is oncogenic, which has spurred development of potent and selective inhibitors as therapeutics. Using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding Akt ATP-competitive and allosteric inhibitors. We compared inhibitors against three different states of Akt1. The allosteric inhibitor caused substantive conformational changes and restricts membrane binding. ATP-competitive inhibitors caused extensive allosteric conformational changes, altering the autoinhibitory interface and leading to increased membrane binding, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and conformational changes induced by Akt inhibitors and has important implications for the design of Akt targeted therapeutics. [Display omitted] •ATP-competitive inhibitors induce weakening of Akt1’s autoinhibitory interface•ATP-competitive inhibitors lead to increased Akt1 membrane binding•Allosteric Akt inhibitors reorient the PH domain and decreases membrane binding Oncogenic mutations in the protein Akt are observed in cancer, leading to both allosteric and ATP-competitive inhibitor development. Here, Shaw et al., have shown how different classes of inhibitors alter the conformation of Akt1, and how this can be incorporated into future inhibitor design.